surface modification of positively and negatively...
TRANSCRIPT
SURFACE MODIFICATION OF POSITIVELY AND
NEGATIVELY CHARGED POROUS SILICON
NANOPARTICLES AND THEIR BIOLOGICAL
APPLICATIONS
Mezbah Uddin
Masterrsquos Thesis
Department of Chemistry
Medicinal Chemistry
4062012
ii
UNIVERSITY OF EASTERN FINLAND School of Pharmacy
Master Degree Program for Research Chemists
MEZBAH UDDIN
Master Thesis 40 p
Supervisors
Senior Scientist Docent Ale Naumlrvaumlnen and Researcher MSc Jussi Rytkoumlnen
September 2012
KEY WORDS Nanotechnology PEGylation Opsonization Zeta potential Fluorescence intensity
Biofunctionalization
Porous silicon nanoparticles (PSiNPs) have drawn significant attention in recent years in the
study of oncology due to their biocompatibility favorable biodistribution and efficient
clearance or biodegradability The clinical use of PSiNPs as nanocarriers in drug delivery
depends on biologically nonspecific adsorption and immunological response Minimization
of the opsonization in the blood increases the half life of the particles and the capability to
target tumors In this project the opsonization of PEGylated porous silicon nanoparticles was
investigated as it is expected that PEGylation of TCPSi-NH2NPs with Me-PEG-COOH can
contribute to minimization of the opsonization PEGylated PSiNPs were also prepared using
single step reaction between terminal amines in thermally carbonized porous silicon
nanoparticles (TCPSi-NH2NPs) and carboxylic acid groups in mPEG-COOH by refluxing
PEGylated particles were characterized by measuring their size and zeta potential and the
opsonized particles were analyzed by SDS-PAGE The surface of TCPSi-NH2NPs was also
conjugated with fluorescent molecule such as fluorescein-5-isothiocyanate (FITC) and by
tailoring of the acid treated thermally hydrocarbonized porous silicon (UnTHCPSi) NPs by
associating with fluoresceinamine isomer-1 (FluorA) activated with DICNHS activator
FITC labeling TCPSi-NH2NPs were further modified by conjugating stearylated NickFect 51
(NF51) cell penetrating peptides (CPPs) and radioactive iodine labeled stearylated NickFect
cell penetrating peptides Fluorescent labeled NPs were characterized with zeta sizer and
fluorescence spectroscopy The surface modified PSiNPs can be loaded with targeted
therapeutic compounds and studied their biodistribution both in vivo and in vitro
iii
ACKNOWLEDGEMENT
I am heartily thankful to my supervisor Ale Naumlrvaumlnen and Jussi Rytkoumlnen whose
encouragement guidance and support from the initial to the final level enabled me to develop
an understanding of the subject
Finally I would like to offer my regards and blessings to all of those who supported me in
any respect during the completion of the project
Mezbah Uddin
iv
Contents Abbreviations vi
1 Introduction 1
11 Nanotechnology 1
12 Porous Silicon (PSi) 2
121 Properties of Porous Silicon 2
122 Structural Properties of Porous Silicon and Their Effect 2
123 Chemical Properties of the Porous Silicon 2
1 3 Surface Functionalization of Porous Silicon 3
14 Advantages of Porous Silicon 3
15 Porous Silicon Nanoparticles 3
151 Amino-derivatized Thermally Carbonized Porous Silicon Nanoparticles (TCPSi-
NH2 NPs) 4
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous Silicon
Nanoparticles (UnTHCPSiNPs) 5
16 Polyethylene Glycol (PEG) Molecule 5
17 Fluorescent Molecules and Their Applications 6
171 Fluorescence Detection 7
18 Cell-penetrating Peptides (CPPs) 7
19 Opsonization 8
110 Role of Zeta Potential in Characterization of Nanoparticles 10
111 Surface Modification 11
112 PEGylation 12
1121 Affecting Factors on PEGylation 13
1122 Different Sized NP 13
1123 Length amp Conformation of PEG Chain 14
1124 Terminal Portion 15
113 Biofunctionalization 16
114 General Conjugation Reactions 17
2 Experimental 20
21 Materials 20
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation) 21
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and NHS as an Activator
Reagent 21
v
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and DIPEA as an
Activator Reagent 21
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of Amino-modified TCPSi-
NH2NPs and MeO-PEG-COOH 21
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different Size (5768kDa
11kDa and 25kDa) 22
225 Opsonization 22
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent
Molecules 23
231 Standard Calibration Curve with FITC and FluorA 23
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC 23
2321 Absorption of Stearylated NickFect 51 (NF51) Cell Penetrating Peptides (CPPs)
onto the Surface of FITC Labeled TCPSi-NH2NPs 23
2322 Conjugation of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (125I-CPPs) to the Surface of FITC Labeled TCPSi-NH2NPs 24
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1 (FluorA) 24
24 Characterization Methods 25
241 Dynamic Light Scattering (DLS) 25
242 Gel Electrophoresis 25
243 Fluorescence Intensity Measurement 25
3 Results and discussion 26
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) 26
32 Opsonization of the PEGylated TCPSi-NH2 NPs 28
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
30
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2)
Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC) 31
341 Characterization of Stearylated Stearylated NickFect 51 (NF51) CPPs Adsorped
FITC Labeled TCPSi-NH2NPs 34
342 Characterization of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51)
Cell Penetrating Peptides Conjugated FITC Labeled TCPSi-NH2 NPs 35
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA 35
4 Conclusion 38
5 References 40
vi
Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane
AFM minus Atomic force microscopy
CPPsminusCell-penetrating peptides
CTminus Cytotoxic ions
DICminusN N-Diisopropylcarbodiimide
DIPEAminusN N-Diisopropylethylamine
DLSminusDynamic Light Scattering
DLVOminusDerjaguin-Landau-Verwey-Overbeek
DMFminusDimethylformamide
DNAminusDeoxyribonuclic Acid
EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
EDTAminus Ethylenediaminetetraacetic acid
EPRminusenhanced permeability and retention
EtOHminusEthanol
FminusFlory radius
Fc minus Fragment crystallizable
FDAminusfood and drug administration
FEminus Field Emission
FESEMminus Field Emission scanning electron microscopy
FITCminusfluorescein isothiocyanate
FluorAminus fluoresceinaminisomer-1
FLminusFluoroskan Ascent
HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate
HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1minusHuman Immunodeficiency Virus 1
HPminus Human plasma
MRIminusMagnetic resonance imaging
NHSminusN-hydroxysuccinimide
NIRminus Near infra red
NPs minus Nanoparticles
PBSminus Phosphate Buffer Saline
PEGminuspolyethylene glycol
PSminus Porous silicon
RESminusReticuloendothelial system
SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis
SEFminus Surface-enhanced fluorescence
SFMminusScanning force microscopy
SOIminusSilicon-on-insulator
SOSminusSilicon on sapphire technology
TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles
TatminusTrans-activating transcriptional activator
TEMminusTransmission electron microscopy
UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon
nanoparticles
vdWminus Van der Waals
X-ray CTminus X-ray computed tomography
ZSminus Zetasizer
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
ii
UNIVERSITY OF EASTERN FINLAND School of Pharmacy
Master Degree Program for Research Chemists
MEZBAH UDDIN
Master Thesis 40 p
Supervisors
Senior Scientist Docent Ale Naumlrvaumlnen and Researcher MSc Jussi Rytkoumlnen
September 2012
KEY WORDS Nanotechnology PEGylation Opsonization Zeta potential Fluorescence intensity
Biofunctionalization
Porous silicon nanoparticles (PSiNPs) have drawn significant attention in recent years in the
study of oncology due to their biocompatibility favorable biodistribution and efficient
clearance or biodegradability The clinical use of PSiNPs as nanocarriers in drug delivery
depends on biologically nonspecific adsorption and immunological response Minimization
of the opsonization in the blood increases the half life of the particles and the capability to
target tumors In this project the opsonization of PEGylated porous silicon nanoparticles was
investigated as it is expected that PEGylation of TCPSi-NH2NPs with Me-PEG-COOH can
contribute to minimization of the opsonization PEGylated PSiNPs were also prepared using
single step reaction between terminal amines in thermally carbonized porous silicon
nanoparticles (TCPSi-NH2NPs) and carboxylic acid groups in mPEG-COOH by refluxing
PEGylated particles were characterized by measuring their size and zeta potential and the
opsonized particles were analyzed by SDS-PAGE The surface of TCPSi-NH2NPs was also
conjugated with fluorescent molecule such as fluorescein-5-isothiocyanate (FITC) and by
tailoring of the acid treated thermally hydrocarbonized porous silicon (UnTHCPSi) NPs by
associating with fluoresceinamine isomer-1 (FluorA) activated with DICNHS activator
FITC labeling TCPSi-NH2NPs were further modified by conjugating stearylated NickFect 51
(NF51) cell penetrating peptides (CPPs) and radioactive iodine labeled stearylated NickFect
cell penetrating peptides Fluorescent labeled NPs were characterized with zeta sizer and
fluorescence spectroscopy The surface modified PSiNPs can be loaded with targeted
therapeutic compounds and studied their biodistribution both in vivo and in vitro
iii
ACKNOWLEDGEMENT
I am heartily thankful to my supervisor Ale Naumlrvaumlnen and Jussi Rytkoumlnen whose
encouragement guidance and support from the initial to the final level enabled me to develop
an understanding of the subject
Finally I would like to offer my regards and blessings to all of those who supported me in
any respect during the completion of the project
Mezbah Uddin
iv
Contents Abbreviations vi
1 Introduction 1
11 Nanotechnology 1
12 Porous Silicon (PSi) 2
121 Properties of Porous Silicon 2
122 Structural Properties of Porous Silicon and Their Effect 2
123 Chemical Properties of the Porous Silicon 2
1 3 Surface Functionalization of Porous Silicon 3
14 Advantages of Porous Silicon 3
15 Porous Silicon Nanoparticles 3
151 Amino-derivatized Thermally Carbonized Porous Silicon Nanoparticles (TCPSi-
NH2 NPs) 4
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous Silicon
Nanoparticles (UnTHCPSiNPs) 5
16 Polyethylene Glycol (PEG) Molecule 5
17 Fluorescent Molecules and Their Applications 6
171 Fluorescence Detection 7
18 Cell-penetrating Peptides (CPPs) 7
19 Opsonization 8
110 Role of Zeta Potential in Characterization of Nanoparticles 10
111 Surface Modification 11
112 PEGylation 12
1121 Affecting Factors on PEGylation 13
1122 Different Sized NP 13
1123 Length amp Conformation of PEG Chain 14
1124 Terminal Portion 15
113 Biofunctionalization 16
114 General Conjugation Reactions 17
2 Experimental 20
21 Materials 20
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation) 21
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and NHS as an Activator
Reagent 21
v
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and DIPEA as an
Activator Reagent 21
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of Amino-modified TCPSi-
NH2NPs and MeO-PEG-COOH 21
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different Size (5768kDa
11kDa and 25kDa) 22
225 Opsonization 22
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent
Molecules 23
231 Standard Calibration Curve with FITC and FluorA 23
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC 23
2321 Absorption of Stearylated NickFect 51 (NF51) Cell Penetrating Peptides (CPPs)
onto the Surface of FITC Labeled TCPSi-NH2NPs 23
2322 Conjugation of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (125I-CPPs) to the Surface of FITC Labeled TCPSi-NH2NPs 24
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1 (FluorA) 24
24 Characterization Methods 25
241 Dynamic Light Scattering (DLS) 25
242 Gel Electrophoresis 25
243 Fluorescence Intensity Measurement 25
3 Results and discussion 26
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) 26
32 Opsonization of the PEGylated TCPSi-NH2 NPs 28
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
30
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2)
Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC) 31
341 Characterization of Stearylated Stearylated NickFect 51 (NF51) CPPs Adsorped
FITC Labeled TCPSi-NH2NPs 34
342 Characterization of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51)
Cell Penetrating Peptides Conjugated FITC Labeled TCPSi-NH2 NPs 35
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA 35
4 Conclusion 38
5 References 40
vi
Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane
AFM minus Atomic force microscopy
CPPsminusCell-penetrating peptides
CTminus Cytotoxic ions
DICminusN N-Diisopropylcarbodiimide
DIPEAminusN N-Diisopropylethylamine
DLSminusDynamic Light Scattering
DLVOminusDerjaguin-Landau-Verwey-Overbeek
DMFminusDimethylformamide
DNAminusDeoxyribonuclic Acid
EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
EDTAminus Ethylenediaminetetraacetic acid
EPRminusenhanced permeability and retention
EtOHminusEthanol
FminusFlory radius
Fc minus Fragment crystallizable
FDAminusfood and drug administration
FEminus Field Emission
FESEMminus Field Emission scanning electron microscopy
FITCminusfluorescein isothiocyanate
FluorAminus fluoresceinaminisomer-1
FLminusFluoroskan Ascent
HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate
HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1minusHuman Immunodeficiency Virus 1
HPminus Human plasma
MRIminusMagnetic resonance imaging
NHSminusN-hydroxysuccinimide
NIRminus Near infra red
NPs minus Nanoparticles
PBSminus Phosphate Buffer Saline
PEGminuspolyethylene glycol
PSminus Porous silicon
RESminusReticuloendothelial system
SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis
SEFminus Surface-enhanced fluorescence
SFMminusScanning force microscopy
SOIminusSilicon-on-insulator
SOSminusSilicon on sapphire technology
TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles
TatminusTrans-activating transcriptional activator
TEMminusTransmission electron microscopy
UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon
nanoparticles
vdWminus Van der Waals
X-ray CTminus X-ray computed tomography
ZSminus Zetasizer
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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Nano 2009 3(1) 197 -206
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H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
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(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
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2000 182 505ndash513
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(b) Walker N J Science 2002 296 557
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(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
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(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
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Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
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39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
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41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
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44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
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46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
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6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
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Sci 2008 320 132
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55 Addison C J amp Brolo A G Langmuir 2006 22 8696
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PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
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67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
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15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
iii
ACKNOWLEDGEMENT
I am heartily thankful to my supervisor Ale Naumlrvaumlnen and Jussi Rytkoumlnen whose
encouragement guidance and support from the initial to the final level enabled me to develop
an understanding of the subject
Finally I would like to offer my regards and blessings to all of those who supported me in
any respect during the completion of the project
Mezbah Uddin
iv
Contents Abbreviations vi
1 Introduction 1
11 Nanotechnology 1
12 Porous Silicon (PSi) 2
121 Properties of Porous Silicon 2
122 Structural Properties of Porous Silicon and Their Effect 2
123 Chemical Properties of the Porous Silicon 2
1 3 Surface Functionalization of Porous Silicon 3
14 Advantages of Porous Silicon 3
15 Porous Silicon Nanoparticles 3
151 Amino-derivatized Thermally Carbonized Porous Silicon Nanoparticles (TCPSi-
NH2 NPs) 4
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous Silicon
Nanoparticles (UnTHCPSiNPs) 5
16 Polyethylene Glycol (PEG) Molecule 5
17 Fluorescent Molecules and Their Applications 6
171 Fluorescence Detection 7
18 Cell-penetrating Peptides (CPPs) 7
19 Opsonization 8
110 Role of Zeta Potential in Characterization of Nanoparticles 10
111 Surface Modification 11
112 PEGylation 12
1121 Affecting Factors on PEGylation 13
1122 Different Sized NP 13
1123 Length amp Conformation of PEG Chain 14
1124 Terminal Portion 15
113 Biofunctionalization 16
114 General Conjugation Reactions 17
2 Experimental 20
21 Materials 20
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation) 21
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and NHS as an Activator
Reagent 21
v
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and DIPEA as an
Activator Reagent 21
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of Amino-modified TCPSi-
NH2NPs and MeO-PEG-COOH 21
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different Size (5768kDa
11kDa and 25kDa) 22
225 Opsonization 22
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent
Molecules 23
231 Standard Calibration Curve with FITC and FluorA 23
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC 23
2321 Absorption of Stearylated NickFect 51 (NF51) Cell Penetrating Peptides (CPPs)
onto the Surface of FITC Labeled TCPSi-NH2NPs 23
2322 Conjugation of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (125I-CPPs) to the Surface of FITC Labeled TCPSi-NH2NPs 24
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1 (FluorA) 24
24 Characterization Methods 25
241 Dynamic Light Scattering (DLS) 25
242 Gel Electrophoresis 25
243 Fluorescence Intensity Measurement 25
3 Results and discussion 26
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) 26
32 Opsonization of the PEGylated TCPSi-NH2 NPs 28
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
30
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2)
Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC) 31
341 Characterization of Stearylated Stearylated NickFect 51 (NF51) CPPs Adsorped
FITC Labeled TCPSi-NH2NPs 34
342 Characterization of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51)
Cell Penetrating Peptides Conjugated FITC Labeled TCPSi-NH2 NPs 35
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA 35
4 Conclusion 38
5 References 40
vi
Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane
AFM minus Atomic force microscopy
CPPsminusCell-penetrating peptides
CTminus Cytotoxic ions
DICminusN N-Diisopropylcarbodiimide
DIPEAminusN N-Diisopropylethylamine
DLSminusDynamic Light Scattering
DLVOminusDerjaguin-Landau-Verwey-Overbeek
DMFminusDimethylformamide
DNAminusDeoxyribonuclic Acid
EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
EDTAminus Ethylenediaminetetraacetic acid
EPRminusenhanced permeability and retention
EtOHminusEthanol
FminusFlory radius
Fc minus Fragment crystallizable
FDAminusfood and drug administration
FEminus Field Emission
FESEMminus Field Emission scanning electron microscopy
FITCminusfluorescein isothiocyanate
FluorAminus fluoresceinaminisomer-1
FLminusFluoroskan Ascent
HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate
HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1minusHuman Immunodeficiency Virus 1
HPminus Human plasma
MRIminusMagnetic resonance imaging
NHSminusN-hydroxysuccinimide
NIRminus Near infra red
NPs minus Nanoparticles
PBSminus Phosphate Buffer Saline
PEGminuspolyethylene glycol
PSminus Porous silicon
RESminusReticuloendothelial system
SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis
SEFminus Surface-enhanced fluorescence
SFMminusScanning force microscopy
SOIminusSilicon-on-insulator
SOSminusSilicon on sapphire technology
TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles
TatminusTrans-activating transcriptional activator
TEMminusTransmission electron microscopy
UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon
nanoparticles
vdWminus Van der Waals
X-ray CTminus X-ray computed tomography
ZSminus Zetasizer
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
iv
Contents Abbreviations vi
1 Introduction 1
11 Nanotechnology 1
12 Porous Silicon (PSi) 2
121 Properties of Porous Silicon 2
122 Structural Properties of Porous Silicon and Their Effect 2
123 Chemical Properties of the Porous Silicon 2
1 3 Surface Functionalization of Porous Silicon 3
14 Advantages of Porous Silicon 3
15 Porous Silicon Nanoparticles 3
151 Amino-derivatized Thermally Carbonized Porous Silicon Nanoparticles (TCPSi-
NH2 NPs) 4
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous Silicon
Nanoparticles (UnTHCPSiNPs) 5
16 Polyethylene Glycol (PEG) Molecule 5
17 Fluorescent Molecules and Their Applications 6
171 Fluorescence Detection 7
18 Cell-penetrating Peptides (CPPs) 7
19 Opsonization 8
110 Role of Zeta Potential in Characterization of Nanoparticles 10
111 Surface Modification 11
112 PEGylation 12
1121 Affecting Factors on PEGylation 13
1122 Different Sized NP 13
1123 Length amp Conformation of PEG Chain 14
1124 Terminal Portion 15
113 Biofunctionalization 16
114 General Conjugation Reactions 17
2 Experimental 20
21 Materials 20
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation) 21
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and NHS as an Activator
Reagent 21
v
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and DIPEA as an
Activator Reagent 21
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of Amino-modified TCPSi-
NH2NPs and MeO-PEG-COOH 21
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different Size (5768kDa
11kDa and 25kDa) 22
225 Opsonization 22
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent
Molecules 23
231 Standard Calibration Curve with FITC and FluorA 23
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC 23
2321 Absorption of Stearylated NickFect 51 (NF51) Cell Penetrating Peptides (CPPs)
onto the Surface of FITC Labeled TCPSi-NH2NPs 23
2322 Conjugation of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (125I-CPPs) to the Surface of FITC Labeled TCPSi-NH2NPs 24
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1 (FluorA) 24
24 Characterization Methods 25
241 Dynamic Light Scattering (DLS) 25
242 Gel Electrophoresis 25
243 Fluorescence Intensity Measurement 25
3 Results and discussion 26
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) 26
32 Opsonization of the PEGylated TCPSi-NH2 NPs 28
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
30
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2)
Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC) 31
341 Characterization of Stearylated Stearylated NickFect 51 (NF51) CPPs Adsorped
FITC Labeled TCPSi-NH2NPs 34
342 Characterization of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51)
Cell Penetrating Peptides Conjugated FITC Labeled TCPSi-NH2 NPs 35
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA 35
4 Conclusion 38
5 References 40
vi
Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane
AFM minus Atomic force microscopy
CPPsminusCell-penetrating peptides
CTminus Cytotoxic ions
DICminusN N-Diisopropylcarbodiimide
DIPEAminusN N-Diisopropylethylamine
DLSminusDynamic Light Scattering
DLVOminusDerjaguin-Landau-Verwey-Overbeek
DMFminusDimethylformamide
DNAminusDeoxyribonuclic Acid
EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
EDTAminus Ethylenediaminetetraacetic acid
EPRminusenhanced permeability and retention
EtOHminusEthanol
FminusFlory radius
Fc minus Fragment crystallizable
FDAminusfood and drug administration
FEminus Field Emission
FESEMminus Field Emission scanning electron microscopy
FITCminusfluorescein isothiocyanate
FluorAminus fluoresceinaminisomer-1
FLminusFluoroskan Ascent
HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate
HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1minusHuman Immunodeficiency Virus 1
HPminus Human plasma
MRIminusMagnetic resonance imaging
NHSminusN-hydroxysuccinimide
NIRminus Near infra red
NPs minus Nanoparticles
PBSminus Phosphate Buffer Saline
PEGminuspolyethylene glycol
PSminus Porous silicon
RESminusReticuloendothelial system
SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis
SEFminus Surface-enhanced fluorescence
SFMminusScanning force microscopy
SOIminusSilicon-on-insulator
SOSminusSilicon on sapphire technology
TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles
TatminusTrans-activating transcriptional activator
TEMminusTransmission electron microscopy
UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon
nanoparticles
vdWminus Van der Waals
X-ray CTminus X-ray computed tomography
ZSminus Zetasizer
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
v
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and DIPEA as an
Activator Reagent 21
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of Amino-modified TCPSi-
NH2NPs and MeO-PEG-COOH 21
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different Size (5768kDa
11kDa and 25kDa) 22
225 Opsonization 22
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent
Molecules 23
231 Standard Calibration Curve with FITC and FluorA 23
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC 23
2321 Absorption of Stearylated NickFect 51 (NF51) Cell Penetrating Peptides (CPPs)
onto the Surface of FITC Labeled TCPSi-NH2NPs 23
2322 Conjugation of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (125I-CPPs) to the Surface of FITC Labeled TCPSi-NH2NPs 24
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1 (FluorA) 24
24 Characterization Methods 25
241 Dynamic Light Scattering (DLS) 25
242 Gel Electrophoresis 25
243 Fluorescence Intensity Measurement 25
3 Results and discussion 26
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) 26
32 Opsonization of the PEGylated TCPSi-NH2 NPs 28
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
30
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2)
Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC) 31
341 Characterization of Stearylated Stearylated NickFect 51 (NF51) CPPs Adsorped
FITC Labeled TCPSi-NH2NPs 34
342 Characterization of Radioactive Iodine Labeled Stearylated NickFect 51 (NF51)
Cell Penetrating Peptides Conjugated FITC Labeled TCPSi-NH2 NPs 35
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA 35
4 Conclusion 38
5 References 40
vi
Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane
AFM minus Atomic force microscopy
CPPsminusCell-penetrating peptides
CTminus Cytotoxic ions
DICminusN N-Diisopropylcarbodiimide
DIPEAminusN N-Diisopropylethylamine
DLSminusDynamic Light Scattering
DLVOminusDerjaguin-Landau-Verwey-Overbeek
DMFminusDimethylformamide
DNAminusDeoxyribonuclic Acid
EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
EDTAminus Ethylenediaminetetraacetic acid
EPRminusenhanced permeability and retention
EtOHminusEthanol
FminusFlory radius
Fc minus Fragment crystallizable
FDAminusfood and drug administration
FEminus Field Emission
FESEMminus Field Emission scanning electron microscopy
FITCminusfluorescein isothiocyanate
FluorAminus fluoresceinaminisomer-1
FLminusFluoroskan Ascent
HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate
HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1minusHuman Immunodeficiency Virus 1
HPminus Human plasma
MRIminusMagnetic resonance imaging
NHSminusN-hydroxysuccinimide
NIRminus Near infra red
NPs minus Nanoparticles
PBSminus Phosphate Buffer Saline
PEGminuspolyethylene glycol
PSminus Porous silicon
RESminusReticuloendothelial system
SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis
SEFminus Surface-enhanced fluorescence
SFMminusScanning force microscopy
SOIminusSilicon-on-insulator
SOSminusSilicon on sapphire technology
TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles
TatminusTrans-activating transcriptional activator
TEMminusTransmission electron microscopy
UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon
nanoparticles
vdWminus Van der Waals
X-ray CTminus X-ray computed tomography
ZSminus Zetasizer
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
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(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
vi
Abbreviations AEASP minus3-(2-Aminoethylamino)propyldimethoxymethylsilane
AFM minus Atomic force microscopy
CPPsminusCell-penetrating peptides
CTminus Cytotoxic ions
DICminusN N-Diisopropylcarbodiimide
DIPEAminusN N-Diisopropylethylamine
DLSminusDynamic Light Scattering
DLVOminusDerjaguin-Landau-Verwey-Overbeek
DMFminusDimethylformamide
DNAminusDeoxyribonuclic Acid
EDACminus1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
EDTAminus Ethylenediaminetetraacetic acid
EPRminusenhanced permeability and retention
EtOHminusEthanol
FminusFlory radius
Fc minus Fragment crystallizable
FDAminusfood and drug administration
FEminus Field Emission
FESEMminus Field Emission scanning electron microscopy
FITCminusfluorescein isothiocyanate
FluorAminus fluoresceinaminisomer-1
FLminusFluoroskan Ascent
HBTUminus O-Benzotriazole-N N N N-tetramethyl-uronium-hexafluoro-phosphate
HEPESminus4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV-1minusHuman Immunodeficiency Virus 1
HPminus Human plasma
MRIminusMagnetic resonance imaging
NHSminusN-hydroxysuccinimide
NIRminus Near infra red
NPs minus Nanoparticles
PBSminus Phosphate Buffer Saline
PEGminuspolyethylene glycol
PSminus Porous silicon
RESminusReticuloendothelial system
SDS-PAGEminusSodium dodecyl sulphate polyacryl amide gel electrophoresis
SEFminus Surface-enhanced fluorescence
SFMminusScanning force microscopy
SOIminusSilicon-on-insulator
SOSminusSilicon on sapphire technology
TCPSi-NH2 NPsminusAmino-treated thermally carbonized porous silicon nanoparticles
TatminusTrans-activating transcriptional activator
TEMminusTransmission electron microscopy
UnTHCPSiNPsminus Undecylenic acid-treated thermally hydrocarbonized porous silicon
nanoparticles
vdWminus Van der Waals
X-ray CTminus X-ray computed tomography
ZSminus Zetasizer
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
1
1 Introduction 11 Nanotechnology
Biomedical process has been remarkably impacted over the preceding decades due to
evolution of micro and nanotechnology Quantum dots controlled released nanoparticles
targeted delivery and cancer nanotechnology are ideal examples Practical employment of
many applications is still difficult but high rate of evolution is raising hope for innovative
biomedical process in near future [1]
Nanotechnology is an interdisciplinary study area comprising physics chemistry biology
engineering and precise diagnosis Treatment of different diseases especially cancer can
adopt with it [2 3] Size of nanoparticle is usually 1 nanometer to few hundreds nm which
fits well with the size of biological molecules such as antibodies Nanoparticles can also have
very good interactions both on surface and interior cells of biomolecules which is very
effective for syndrome diagnosis and treatment used in nanomedicine
Nanodevices such as nanochips nanosensorss etc are evident for in vitro and ex vivo
purposes [4 5] Biocompatibility in vivo kinetics capability to break out the
reticuloendothelial system (RES) targeting efficiency severe persistent toxicity and cost
effectiveness are main challenges in preclinical animal models and clinical translation of
nanotechnology for in vivo purposes Nanoparticles can specially work in oncology where
the absorbent tumor vasculature can let for superior tissue permeation than normal organs
Nanoparticles can help to carry diagnostic or therapeutic agents to enter in cells intervening
molecular interactions and to identify molecular changes in a susceptible approach
Nanotechnology made a great progress in biological imaging and drug delivery They are
now employed as fluorescent probes drug carriers and contrasts agents in therapeutic and
diagnostic sectors [6-10] Gold nanoparticles silver nanoparticles silicon nanoparticles and
many more are used in nanomedicine We are interested to monitor biological characteristics
of porous silicon nanoparticles
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
2
12 Porous Silicon (PSi)
Porous silicon has holes in its nanostructure with a large surface to volume ratio (500 -
m2cm
3) Porous silicon was first prepared in 1956 by Arthur Uhlir Jr at the Bell Labs in the
VS Porous silicon has many applications in drug delivery biological imaging photonics
chemical sensing and so on
121 Properties of Porous Silicon
122 Structural Properties of Porous Silicon and Their Effect
Basic structural parameters are pore size porosity and porous thickness Field emission
scanning electron microscopy (FESEM) transmission electron microscopy (TEM) atomic
force microscopy (AFM) or scanning force microscopy (SFM) etc are usually used to study
structure of porous silicon Porous silicon can be bio-inert bioactive and resorbable
depending on porosity [11] In in-vitro study porous silicon samples were exposed to
simulate body fluid containing similar ion concentration as in human blood They were
investigated for longer time High porosity mesoporous layers were completely removed
within a day but low or medium porosity micro-porous layer were stable with hydroxyapatite
growth
123 Chemical Properties of the Porous Silicon
Porous silicon has high surface area with high density of Si dangling bonds and adulterations
like hydrogen and fluorine Adulterations are usually formed during PSi formation from
electrolytes Initially porous layer surface is covered by SiHx (x= 1 2 3) bond It fades
during annealing (300-5000C) PSi surface oxidizes gradually in atmosphere and finally
whole surface is oxidized Light and elevated temperature increases oxidation A blue shift in
luminescence spectra is observed after oxidation [12] and electrical conductivity and optical
properties are also influenced Level of impurity of fluorine depends on type of electrolyte
and identified in freshly prepared porous silicon Fluorine exists as SiFx (x= 1 2 3) and it is
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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H18ndashH40
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1555
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2000 182 505ndash513
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(b) Walker N J Science 2002 296 557
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(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
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Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
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39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
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41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
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46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
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6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
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20 2677
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114 15
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Sci 2008 320 132
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PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
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67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
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15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
3
replaced by SiOH during reactions with water vapour of atmosphere So usually fluoride
concentration decreases with time
1 3 Surface Functionalization of Porous Silicon
PSi is not good for commercial purposes because it is unstable with meta-stable Si-Hx
termination Meta-stable hydro-silicon can oxidize and degrades surface structure So
passivation of PSi surface is important to have good electrical contacts on PSi
14 Advantages of Porous Silicon
Porous silicon micro-particles are very useful for controlled drug delivery because they are
less toxic They are good for chronic use as they disintegrate in body whereas carbon
nanotubes degrade Secondly desired surface area free volume and pore size (few
nanometers to several hundreds of nanometers) can be achieved by electrochemical
fabrication Thirdly the surface of porous silicon is tailored with wide variety of organic or
biological molecules (drugs peptides antibodies proteins etc)
Fourthly porous silicon offers excellent optical properties because it exhibits fluorescence
deriving from silicon quantum dot structures [14] It is useful for diagnostic or therapeutic
purposes Additionally porous silicon is simple enough to replace more complicated clinical
devices [15-17]
15 Porous Silicon Nanoparticles
Porous silicon nanoparticle is commonly used as nanomedicine They have excellent
properties like biocompatibility favorable bio-distribution biodegradability for controlled
drug delivery Nanoparticles have large pay loads stability avidity and enhancement They
can be used in multiple purposes because of their unique size and on elevated ratio of surface
to volume tunable optical electric magnetic and biological features They can have various
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
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2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
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18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
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19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
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(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
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(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
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29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
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(b) Walker N J Science 2002 296 557
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Applications Kluwer Academic Publishers New York 1998
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(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
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(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
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(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
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Artemyev M Nano Lett 2002 2 1449
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(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
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35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
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(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
4
sizes and shapes chemical constituents surface chemical features and hollow or solid
structures So they are suitable for drug delivery vehicles distinction agents and diagnostic
devices NPs are smaller than cells and can effect in vivo applications For example in cancer
tissue NPs can remain in the area even after extravasating from leaky tumor and shows
enhanced permeability and retention effect [18] for signaling or therapy [19]
Moreover SiNPs are commercially available and easily separable from aqueous suspension
Large specific surface of most SiNPs can be easily functionalized and delivered into living
cell
151 Amino-derivatized Thermally Carbonized Porous Silicon
Nanoparticles (TCPSi-NH2 NPs)
The most important characteristics of TCPSi-NH2-NPs are summarized in following table
Table 11
Table 11 Features of the plain TCPSi-NH2
NPs
Sample Surface Area
(m2g)
Pore Volume
(cm3g)
Pore Size (nm) Particle Size
Distributions
(nm)
TCPSi-
NH2
970372 0508017 926 170
Figure 11 Chemical structure of the TCPSi-NH2
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
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9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
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14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
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18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
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23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
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(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
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Birkhauser Verlag Berlin Germany 1997
41
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Birkhauser Verlag Berlin Germany 1997
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(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
5
TCPSi-NH2-NPs contain both primary and secondary amine (figure 11) Primary amine is
more reactive than secondary amine Surface of TCPSi-NH2NPs can be modified when
primary amine forms amide bond with succinimidyl ester terminated surface layer is formed
during reaction between carboxyl acid (-COOH) and N-hydroxysuccinimide Surface of
TCPSi-NH2NPs can also be modified by covalently attaching amine specific ndashN=C=S group
to primary amine
152 Carboxyl Acid-derivatized Thermally Hydrocarbonized Porous
Silicon Nanoparticles (UnTHCPSiNPs)
Our investigated UnTHCPSiNPs had pore size ~10 nm with 135-140 nm particle size
distribution They contain carboxyl group (-COOH) (figure 12) which can react with NHS in
presence of di-isopropyl carbodimide (DIC) to form succinimidylester (- COOSuc) surface
This terminated surface further reacts with primary amine to form amide bond
Figure 12 Chemical structure of the UnTHCPSi
16 Polyethylene Glycol (PEG) Molecule
PEG has repeating ethylene ether units and very attractive for conjugation It is most
frequently used in biomedical applications due to high solubility in water PEG is very
flexible and can be cleared from body
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
6
17 Fluorescent Molecules and Their Applications
Derivatized fluoresceins such as fluoresceinamine isomer-1(FluorA) or fluorescein
isothiocyanate (FITC) are often utilized in many biological purposes These derivatives
contain a reactive substituent on the phenyl ring for coupling reactions
Figure 13 Stucture of fluorescein isothiocyanate (FITC) and fluoresceinaminismer-1(FluorA)
FITC is the isothiocyanate (-N=C=S) derivative of the original fluorescein molecule and (-
N=C=S) is a reactive group replacing a hydrogen atom on the bottom ring of the structure
This derivative can easily react with nucleophiles including amine and sulfhydryl groups
FluorA is the amine derivative of the original fluorescein molecule which reacts with
carboxyl acid ester groups to form amide bond Their excitation and emission spectrum peak
wavelengths are approximately 495 nm521 nm Like most fluorochromes it has a tendency
to photobleaching This feature is challenging so it has to be overcome for the biological
applications where greater photostability higher fluorescence intensity or different
attachment groups are needed
Biomolecules cells tissue sections [20] and fluorescent molecular probes are widely used for
labeling These probes are used in vivo purposes for example to image target tissue in live
animals or as blood pool contrast in human subject Green emitting fluorescein dyes are used
for retinal angiography in human subject [21] Cerebral vertebral arteries can be visualized
with near infra red (NIR) emitting indocyanine green [22 23] Labeled fluorescent dyes
fluorescent micro-particles and nanoparticles are used in many imaging techniques
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
7
171 Fluorescence Detection
Fluorescence detection is useful for biological assays [24 25] but higher sensitivity is
required to identify low concentrated objective molecule [26-29] There are many methods
for detection [30 31] like surface enhanced fluorescence (SEF) on metal nanostructures [32]
When a fluorophore is confined near to surface of metallic nanoparticles [33 34]
fluorescence enhances Conjugation of fluorophore with radiating Plasmon from metallic
particles causes SEF When fluorophore is near to metal core then fluorescence quenched
completely Highest enrichment occurs when fluorophore is 10 nm from metal surface [48]
Small particles quench fluorescence due to absorption and big particles increases
fluorescence due to dispersion [35]
18 Cell-penetrating Peptides (CPPs)
CPP carries cargo into cells usually via endocytosis to the endosomes of living mammalian
cells Cargoes are from small chemical molecules to nanosize particles and large fragments of
DNA Cargo conjugates with CPPs either through chemical linkage via covalent bonds or
through non-covalent interactions
CPPs have great potential as in vitro and in vivo delivery vectors CPPs pose an amino acid
composition of high relative abundance of positively charged amino acids or non polar
hydrophobic amino acids Initially it was found that trans-activating transcriptional activator
(TAT) from Human Immunodeficiency virus 1 (HIV-1) internalized from surrounding media
by numerous cell types in culture [36] Many CPPs have been identified and small synthetic
analogues with more effective protein transduction properties have been established [37]
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
8
Figure 14 Minimizing proteolysis effects for CPP (httpenwikipediaorgwiki Mechanism_ cell _uptake_
for_CPPjpg 26 April 2009)
Extracellular materials are gene or nanoscale synthetic delivery system They can not
transport through plasma membrane of a cell Various cell penetrating peptides (CPPs) can
overcome this problem through receptor independent pathway CPP mediated NPs can be
internalized by a cell and have high potential for delivering imaging and therapeutic agents
into the cells
19 Opsonization
A process in which pathogens are layered and neutralized by immune system is called
opsonization An opsonized pathogen is killed either by uptaking or direct neutralization
RES is an immune system where circulating and macrophages liver kupffer cells and spleen
and other lymphatic vessels neutralize foreign material like bacteria or viruses [38] Opsonin
protein conjugate with foreign bodies and coat it`s surface (figure 15 A B) [39 40]
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
9
Figure 15(A) Polyethylene glycol reduces reticuloendothelial system uptake (A1) opsonized NPs (A2)
opsonized NPs conjugated with macrophages (A3) and transported to the liver (A4) (Jokerst JV Lobovkina T
Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash
728)
Phagocytic cell uptake the material and deliver it to the liver or spleen for degradation
(Figure 15 A3ndashA4) Extra phagocytic macrophages are eternally sited in the liver For
example Kupffer cells serve as major filter for many types of NPs with major interference
(long t12) [41]
Figure 15(B) (B) PEGylated NPs (B1) minimize opsonization (B2) and reduction of NP accumulation in liver
(B3) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
PEG polymer on a NP surface minimize opsonization (Figure 15B2) and increases t12 It
protects recognition by monocytes and macrophages and thus NPs circulates [39 40]
Hydrophilic PEG can reduce hydrophobic particles Aggregation causes poor t12 Initially
NPs aggregate because attraction between particles is stronger than solvent [40 42]
According to Derjaguin-landau-Verwey-Overbeek (DLVO) theory NPs aggregates due to
their high surface energy [43 44] Electrostatic repulsive potential [44] and Vander Walls
attraction potential is important factor for aggregation of spherical NPs PEG reduces surface
energy of NPs and Vander Waals attraction [45-47]
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
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(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
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(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
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(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
10
110 Role of Zeta Potential in Characterization of Nanoparticles
Figure 16 Schematic representation of the zeta potential (httpwwwhoribacomscientific products particle-
characterizationtechnologyzeta-potential)
Zeta potential is the electric potential at the slip plane between the bound layer of diluents
molecules surrounding the particle and bulk solution It depends on particles surface charge
and diluents solution Higher zeta potential means greater electrostatic repulsion between
particles and less aggregation
Zeta potential is used to characterize nanoparticles Size zeta potential and size distribution
can be measured with dynamic light scattering Zeta potential determines stability of colloidal
system and particle ndashparticle interactions within suspension
Minimum value of zeta potential to prevent aggregation depends on particular sample but
usually it is between -30 mV to +30 mV To reduce aggregation of a sample for drug delivery
and pharmaceutical applications higher zeta potential is required For removing too small
particles in water treatment applications low zeta potential is necessary
According to Derjaguin Landau Verwey Overbeek (DLVO) theory dispersion stability
depends on attractive Vander Waals and repulsive electrostatic forces If repulsive
electrostatic force overcomes attractive vdw force nanoparticles will be stable and free of
aggregation So nanoparticles with adequate density of surface charge will not aggregate By
screening charges with counter ions can favor vdw forces which are crucial for surface
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
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2000 182 505ndash513
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(b) Walker N J Science 2002 296 557
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(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
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Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
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41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
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46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
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48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
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PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
11
functionalization such as biocompatibility of fluorescent nanoparticles nanoparticle
clustering non specific adsorption of proteins (p) release cytotoxic ions (CT)
Biofunctionalization can cause surface charge neutralization which decreases dispersion
Zeta potential can identify interaction between active substance and carrier It ensures
whether drug encapsulated in body or adsorbed on surface Adsorbed drug can not be
protected from enzymatic degradation or released fast after degradation
111 Surface Modification
NPs have some shortcomings for clinical use They can be uptaken by reticuloendothelial
system (RES) and thus they are quickly shuttled out of circulation to liver spleen or bone
marrow Another disadvantage is binding of NPs to non-targeted spots Due to RES
accumulation NPs become toxic NPs have tendency to aggregate and can cause entrapment
in liver lungs or elsewhere due to capillary occlusion [48] So surface modification of NPs is
necessary to overcome these problems and it can be done with PEGylation
Fluorescence molecular probes are limited for real time imaging studies due to several
limitations such as short stokes shift poor photochemical stability sensitivity to buffer
composition photo-bleaching and decomposition non-targeted accumulation and faster rate
of photo-bleaching still they are used due to low cost commercial availability and ease of
use Organic dyes such as fluorescein [49 50] rhodamin [51 52] cyanine [50 53] alexa
dyes [50 54] oxazines [55 56] porphyrin [57] phthalocyanins [58] show better chemical
and optical properties Usually organic fluorophores are used as luminescent bio-pointer but
they have some limitations Reliable determination of cancer biopointers needs extremely
sensitive and photo-stable probes with sophisticated fluorescent imaging detection system
Optical based in vivo imaging is more challenging than X-ray computed tomography (X-ray
CT) or magnetic resonance imaging (MRI) due to many reasons such as sharp absorption of
optical signal by body tissue and fluid in UV and visible range weak tissue penetration
depth light dispersion by tissues weak spatial image resolution
Optimized particle sized of nanoscales materials is necessary because they are easily
consumed by cells Quantum dots and dye loaded nanoparticles are commonly used for real
time imaging of cells QDs exhibit less photo-bleaching Most of them are cadmium based
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
12
and have cytotoxicity due to heavy metal ions [59 60] They have less quantum yield than
organic dyes QDs are hydrophobic and cause poor solubility into biological buffers and
quenching in aqueous environment Under intense excitation of light they exhibit irreversible
photo-degradation So fluorescence molecule needs to be functionalized Our aim is to use
fluorescence molecule with porous silicon nanoparticles to overcome those limitations
Covalent and non-covalent bonding processes are two usual chemical approaches for
conjugating molecules into silicon naoparticles Dye can be covalently bonded with silicon
nanoparticles or entrapped into silicon naoparticles by electrostatic interactions Covalently
bound PEG chains are more effective because they have longer blood circulation half lives
Covalently bound fluorescent molecule can reduce dye leakage
112 PEGylation
Covalently grafting entrapping or adsorbing a particle surface with polyethylene glycol
(PEG) chains is PEGylation It minimizes opsonization which affects system uptake and
pharmacokinetics It reduces rate of mononuclear phagocyte system uptake and increases
circulation half life of porous silicon nanoparticles and encapsulated drugs Cellular
interactions and bio-barrier transport may prove superior with targeting ligands like
antibodies Antibodies favor uptake by macrophages of reticuloendothelial system (RES)
PEG chains can conjugate with biodegradable nanoparticles as copolymer Numbers of PEG
chains are constantly available PEG chains can attach to NP surface or other candidate
molecule and reduce some challenges (fig 17)
Figure 17 PEGylated NP containing yellow colored metallic or polymeric core with red colored cloud of PEG
chain (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and therapy
Nanomedioine 2011 6 (4) 715ndash728)
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
13
PEGylated nanoparticles reduce RES uptake and increases circulation time than uncoated
NPs [40] It is helpful in drug delivery and imaging purposes
Coagulation decreases due to passivity of surfaces and non-targeted conjugation is also
reduced Charged based contact of proteins and small molecule are diminished Solubility
increases in buffer and serum due to hydrophilic ethylene glycol EPR effect changes due to
PEGylated NPs [61 62] For these reasons PEGylated nanoparticles store very less in liver
than non-PEGylated NPs and they are also exhibit higher tumor accumulation [63]
1121 Affecting Factors on PEGylation
Figure 18 Polymerization of ethylene glycol to form PEG which contains linkage group (R1) and terminus
group (R2) (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging and
therapy Nanomedioine 2011 6 (4) 715ndash728)
Most PEG molecules have two ends One end (R1) attaches to NP surface and other end (R2)
interact with solvent Longer PEG is represented by m-PEG and PEG is often illustrated as
polyethylene oxide The behavior of PEG-NP construct is affected with increased t12
1122 Different Sized NP
t12 depends on type of NP to be PEGylated like their size charge and composition For liver
accumulation ganglioside liposomes are size dependent but phosphatidylserine liposomes are
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
14
size independent Positively charged NPs with size more than 100 nm can be rapidly cleared
from circulation [64 65] NP composition is also very important for PEGylation
1123 Length amp Conformation of PEG Chain
Grafted PEG layer on NP surface and polymer conformation are related
F= αn35
Here F represents Flory radius Polymer conformation can be described on basis of Flory
radius n represents the number of monomer per polymer chain and length of one monomer in
Angstrom is α [66 67]
Figure 19 Flory radius VS no of monomers (n) for PEG (Jokerst JV Lobovkina T Zare RN Gambhir SS
Nanoparticle PEGylation for imaging and therapy Nanomedioine 2011 6 (4) 715ndash728)
PEG chains can have two main conformations on basis of surface coverage
Figure 110 Gold nanoparticles with PEG on surface (A) Contains mushroom configuration and (B) contains
brush configuration (Jokerst JV Lobovkina T Zare RN Gambhir SS Nanoparticle PEGylation for imaging
and therapy Nanomedioine 2011 6 (4) 715ndash728)
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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Nano 2009 3(1) 197 -206
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H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
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(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
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2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
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(b) Walker N J Science 2002 296 557
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(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
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Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
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39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
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41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
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46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
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48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
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Sci 2008 320 132
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PhysChem B 2006 110 11399
43
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59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
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67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
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71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
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15(1) 79ndash86
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5605ndash5620
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75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
15
PEG chains with lower surface density are called mushroom conformation where distance
between attachment points of polymer is larger than F (Figure 110A) In mushroom
conformation polymer chain have roughly sphere with Flory radius Polymer with bigger
graft density DltF) is called brush room configuration In brush configuration long thin
bristles of PEG enlarge from NP surface (figure 110B) [66 67] Number of repeating units
necessary to transform from mushroom to brush arrangement depends on types of PEG and
NP When distance of PEG molecules on surface close to F then mushroom conformation
coverts to brush conformation NPs containing brush PEG have denser coating and they
shield NPs from RES better Usually brush PEG conformation has longer circulation time
Typically larger PEG is used for smaller therapeutic materials It avoids excretion by kidneys
and maintains high concentration for longer time by increasing recirculation So oligos and
small molecules are coated with PEG 20000-50000 Da It increases hydrodynamic radius
and reduces t12 So larger NP ranges from 50-100 nm are usually coated with smaller PEG
(3400-10000 Da) Kidney excretion of non-conjugated PEG reduces with bigger molecular
weight but liver uptake increases For example liposome circulation with PEG 5000 is very
long (7 h) than PEG 2000 or PEG 750 (~1h) Longer molecular weight PEG has longer t12 on
QD [70-72] In mushroom configuration If PEG is larger then less copy number can be
loaded on a NP
1124 Terminal Portion
In vivo behavior is influenced by terminal end of PEG Methoxy or alcohol terminal groups
diminish non specific binding of lysosyme and fibrinogen [73 74] mPEG is usually very
ubiquitous NPs with negative charge (thiol or carboxy) makes surface of cell negatively
charged and causes fewer phagocytic events Simple hydroxyl group can diminish non
specific binding [75] Succinimide maleimide or alkyne reactive group assists binding of
secondary targeting ligand [76] but it is challenging due to steric hindrance
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
16
113 Biofunctionalization
NPs conjugate with biomolecules as address tags They conduct NPs to exact sites of body
exact organelles in body or transform protein or RNA in living cells Monoclonal antibodies
aptamers streptavidin or peptides are common address tags They are covalently coupled and
should be in controlled number per nanoparticles
For successful biofunctionalization reactions [77] few requirements are very important
Activity of biomolecules must be removed Signal of nanoparticles should not be slowed
down Linkage sites of NP surface should be covered and stability of biomolecule-
nanoparticle coupling should be controlled Finally width of nanoparticle shell should stay as
tiny as nanoparticles
Figure 111 Different approach of association of biomolecules to nanoparticles comprise direct physisorption
(A) facilated physisorption (B) chemically connected biomolecules with cross-linkers adsorbed on NP (C) direct
chemical coupling with NPs (D) biotin-streptavidin targeted coupling of biotinylated biomolecules (M Murcia
amp C Naumann Biofunctionalization of Fluorescent Nanoparticles (Nanotechnologies for the Life Sciences
Vol1Edited by Challa S S R Kumar Copyright 8 2005 WILEY-VCH Verlag GmbH amp Co KGaA
Weinheim ISBN 3-527-31381-8
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
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10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
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(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
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14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
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16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
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19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
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(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
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(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
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29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
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(b) Walker N J Science 2002 296 557
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(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
17
There are several strategies to bioconjugate nanoparticles Simple adsorption of biomolecules
to nanoparticle surface is least demanding (figure 111A) Activity of adsorbed biomolecules
change and it is difficult to control amount of adsorbed biomolecules per nanoparticle
Physisorption or noncovalent coupling of biomolecules to other biomolecules is another
technique (figure 111B) Here biomolecules are connected in appropriate direction Reactive
groups of biomolecules can also conjugate with cross-linker molecules by physisorption or
chemisorptions (figure 111C) Chemical conjugation is possible for coupling of
oligonucleotides to nanoparticles through mercapto groups (figure 111D) [78-80] To control
number of biomolecules per nanoparticle a separation step is needed [81] Usually it is done
by gel electrophoresis Linkage of biotinylated ligands or target biomolecules to streptavidin
or avidin-tailored nanoparticles is very specific (figure 111E) [82 83] Biotin binding
proteins avidin or streptavidin is used as linker molecules
114 General Conjugation Reactions
Nanoparticles are conjugated with covalent coupling One popular coupling process is
reaction between primary amine and carboxylic acid group
Initially a carboxylic acid reacts with 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide
(EDAC) and N-hydroxysuccinimide (NHS) to form an acyl amino ester Then this ester
reacts with primary amine to form a stable amide bond
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
18
Another approach is reaction between thiol and maleimide groups At certain pH a good
yield of stable thioester bond is formed It directly connects maleimide functional ligands to
thio groups of proteins Thiols can be prepared with heterobio-functional crosslinkers with
one thiol end group or by reducing disulfide bonds within the target protein
Coupling of two thiols to form a disulfide linkage is another approach Disulfide bond is
usually labile in biological fluids
Covalent linkage between aldehyde and amine groups form hydrazide bond Mild oxidation
of carbohydrate forms reactive aldehyde group
Chemical reaction between two primary amines is applied for bioconjugation
Homobiofunctional crosslinkers like glutaraldehyde can precede coupling reaction [84 85]
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
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(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
19
In this study one of our goal was to ensure efficient surface modification of the positively
charged amino modified thermally carbonized porous silicon nanoparticles (TCPSi-NH2NPs)
which has primary amine on the surface through PEGylation so that resulting particles can
minimize the opsonization which involved in mononuclear phagocytic uptake of
nanoparticles for optical data storage and other technical applications in related fields Our
another goal was surface functionalization of negatively charged acid treated thermally
hydrocarbonized porous silicon nanoparticles (UnTHCPSiNPs) having carboxyl acid group
on the surface with fluoresceinamine isomer-1(FluorA) and TCPSi-NH2NPs with
Fluorescein-5-isothiocyanate (FITC) so that producing fluorescent labeling particles can
optimize photostability sensitivity and photbleaching and eventually they can act as an
effective bioimaging probe Subsequently stearylated NickFect 51 (NF51) CPPs was
introduced to the FITC labeled TCPSi-NH2NPs and conjugating radioactive iodine labeled
stearylated NickFect CPPs to check the CPPs conjugation
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
20
2 Experimental
21 Materials
Undecylenic acid modified thermally hydrocarbonized porous silicon (UnTHCPSi)
nanoparticles (NPs) were prepared by Docent Jarno Salonen department of Physics and
Astronomy University of Turku Amino-modified thermally carbonized porous silicon
(TCPSi-NH2) nanoparticles (NPs) were fabricated by Wujung Ju Department of applied
Physics at the University of Eastern Finland Alpha-methoxy-omega-carboxylic acid
poly(ethylene glycol) (MeO-PEG-COOH) (Iris Biotech -GmbH) NN-
Diisopropylcarbodiimide (DIC) (perceptive Biosystems GmbH) N-Hydroxy-succinimide
(NHS) (Fluka) O-Benzotriazole-NNNrsquoNrsquo-tetramethyl-uronium-hexafluoro-phosphate
(HBTU) (GL BioCHEM LTD) NN-Diisopropylethylamine (DIPEA) (Fluka) Fluorescein-5-
isothiocyanate (FITC) Fluoresceinamine isomer-1(FluorA) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from SIGMA ALDRICH Cell
penetrating peptide (CPP) was provided by University of Tartu cell culture medium (FCS
PSG) Phosphate Buffer Saline (PBS) Dimethylformamide (DMF) Human EDTA Plasma
Ethanol (EtOH)
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
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14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
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19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
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(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
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29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
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(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
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(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
21
22 Surface Functionalization of TCPSi-NH2 with MeO-PEG-COOH (PEGylation)
221 Preparation of PEGylated TCPSi-NH2NPs by Using DIC and
NHS as an Activator Reagent
To optimize the activation concentrations of DIC and NHS the carboxyl group 780microL of
5768 kDa MeO-PEG-COOH (10 mgmL in DMF) was activated separately by adding 625
125 1875 25 50 75 100 125 150microL of DIC(100 mgmL in DMF) and 575 115 1725
23 46 69 92 115 and 138 microL of NHS (100 mgmL in DMF) at room temperature to
prepare an active succinimidyl ester-terminated intermediate After activation 02 mg
(10526microL) of amino-modified TCPSiNPs (19 mgmL in DMF) was added into the reaction
system to produce the PEGylated TCPSi NPs Paparticles were mixed by using end over end
mixer (BIOSAN Bio Rotator RS-Multi) overnight at room temperature to complete the
reaction and resulting PEGylated TCPSiNPs were washed by six cycles of centrifugation
(Eppendorf centrifuge 5415D 13200 rpm for 20 min) and washing (three times with DMF
and three times with water) to remove unreacted MeO-PEG-COOH DIC and NHS
222 Preparation of PEGylated TCPSiNH2NPs by Using HBTU and
DIPEA as an Activator Reagent
In order to optimize the activation concentrations of HBTU and DIPEA for the preparation of
PEGylated TCPSiNPs the carboxyl group of 390 microL of 5768 kDa MeO-PEG-COOH (10
mgmL in DMF) was activated separately by adding 379 758 1516 3032 4548 6064
758 microL of HBTU (100 mgmL in DMF) and 129 258 516 1032 1548 258 microLof DIPEA
(100 mgmL in DMF) at room temperature After activation 01mg of amino-modified
TCPSiNPs (19 mgmL in DMF) were added to produce PEGylated TCPSiNPs Particles
were washed as described above
223 Preparation of PEGylated TCPSi-NH2NPs by Refluxing of
Amino-modified TCPSi-NH2NPs and MeO-PEG-COOH
In the third method of PEGylation 03 mg of TCPSi-NH2NPs and 1170 microL of MeO-PEG-
COOH (10 mgmL) were taken into 10 mL round bottom flask refluxed overnight stirring
with a magnetic stirrer and finally cooled at room temperature The unreacted Me-PEG-
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
22
COOH was removed by washing three times with 1mL of DMF and then three times with
1mL of water as described above After each centrifugation an ultrasonic bath was used to re-
suspend the PEGylated-TCPSiNPs in solution
224 PEGylation of TCPSi-NH2 with MeO-PEG-COOH of Different
Size (5768kDa 11kDa and 25kDa)
780 microL of 5768 11 and 25 kDa of MeO-PEG-COOH (10 mgmL in ethanol) were taken
into three separate Eppendorf tubes and 50 microL of DIC (100 mgmL in ethanol) and 46 microL of
NHS (100 mgmL in EtOH) were added followed by 02 mg (10526 microL) of TCPSi-NH2NPs
(19 mgmL in EtOH) into each tube Particles were mixed using end over end mixer
overnight at room temperature to complete the reaction Centrifugation was performed to
remove the supernatant and particles were washed three times with 1mL of EtOH and finally
washed three times with 1mL of water as described above After washing 50 microg of sample
was taken from each tube and diluted with water to 3 mL for size and zeta potential
measurement
225 Opsonization
150 microg of PEGylated TCPSi-NH2 nanoparticles were suspended into 200 microL of a solution
containing 11 ratio of PBS (pH = 74) and human plasma (HP) Particles were incubated for
15 minutes at 37oC in Termaks incubator centrifuged for 10 minutes and washed three times
with 1mL of water as above Finally 50 microg PEGylated TCPSi-NH2 nanoparticles were diluted
to 3 mL with water for the size and zeta potential measurement as mentioned earlier
Opsonized particles were also analyzed with sodium dodecyl sulphate polyacryl amide gel
electrophoresis (SDS-PAGE)
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
23
23 Surface Functionalization of TCPSi-NH2 and UnTHCPSi NPs with Fluorescent Molecules
231 Standard Calibration Curve with FITC and FluorA
Fluorescence intensity was measured with Fluoroskan Ascent FL (Thermo Lab systems) and
standard calibration curve was plotted with fluorescence intensity versus concentration of
FITC and FluorA
232 Labeling of TCPSi-NH2 Nanoparticles (NPs) with FITC
FITC labeled NPs were prepared by addition of 02 mg of FITC (10 mgmL in ethanol) into
02 mg (12658 microL) of TCPSi-NH2ndashnanoparticles (158 mgmL in ethanol) in a Eppendorf
tube and sonicated for about 3 hours in the dark followed by mixing with end-over-end mixer
overnight at 37oC in Termaks incubator The resulting FITC labeled TCPSiNPs were washed
by six cycles of centrifugation (20 min) NPs were washed three times with 1mL ethanol and
three times with 1mL 50mM HEPES (pH 72) to remove unreacted FITC An ultrasonic bath
(Branson 2510 E-MT Danbury CT USA) was used to re-suspend the FITC labeled TCPSi-
NH2 NPs in solution after each centrifugation Finally they were suspended in 200 μL of 50
mM HEPES (pH 72) and stored at +40C
2321 Absorption of Stearylated NickFect 51 (NF51) Cell
Penetrating Peptides (CPPs) onto the Surface of FITC Labeled
TCPSi-NH2NPs
Prior to CPP absorption 180μL (180μg) of FITC labeled NPs were centrifuged for 20 min at
13200 rpm and the supernatant was discarded FITC labeled TCPSi-NH2NPs were washed
two times with cell culture medium (FCS PSG) An ultrasonic bath was used to resuspend
the FITC-TCPSi-NH2 in solution after each centrifugation Then 18 microL (57 mgmL) of CPPs
were added into sample After adding CPPs the solution was sonicated for 3 hours followed
by centrifugation and discarding of supernatant The particles were washed thrice with
HEPES and resuspended into 180 microL of HEPES Fluorescence intensity was measured in a
similar way as mentioned earlier by taking 20 microL (1microgmicroL) of sample
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
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3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
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7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
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9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
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14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
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19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
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Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
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(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
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(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
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(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
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(b) Walker N J Science 2002 296 557
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Applications Kluwer Academic Publishers New York 1998
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Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
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32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
24
2322 Conjugation of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides (125I-CPPs) to the
Surface of FITC Labeled TCPSi-NH2NPs
Prior to 125
I-CPPs absorption 130 μL (1microgmicroL) of FITC labeled TCPSi-NH2NPs were
centrifuged for 20 min at 13200 rpm and the supernatant was discarded FITC labeled TCPSi-
NH2NPs were washed two times with 500 microL of cell culture medium An ultrasonic bath was
used to resuspend the FITC labeled TCPSi-NH2 in solution after each centrifugation Then
13microL (57 mgmL) of 125
I-CPPs (9KBq) were added into sample After adding 125
I-CPPs the
solution was mixed with end-over-end mixture overnight at room temperature Then the
mixture was centrifuged and the supernatant was discarded The particles were washed thrice
with 500 microL of 10mM HEPES (pH 74) and finally modified particles were resuspended into
500 microL of 10mM HEPES All of the supernatants were collected into a gamma-counter tube
The radioactivity of 125
I-CPPs conjugated particles was measured with Gammacounter (LKB-
Wallac Clingamma 1272 Turku Finland)
233 Labeling of UnTHCPSiNPs with Fluoresceinamine Isomer-1
(FluorA)
The carboxyl group of 02mg of UnTHCPSiNPs (72 mgmL in ethanol) was activated by
adding 40mM of DIC (50microL) and NHS (46microL) in ethanol at room temperature to give an N-
hydroxysuccinimide ester -terminated intermediate 40micromol (1388mg) of FluorA in ethanol
was added on NPs and the mixture was protected from light by wrapping with tinfoil By
using end over end mixer (BIOSAN Bio Rotator RS-Multi) NPs were mixed overnight at
37oC in Termaks incubator followed by centrifuging for 20 min and discarding the
supernatant The resulting PEGylated TCPSiNPs were separated by six cycles of
centrifugation (20 min) and washing PEGylated TCPSiNPs were washed three times with
1mL of ethanol and then three times with 1mL of 50 mM HEPES (pH 72) to remove
unreacted FluorA An ultrasonic bath was used to resuspend the UnTHCPSi-FluorA in
solution after each centrifugation Finally labeled NPs were suspended into 200 μL of 50 mM
HEPES (pH 72) and fluorescence intensity was measured in a similar manner as mentioned
earlier
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
25
24 Characterization Methods
241 Dynamic Light Scattering (DLS)
Size and zeta potential was measured at room temperature (230C) with Zetasizer Nano-ZS
by Malvern Instruments USA by taking 50 microg of PEGylated-TCPSi-NH2NPs which was
diluted to 3mL with water Before measuring the particles were sonicated for a while
242 Gel Electrophoresis
SDS-PAGE was used for separation of nanoparticles and proteins based on their size Nine
samples containing 50microg opsonized NPs human plasma (1200) and molecular weight size
markers were loaded into adjacent wells in the gel In the gel electophoresis part the samples
were suspended into 15microL buffer containing 125mM Tris-HCl (pH 68) 2 SDS 5
glycerol and 0002 bromophenol blue and were heated about 5 minutes at 100oC in block
heater before loading to the wells The running buffer was 1 x SDS electrophoresis buffer
containing 25 mM Tris-HCl 200 mM Glycine and 01 Sodium dodecyl sulfate 10 gel
with 4 stacking gel was used to run in 100 V 400mA and approximately 100 minutes
Every lane showed separation of the plasma proteins from the original mixture After running
the gel was stained with 0025 Coomassie Brilliant Blue overnight Finally they were
washed with water and molecular sizes of the stained proteins were compared to the
molecular weight standards
243 Fluorescence Intensity Measurement
20 μL (1μgμL) of suspended nanoparticle solution was pipetted into a well (96 plate well)
and diluted with 180 μL of PBS for the fluorescence measurement The concentration of the
particles in the first well was 01μgμL in Phosphate buffer saline (PBS) 100 μL of FITC-
TCPSi-NH2 NPs were titrated serially 12 with PBS into next four wells Fluorescence was
measured using excitation wave length of 488 nm and emission wave length of 520 with
Fluoroskan Ascent FL (Thermo Lab systems)
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
26
3 Results and discussion
To functionalize the surface of the porous silicon nanoparticles with FITC and MeO-PEG-
COOH we have employed amino thermally carbonized porous silicon nanoparticles (TCPSi-
NH2) Fluoresceinamine isomer-1 (FluorA) has been used for the modification of undecylenic
acid dervatized thermally hydrocarbonized porous silicon nanaoparticles (UnTHCPSi)
31 Surface Modification of TCPSi-NH2 with MeO-PEG-COOH (5768 kDa) Polyethylene glycol conjugation to amino derivated silicon nanoparticles (amide bond) was
Figure 31 Formation of amide through the reaction of terminal ndashNH2 groups in TCPSi-NH2 with ndashCOOH
groups in MeO-PEG-COOH
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
27
achieved via the formation of N-hydroxysuccinimide ester or HBTU as a leaving group
(reaction scheme shown with DICNHS Figure 31)
Prior to any modification of the surface amino groups are responsible for the positive zeta
potential of the core particle The size of the original nanoparticles was 170 nm and the zeta
potential was +70 mV When using DICNHS as activators the zeta potential shifted to lower
values (mostly negative zeta potential) Although the zeta-potential and the size changed
during the conjugation there was no direct correlation between size zeta potential and the
concentrations (Table 31) The samples containing 20 and 40 mM of DIC and NHS is
suggested to be aggregated In conclusion the changes in size and zeta potential of the
particles suggest that the surface conjugation of MeO-PEG-COOH to the TCPSi-NH2 NPs
were successful
Table 31 Size and zeta potential values of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as
a surface modifier with different concentration of DIC and NHS as an activator
Sample Name Size (nm) Zeta potential (mV)
Original (plain) NPs 170 +70
NP+5mM of DIC and NHS 196 -287
NP+10mM of DIC and NHS 183 +163
NP+20mM of DIC and NHS 922 -185
NP+40mM of DIC and NHS 666 -165
NP+60mM of DIC and NHS 181 -252
NP+80mM of DIC and NHS 1716 -308
NP+100mM of DIC and NHS 1761 -27
NP+120mM of DIC and NHS 219 -223
The coupling efficiency was further tested using HBTU as a coupling reagent The size and
the zeta-potential of the PEGylated nanoparticles by using HBTU and DIPEA had
insignificant changes (table 32) suggesting insufficient conjugation of PEG
Thirdly PEG was also conjugated to the nanoparticles by refluxing The zeta potential and
size of refluxed nanoparticles showed significant change indicating successful conjugation
(Table 32 last row)
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
28
Table 32 Size and zeta potential of PEGylated TCPSi-NH2 nanoparticles by using MeO-PEG-COOH as a
surface modifier and different concentration of HBTU and DIPEA as an activator except last row
Sample Size (nm) Zeta potential (mV)
NP+ 10mM of HBTU and DIPEA 188 +52
NP+ 40mM of HBTU and DIPEA 1857 +532
NP+80mM of HBTU and DIPEA 1701 +494
NP+120mM of HBTU and DIPEA 196 +546
NP+ 160mM of HBTU and DIPEA 181 +483
NP+200mM of HBTU and DIPEA 194 +514
NP+ MeO-PEG-COOH (reflux) 240 -300
32 Opsonization of the PEGylated TCPSi-NH2 NPs
Polyethyleneglycol is a relatively inert hydrophilic polymer that provides good steric
hindrance for preventing protein binding Comparing the obtained size and zeta potential
after the opsonization of the PEGylated and original TCPSi-NH2 nanoparticles lower protein
adsorption was observed for the PEGylated than for the non-PEGylated particles (Table 33)
Table 33 Size and zeta potential values of the investigated opsoninated NPs prepared with DIC and NHS
Sample Size(nm) Zeta-potential(mV)
Plain NPs 357 -313
NP+5mM of DIC and NHS 428 -19
NP+10mM of DIC and NHS 868 -272
NP+20mM of DIC and NHS 445 -291
NP+40mM of DIC and NHS 358 -312
NP+60mM of DIC and NHS 774 -169
NP+80mM of DIC and NHS 344 -327
NP+100mM of DIC and NHS 377 -278
NP+120mM of DIC and NHS 338 -215
The zeta potential of the non-PEGylated particles as well as PEGylated particles with positive
zeta potential was changed to the negative values after opsonization Nanoparticles
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
29
conjugated with 120 micromol of DIC and NHS showed the lowest value in the size and zeta
potential after opsonization suggesting the optimal shielding properties
Figure 32 The SDS-PAGE analysis of original and PEGylated nanoparticles MW = molecular marker NP=
only nanoparticles R= refluxed sample 05=5mM of DIC and NHS+ NP + m -PEG-COOH 10=10mM of DIC
and NHS +NP + m-PEG-COOH 15=15mM of DIC and NHS +NP + m-PEG-COOH 20=20 mM of DIC and
NHS +NP + m-PEG-COOH 40=40 mM of DIC and NHS+ NP + m-PEG-COOH 60= 60mM of DIC and NHS
+ NP + m-PEG-COOH 80=80 mM of DIC and NHS+NP+m-PEG-COOH100=100 mM of DIC and
NHS+NP+m-PEG-COOH120=120 mM of DIC and NHS +NP + m-PEG-COOH Plasma= human plasma
The molecular weight and relative adsorption rates of the human plasma proteins were
analyzed with PAGE (Figure 32) In gel-1 wells containing (40 mM of DIC NHS and 60
mM of DIC NHS) showed the lower adsorption rate opsonization than other wells In gel-2
well containing (120 mM of DIC and NHS) showed the less opsonization From gel-3 we
cannot find significant difference like gel-1 and gel-2 If we compare the gel-1 and gel-2 we
can conclude that well containing 40 mM of DIC NHS and 60 mM of DIC NHS slightly
better than the well containing 120 mM of of DIC NHS of gel-2 Using relative high
concentration ie 40 - 120 mM of of DIC and NHS the lowest opsonization degree was
achieved suggesting the maximal coverage of the nanoparticles with PEG-molecules From
the aforementioned discussion we can conclude that the concentration of NHS and DIC
influenced the activation and the optimum result was found with 40 and 60 mM of DIC and
NHS From this point of view 40 mM concentration of DIC and NHS were used for the
further PEGylation The proteins which were adsorbed to the surface of TCPSi-NH2 NPs
during opsonization may be fibrinogen Immunoglobulin G (IgG) and Human serum albumin
(HSA)
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
30
33 Surface Functionalization of TCPSi-NH2 by Using Different Size of MeO-PEG-COOH
Surface functionalization of TCPSi-NH2 with different sizes of MeO-PEG-COOH was
investigated by measuring size and zeta potential The pegylated-TCPSi-NH2NPS with
different size of PEG molecules were prepared several times but surprisingly the consistent
results (size and zeta potential) were not found We got different results every time Two of
test series are shown in the table 34(a) and 34(b)
Table 34a Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 210 -163
PEG 11kDa 243 -134
PEG 25kDa 245 -78
Table 34b Zeta potential values of the investigated PEG-TCPSi-NH2 NPs
Sample Size(nm) Zeta potential (mV)
PEG 5768kDa 1662 -343
PEG 11kDa 1761 -140
PEG 25kDa 187 -201
Three PEG molecules with different molecular weights were 5768 11 and 25kDa No direct
correlation was also observed between the molecular weight of the PEG molecule and the
zeta potential values (Table 34b) though all PEG molecules bound to 3-(2-
Aminoethylamino)propyldimethoxymethylsilane (AEASP) amine groups resulting diminish
of the positive charges introduced and thus causing a negative zeta potential which could be
partially interpreted by the charge-shielding effect of PEG backbones A significant
correlation was observed between the molecular weight of the PEG molecule and the size
values Size was increased with increasing the length of the PEG molecule
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
31
34 Surface Modification of Amino Thermally Carbonized Porous Silicon (TCPSi-NH2) Nanoparticles (NPs) with Fluorescein-5-isothiocyanate (FITC)
FITC is an organic fluorophore with mainly hydrophobic behavior which is a popular amine
labeling reagent forming a thiourea bond upon reaction The particles are conjugated with
FITC by a covalent attachment of the dye with an appropriate functional group Using FITC
the fluorochrome was covalently bound to AEASP through the isothiocyanate group in the
dye and the primary amino group of the TCPSi-NH2 (Figure 33)
Figure 33 Represents covalent conjugation of isothiocyanate with a primary amine
Mechanism of the reaction
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
32
The lone pair of nitrogen in primary amine attacks on the carbon of the isothiocyanate group
resulting negative charge on nitrogen of isothiocyanate group and positive charge on nitrogen
of primary amine Finally negatively charged nitrogen abstracts a proton from the positively
charged nitrogen and consequently thiourea bond is formed
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
In addition non-covalent conjugation of FITC to the surface may be observed Non-covalent
adsorption of FITC varied Centrifugation and washing cycles totally removed adsorbed
fluorophores which was confirmed by fluorescence spectroscopy The surface of covalently
conjugated SiNPs was considered free of adsorbed labels because they did not fade during
washing
The fluorescence intensity of FITC-labeled silicon NPs were determined in standard PBS
solutions (excitation wave length at 488 nm emission wavelength of 520 nm) The maximum
emission for FITC-labeled particles is at ~520 nm The fluorescence intensity of FITC-
labeled NPs is extremely dependent on pH of the solution screening enhanced intensity in
basic conditions
The determination of a number of FITC conjugated inside the NPs is challenging due to the
potential effect of self-quenching of dye molecules in close proximity Calibration curves
with the pure fluorophore were performed in order to elucidate the number of FITC
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
33
molecules trapped inside the matrix (Figure 34) Linearly dependent fluorescence intensity on
FITC concentration was observed with regression coefficient 0995
Figure 34 Fluorescence intensity vs FITC concentration
Figure 35 Fluorescence intensity vs FITC labeled TCPSi-NH2NPs concentration
Linearly dependent fluorescence intensity on FITC labeled NPs concentration was also
observed (Figure 35) with regression coefficient 09943
y = 34477x Rsup2 = 09959
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty o
f FI
TC
Concentration of FITC(micro-mol)
y = 15057x Rsup2 = 09943
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
Flu
ore
sce
nce
inte
nsi
ty
Concentration of FITC labeled NPs (microg)
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
34
The fluorescence intensity of FITC labeled TCPSi-NH2NPs (10microg10microL) was 147 mV In
comparison to the calibration curve 4255 nmol of FITC was conjugated to the amino-
modified NPs The obtained fluorescence intensity of the particles reveals that the surface
conjugation of FITC to the TCPSi-NH2 NPs were successful
After consecutive washing of FITC labeled TCPSi-NH2NPS with both ethanol and HEPES
the fluorescence of the supernatant was measured (data not showed) but surprisingly
fluorescence of the supernatant was higher than the precipitate NPs interacted with
fluorescence material and lowered their intensity and this may be a reason for lower
fluorescence intensity in suspension compared to supernatant as in supernatant there are no
NPs and for that such interaction is not expected there
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
FITC conjugated nanoparticles Observed single peak reveals uniform size distribution and
monodispersity of the particles The size of the Plain TCPSi-NH2 NPs was 170 Significant
change in particle size was not shown with a mean diameter around (176 nm) in deionized
water at ambient temperature after fluorescence labeling The zeta potential of the plain
TCPSi-NH2 was +70 mV due to the presence of amino groups but after modification with
FITC the potential drops to -26 mV which indicates conjugation of FITC to surface Plain
NPs have some silanol groups which were remained after FITC labeling and higher negative
charged was observed on account of silanol groups that were dissociated or deprotonated in
de-ionized water or oxidation of silanol groups
341 Characterization of Stearylated Stearylated NickFect 51
(NF51) CPPs Adsorped FITC Labeled TCPSi-NH2NPs
Stearylated NickFect 51 (NF51) CPPs conjugated FITC labeled NPs were investigated by
measuring fluorescence intensity with florescence spectroscopy Fluorescence intensity (~30)
of CPPs conjugated FITC labeled NPs were higher than Fluorescence intensity (~14 mV) of
the FITC labeled NPs which suggests the conjugation of CPPs to the FITC labeled NPs were
flourishing It is not conclusive So for further confirmation radioactive iodine labeled CPPs
(125
I-CPPs) were used for the conjugation onto the surface
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
35
342 Characterization of Radioactive Iodine Labeled Stearylated
NickFect 51 (NF51) Cell Penetrating Peptides Conjugated FITC
Labeled TCPSi-NH2 NPs
The detection of radioactive materials after the radiolabeling was counted for 1 min by a γ-
counter The radioactivity was 88 It reveals that 88 of the added peptide was adsorped to
NPs surface
35 Surface Modification of UnTHCPSi Nanoparticles (NPs) with FluorA
FluorA is an organic dye containing primary amine group which is used to carboxyl acid
labeling reagent forming an amide bond during reaction Fluoresceinamine isomer-
1(FluorA) an amino derivative of fluorescein was covalently conjugated to the carboxylic
acid group of the UnTHCPSiNPs with NHS and DIC by mixing overnight at 370C in EtOH
through reaction route in Figure 36
Figure 36 Coupling of a carboxylic acid and a primary amine via O-acylisourea ester and NHS ester
intermediates
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
36
As mentioned in figure 36 the carbodiimide-mediated conjugation of a carboxylic acid (1)
with a primary amine first proceed through an unstable intermediate an O-acylisourea ester
(3) produced by the reaction of the carbodiimide (2) with the carboxylic acid (1) Since this
intermediate is unstable it is often the case that NHS (N-hydroxysuccinimide) (4) is added to
the carboxylic acid together with the carbodiimide in order to form an NHS-ester (5) which
is a much more stable that remains reactive with amines Upon the final addition of an amine
(6) the preferred coupling product an amide (7) is formed by displacement of the NHS
Thus in this case the FluorA was covalently bound to UnTHCPSiNPs through the amine
group in the dye and the carboxyl acid group of UnTHCPSiNPs The conjugation of FluorA
to the UnTHCPSiNPs was investigated by measuring fluorescence intensity
The fluorescence of the FluorA labeled UnTHCPSiNPs was monitored by comparing the
fluorescence signal of the NPs solution with a calibration curve of the pure dye under the
same conditions Though it is not possible to determine the accurate number of dye molecules
because of the significant effect of self-quenching (proved) of dye molecules in close
proximity the determination of the average fluorescence of the NPs is possible Figure 37
and 38 shows a correlation between fluorescence intensity and concentration of the pure dye
and dye labeled NPs Linearly dependent fluorescence intensity on FluorA and FluorA
labeled UnTHCPSiNPs concentration were observed with regression coefficient 09933 and
09901
Figure 37 Fluorescence intensity vs concentration of FluorA
y = 34959x Rsup2 = 09933
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5
Flu
ore
sce
nce
inte
nsi
ty
Concentration of fluoresceinamine (microM)
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
37
Figure 38 Fluorescence intensity vs concentration of UnTHCPSi-FluorA NPs
The fluorescence intensity of FlourA labeled UnTHCPSiNPS (10microg10microL) was 298 mV In
comparison to the calibration curve 85243 nmol of FlourA was covalently attached to the
acid-modified UnTHCPSiNPs The investigated fluorescence intensity of the particles reveals
that the surface conjugation of FlourA to the UnTHCPSiNPs was also successful
Dynamic light scattering (Zetasizer) was used to investigate the size and zeta potential of the
fluoresceinamine labeled UnTHCPSi nanoparticles The single peak obtained reveals uniform
size distribution and monodispersity of the particles The size of the Plain UnTHCPSiNPs
was (135-140 nm) The NPs showed a little bit change in particle size with a mean diameter
around (148 nm) in deionized water at ambient temperature after fluorescence labeling The
zeta potential of the plain UnTCPSiNPs was -30 mV due to the presence of carboxyl acid
groups Following modification with fluorA the potential changes to -36 mV
y = 0305x Rsup2 = 09901
0
05
1
15
2
25
3
35
0 2 4 6 8 10 12
Flo
ure
sce
ne
inte
nsi
ty
microg of NPs well
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
38
4 Conclusion
Surface activation of the three different sized (5768 11 and 25 kDa) of Me-PEG-COOH was
carried out by acid termination into succinymidyl (activated) ester by using DICNHS action
Different concentrations were used to activate the carboxyl acid group (only 5kDa Me-PEG-
COOH) but the concentrations of DIC and NHS must be carefully chosen to ensure optimal
activation The activated PEG polymers were conjugated to the amine groups of the amino
derivatized thermally carbonized porous silicon nanoparticles through the covalent amide
bond Aminated thermally carbonized porous silicon nanoparticles were successfully
functionalized with PEG layer using DICNHS as activators but not with HBTUDIPEA
reagents In addition we tried to PEGylate the nanoparticles by thermal decomposition which
was also successful The degree of the conjugation was approximated by size change and zeta
potential measurements Potential aggregation was observed in PBS for coated nanoparticles
after few hours using 20 ndash 40 mmolar concentrations of DICNHS The efficiency of the
PEGylated nanoparticle surfaces in preventing biologically nonspecific adsorption of human
plasma proteins was investigated with opsonization (with 5768 kDa PEG molecule) High
degree of PEGylation may change the balance of hydrophilicity and hydrophobicity of the
nanoparticles The DICNHS activated particles showed lowest opsonization degree
according to SDS-PAGE analysis In addition aggregation caused by 20 - 40 mmolar
concentration was not observed after opsonization suggesting that plasma proteins or other
plasma components interrupt the aggregation
Desirable surface modification of the acid derivated thermally hydrocarbonized porous
silicon nanoparticles were successfully tailored by flouoresceinamine isomer-1(FluorA)
through the amide bond formation and the surface of the aminated thermally carbonized
porous silicon nanoparticles were functionalized with FITC through the thiourea bond
formation Compared to the FITC labeled nanoparticles fluorA labeled nanoparticles were
screened lower fluorescence intensity because of the self quenching The stearylated
NickFect 51 (NF51) CPPs moiety was also successfully adsorbed to FITC labeled TCPSi-
NH2NPs which was screened higher fluorescence intensity and to check the CPPs
conjugation to the surface of FITC labeled TCPSi-NH2NPs radioactive iodine labeled
stearylated NickFect 51 cell penetrating peptides was conjugated to FITC labeled TCPSi-NH2
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
39
NPs which was ensured by Gammacounter Both fluorescent nanoparticles and stearylated
NickFect 51 (NF51) cell penetrating peptides conjugated FITC labeled TCPSi-NH2NPs can
be used as an effective bioimaging probe
Complete deployment of PEGylated and fluorescent labeled NPs still hampers due to a
number of limitations and challenges Size and specifically zeta potential was not consistent
for both PEGylated and fluorescent labeled NPs The higher zeta potential of modified NPs
was surprising The minimization of the opsonization was monitored but total minimization
still to be observed The fluorescence intensity was higher in the supernatant for fluorescent
labeled NPs which can be overcome by performing further study To summarize our
experiment turned out successful in minimization of opsonization and obtaining desired
surface modification of PSiNPs and now next turn is to check out effectiveness these
modified PSiNPs and continue further research to make those achieved results better
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
40
5 References
1 Salonen J Kaukonen A M Hirvonen J amp Lehto V P J Pharm Sci 2008 97 632-653
2 Cai W Chen X Small 2007 3 1840
3 Cai W Gao T Hong H amp Sun J Nanotechnol Sci Appl 2008 1 17
4 Sahoo S K Parveen S amp Panda J J Nanomedicine 2007 3 20
5 Grodzinski P Silver M amp Molnar L K Expert Rev Mol Diagn 2006 6 307
6 Larson D R Zipfel W R Williams R M Clark S W Bruchez M P Wise F W amp Webb W W
Science 2003 300(5624) 1434ndash1436
7 Babes L Denizot B Tanguy G Jeune J J L amp Jallet P J Colloid Interf Sci 1999 212(2) 474ndash482
8 Rosenholm J M Meinander A Peuhu E Niemi R Eriksson J E Sahlgren C amp Linden M ACS
Nano 2009 3(1) 197 -206
9 Barreto J A OrsquoMalley W Kubeil M Graham B Stephan H amp Spiccia L Adv Mater 2011 23(12)
H18ndashH40
10 Wu H Huo Q Varnum S Wang J Liu G Zimin N Liu J amp Lin Y Analyst 2008 133(11) 1550ndash
1555
11 Canham L T Advanced Materials 1995 7(12) 1033
12 (a) Hossain S M Chakraborty S Dutta S K Das J amp Saha H J Lumin 2000 91 195-202
(b) Karacali T Cakmak B amp Efeoglu H Optics Express 2003 11 1237-1242
13 Astrova E V Voronkov V B Remenyuk A D amp Shuman V B Semiconductors 1999 33(10)
1149-1155
14 Collins R T Fauchet P M amp Tischler M A Phys 1997 50 24ndash31
15 Mayne A H Bayliss S C Barr P Tobin M amp Buckberry L D Phys Status Solidi AmdashAppl Res
2000 182 505ndash513
16 Stewart M P amp Buriak J M Angew Chem Int Ed Engl 1998 37 3257ndash3260
17 Nassiopoulos A G Grigoropoulos S amp Canham L et al Thin Sol Films 1995 255 329ndash333
18 Maeda H Wu J Sawa T Matsumura Y amp Hori K a review J Control Release 2000 65(1ndash2) 271ndash
284
19 Jain P K Huang X El-Sayed I H amp El-Sayed M A Acc Chem Res 2008 41(12) 1578ndash1586
20 Santra S Xu J S Wang K M amp Tan W H J Nanosci Nanotechnol 2004 4 590ndash599
21 Kwan A S Barry C McAllister I L amp Constable I Clin Experiment Ophthalmol 2006 34 33ndash38
22 D e Oliveira J G Beck J Seifert V Teixeira M J amp Raabe A Neurosurgery 2008 62 1300ndash1309
23 Raabe A Beck J Gerlach R Zimmermann M amp Seifert V 2003 52 132ndash139
24 (a) Lakowicz J R Topic in Fluorescence spectroscopy Lakowicz J R Ed Plenum Press New York
1994 4
(b) Lakowicz J R Soper S amp Thompson R B Eds SPIE San Jose CA 1999
25 (a) Frontiers in Biosensorics I Fundamental Aspects Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
41
(b) Frontiers in Biosensorics II Practical Applications Scheller FW Schubert F Fedrowitz J Eds
Birkhauser Verlag Berlin Germany 1997
26 (a) Ulman A Chem Rev 1996 96 1533
(b) Taton T A Mucic R C Mirkin C A amp Letsinger R L J Am Chem Soc 2000 122 6305
(c) He L Musick M D Nicewarner S R Salinas F G Benkovic S J Natan M J amp Keating C D J
Am Chem Soc 2000 122 9071
(d) Albrecht M G amp Creighton J A J Am Chem Soc 1977 99 5215
(e) Nam J -M Park S -J amp Mirkin C A J Am Chem Soc 2002 124 3820
(f) Wang G Zhang J amp Murray R W Anal Chem 2002 74 4320
27 (a) Kneipp K Kneipp H Itzkan I Dasari R R amp Feld M S Chem Rev 1999 99 2957
(b) Leopold N amp Lendl B A J Phys Chem B 2003 107 5723
(c) Mulvaney S P Musick M D Keating C D amp Natan M J Langmuir 2003 19 4784
(d) Goulet P J G Pieczonka N P W amp Aroca R F Anal Chem 2003 75 1918
28 (a) Ho W J Phys Chem B 2002 117 11033
(b) Xu H X amp Kall M Phys Rev Lett 2002 89 246802
(c) Tian Z Q Ren B WuD Y J Phys Chem B 2002 106 9463
(d) Lee S J amp Kim K Chem Commun 2003 2 212
29 Lakowicz J R Principles of Fluorescence Spectroscopy 2nd
Ed Kluwer AcademicPlenum Publishers
New York 1999
30 (a) Gosling J P A Cell 1990 36 1408
(b) Walker N J Science 2002 296 557
31 (a) Kronick M N J Immunol Methods 1986 92 1
(b) Daehne S Resch-Genger U amp Wolfbeis O S Eds Near-Infrared Dyes for High Technology
Applications Kluwer Academic Publishers New York 1998
(c) Casay G A Shealy D B amp Patonay G Near-infrared fluorescence probes In Topics in
Fluorescence Spectroscopy Probe Design and Chemical Sensing Lakowicz J R Ed Plenum Press
New York 1994 4 183-222
(d) Loumlvgren T amp Pettersson K In Fluorescence Immunoassay and Molecular Applications Van Dyke
K Van Dyke R Eds CRC Press New York 1990 234-250
32 (a) Lakowicz J R Anal Biochem 2005 337 171
(b) Lakowicz J R Anal Biochem 2001 298 1
(c) Gryczynski I Malicka J Shen Y B Gryczynski Z amp Lakowicz J R J Phys Chem B 2002 106
2191
(d) Malicka J Gryczynski I Fang J Kusba J amp Lakowicz J R Anal Biochem 2003 315 160
(e) Gersten J I amp Nitzan A Surf Sci 1985 158165
33 (a) Kummerlen J Leitner A Brunner H Aussenegg F R amp Wokaun A Mol Phy 1993 80 1031
(b) Sokolov K Chumanov G amp Cotton T M Anal Chem 1998 70 3898
(c) Antunes P A Constantino C J L Aroca R F Duff J Langmuir 2001 17 2958
(d) Kamat P V J Phys Chem B 2002 106 7729
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
42
(e) Kulakovich O Strekal N Yaroshevich A Maskevich S Gaponenko S Nabiev I Woggon U amp
Artemyev M Nano Lett 2002 2 1449
34 (a) Geddes C D Cao H Gryczynski I Gryczynski Z Fang J Y amp Lakowicz J R J Phys Chem A
2003 107 3443
(b) Geddes C D Parfenov A Roll D Fang J Y amp Lakowicz J R Langmuir 2003 19 6236
(c) Parfenov A Gryczynski I Malicka J Geddes C D Lakowicz J R J Phys Chem B 2003
107 8829
35 (a) Kreibig U Vollmer M Optical Properties of Metal Clusters Springer-Verlag Berlin and
Heidelberg Germany 1995
(b) Kerker M amp Blatchford C G Phys Rev B 1982 26 4082
(c) Zhang J Whitesell J K amp Fox M A J Phys Chem B 2003 107 605
36 Wagstaff K M amp Jans D A Current Medicinal Chemistry 2006 13(12) 1371-1387(17)
37 Okuyama M Laman H Kingsbury S R Visintin C Leo E Eward K L Stoeber K Boshoff C
Williams G H amp Selwood D L Nature Methods 2007 4(2) 153-159
38 Saba T M Arch Intern Med 1970 126(6) 1031ndash1052
39 Owens D 3rd
amp Peppas N Int J Pharmaceut 2006 307(1) 93ndash102
40 Van Vlerken L E Vyas T K amp Amiji M M Pharm Res 2007 24(8) 1405ndash1414
41 Decker K Eur J Biochem 1990 192(2) 245ndash261
42 Zolnik B S amp Sadrieh N Adv Drug Deliv Rev 2009 61(6) 422ndash427
43 Guzman K Finnegan M Banfield J Environ Sci Technol 2006 40(24) 7688ndash7693
44 Yang P Ando M amp Murase N Langmuir 2010 27(3) 895ndash901
45 Jun Y Casula M Sim J et al J Am Chem Soc 2003 125(51) 15981ndash15985
46 Foumlrster S amp Antonietti M Adv Mat 1998 10(3) 195ndash217
47 Zhao W Brook M amp Li Y Chem Bio Chem 2008 9(15) 2363ndash2371
48 Knop K Hoogenboom R Fischer D amp Schubert U S Angew Chem Int Ed Engl 2006 49(36)
6288ndash6308
49 Wang F Tan W B Zhang Y Fan X amp Wang M Nanotechnology 2006 17 R1
50 Ow H Larson D R Srivastava M Baird B A Webb W W amp Wiesner U Nano Lett 2005 5 113
51 Larson D R Ow H Vishwasrao H D Heikal A A Wiesner U amp Webb W W Chem Mater 2008
20 2677
52 Ethiraj A S Hebalkar N Kharrazi S Urban J Sainkar S R amp Kulkarni S K J Luminescence 2005
114 15
53 Bringley J F Penner T L Wang R Harder J F Harrison W J amp Buonemani L J Colloid Interface
Sci 2008 320 132
54 Aslan K Wu M Lakowicz J R amp Geddes C D J Am Chem Soc 2007 129 1524
55 Addison C J amp Brolo A G Langmuir 2006 22 8696
56 Shibata S Taniguchi T Yano T amp Yamane M J Sol-Gel Sci Technol 1997 10 263
57 Imahori H Mitamura K Shibano Y Umeyama T Matano Y Isoda S Araki Y amp Ito O J
PhysChem B 2006 110 11399
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971
43
58 Chen X Zou J Zhao T amp Li Z J Fluorescence 2007 17 235
59 Bailey R E Smith A M amp Shuming N Phys E 2004 25(1) 1ndash12
60 Li K G Chen J T Bai S S Wen X Song S Y Yu Q Li J amp Wang Y Q Toxicol Vitr 2009
23(6) 1007ndash1013
61 Kanaras A G Kamounah F S Schaumburg K Kiely C J amp Brust M Chem Commun 2002 20
2294ndash2295
62 Kwon G S Crit Rev Ther Drug Carrier Syst 2003 20(5) 357ndash403
63 Gref R Minamitake Y Peracchia M T et al Science 1994 263(5153) 1600ndash1603
64 Alexis F Pridgen E Molnar L K amp Farokhzad O C Mol Pharm 2008 5(4) 505ndash515
65 Ishida O Maruyama K Sasaki K amp Iwatsuru M Int J Pharm 1999 190(1) 49ndash56
66 Degennes P G Adv Colloid Interface Sci 1987 27(3ndash4) 189ndash209
67 Degennes P G Macromolecules 1980 13(5) 1069ndash1075
68 Levin C S Bishnoi S W Grady N K amp Halas N J Anal Chem 2006 78(10) 3277ndash3281
69 Lee H de Vries A H Marrink S J amp Pastor R W J Phys Chem B 2009 113(40) 13186ndash13194
70 Daou T J Li L Reiss P Josserand V amp Texier I Langmuir 2009 25(5) 3040ndash3044
71 Choi H S Ipe B I Misra P et al Nano Lett 2009 9(6) 2354ndash2359
72 Ballou B Lagerholm B C Ernst L A Bruchez M P amp Waggoner A S Bioconjug Chem 2004
15(1) 79ndash86
73 Ostuni E Chapman R G Holmlin R E Takayama S amp Whitesides G M Langmuir 2001 17(18)
5605ndash5620
74 Prime K Whitesides G Science 1991 252(5009) 1164ndash1167
75 Kairdolf BA Mancini MC Smith AM Nie S AnalChem 2008 80 (8) 3029ndash3034
76 Parrish B Breitenkamp RB Emrick T J Am Chem Soc 2005 127(20) 7404ndash7410
77 Bagwe R P Zhao X amp Tan W J Dispers Sci Technol 2003 3amp4 453ndash464
78 Mitchell G P Mirkin C A amp Letsinger R L J Am Chem Soc 1999 121(35) 8122ndash8123
79 Zhang C Y Ma H Nie S M Ding Y Jin L amp Chen D Y Analyst 2000 125 1029ndash1031
80 Willard D M Carillo L L Jung J amp Orden A V Nano Lett 2001 1(9) 469ndash474
81 Parak W J Gerion D Pellegrino T Zanchet D Micheel C Williams S C Boudreau R Le Gros M
A Larabell C A amp Alivisatos A P Nanotechnology 2003 14
82 Goldman E R Balighaian E D Mattoussi H Kuno M K Mauro J M Tran P T amp Anderson G P
Avidin J Am Chem Soc 2002 124 6378ndash6382
83 Baeumle M Stamou D Segura J M Hovius R amp Vogel H Langmuir 2004 314(2) 529ndash534
84 Torchilin V P Khaw B A Smirnov V N amp Haber E Biochem Biophys Res Commun 1979 85
1114ndash1119
85 Magnani P Paganelli G Modorati G Zito F Songini C Sudati F Koch P Maecke H R Brancato
R Siccardi A G amp Fazio F J Nucleic Med 1996 37 967ndash971