Download - GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES: …
GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES:
SYNTHESIS, PROPERTIES, CELLULAR INTERACTIONS
AND CYTOTOXICITY STUDIES
A Thesis presented to the Faculty of the Graduate School University of Missouri
In Partial Fulfillment Of the Requirements for the Degree
Master of Science
By
SWAPNA MEKAPOTHULA
Dr. Chada S. Reddy and Dr. Kattesh V. Katti, Thesis Supervisors
MAY 2008
The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled
GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES: SYNTHESIS, PROPERTIES, CELLULAR INTERACTIONS AND CYTOTOXICITY STUDIES
presented by Swapna Mekapothula,
a candidate for the degree of Master of Science,
and hereby certify that, in their opinion, it is worthy of acceptance.
Dr. Chada S. Reddy
Dr. Kattesh V. Katti
Dr. Wade V. Welshons
Dr. Stan W. Casteel
Dr. Raghuraman Kannan
Dedicated to my dear husband
ii
ACKNOWLEDGEMENTS
First and foremost I wish to thank Dr. Kattesh V. Katti for his support and
guidance throughout the project and for giving me this great opportunity to work in the
field of cancer treatment which is my goal in life. I am indebted to Dr. Chada S. Reddy
who has been a guide at every turn and was always there encouraging me with his very
valuable suggestions. This research would not have been fruitful without the
encouragement and in depth knowledge provided by Dr. Raghuraman Kannan.
Dr. Wade V. Welshons and Dr. Stan W. Casteel were gracious in providing me
with the laboratory facilities necessary to pursue the thoughts and ideas into practical
existence. John A. Flanders, Kavita Katti, Nripen Chanda, Nune Satish, Ravi Shukla,
Rajesh Kulkarni, Jeanne Muse, Randy Tindall and Cheryl Jensen were very helpful at
different stages of the project giving me a very good insight into my research.
It would have been a daunting task if it were not for the people in my personal
life. Constant encouragement and love of my parents and my dear sister always helped
me to believe in me and my dreams. Lastly, and most importantly, I wish to thank my
husband whose love and support made all this possible.
I cannot end without thanking Mr & Mrs Reddy and their family who made me
almost forget my home and no amount of gratitude is enough for them. I also wish to
thank my little angel Ankush, who has always put a smile on my face with his cute
mannerisms.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...............................................................................................ii LIST OF ILLUSTRATIONS .............................................................................................vi LIST OF TABLES ..........................................................................................................viii LIST OF ABBREVIATIONS.............................................................................................ix ABSTRACT …....................................................................................................................x
CHAPTER 1 INTRODUCTION ………………………………………………………………………..1 1.1 Gold nanoparticles and their properties……………………………….....................1
1.2 Synthesis of Gold Nanoparticles …………………………………………………...4
1.3 Gold nanoparticle based targeted delivery systems………………………………...5
CHAPTER 2 BOMBESIN CONJUGATED GOLD NANOPARTICLES FOR TARGETTED DELIVERY…......................................................................................................................7 2.1 Introduction…………………………………………………………………………..7 2.2 Materials and Methods ………………………………………………………………8 2.3 Synthesis of Bombesin conjugated Gold nanoparticles ……………………………..9
2.3.1. Synthesis of Starch stabilized Gold Nanoparticles………………………....9 2.3.2. Filtration …………………………………………………………………..10 2.3.3. Conjugation of Thioctic acid-BBN conjugate to S-AuNps……………….10
2.4. Cell internalization procedure…………………………………………………….10 2.5. Results and Discussion ……………………………………………….. ………...12
iv
CHAPTER 3 ANNEXIN V – GOLD NANOPARTICLES AS APOPTOSIS DETECTION PROBES…………………………………………………………………………………25 3.1 Introduction……………………………………………………………………….25 3.2 Materials and Methods …………………………………………………………...26 3.3 Synthesis of Annexin V-Gold Nanoparticles ………………………….. ………..26
3.3.1 Synthesis of Thioctic acid analogue of Annexin V………………………....26 3.3.2 Starch coated AuNps………………………………………………………..27 3.3.3 Conjugation reaction of Annexin V-TA with S-AuNps………………….....27
3.4 Interaction of Annexin-V-AuNps with Apoptotic Cells…………………………..28 3.5 Scanning Electron Microscopy……………………………………………………29 3.6 Results and Discussion…………………………………………………………... 30 CHAPTER 4 GREEN GOLD NANOPARTICLES……………………………………………………37 4.1 Introduction………………………………………………………………………..37 4.2 Materials and Methods …………………………………………………………...38 4.2.1 Synthesis of Soybean Gold Nanoparticles …………………………………38 4.2.2 Synthesis of Tea Gold Nanoparticles ………………………………………38 4.2.3 Synthesis of Cinnamon gold nanoparticles ………………………………...39 4.3 Cellular Interaction studies …………………………………………………….39 4.3 Cytotoxicity Studies ………………………………………………………………39 4.4 Results and Discussion…………………………………………………………....40
v
CHAPTER 5
SUMMARY AND FUTURE WORK
5.1 Summary …………………………………………………………………………..53
5.2 Future work………………………………………………………………………...54 BIBILOGRAPHY………………………………………………………………………..55
vi
LIST OF ILLUSTRATIONS
Figure Page
1.1 Gold Nanoparticles and their electronic properties……………………………..1 1.2 Theory of Surface Plasmonics…………………………………………………..2
1.3 General Synthesis protocol of Gold Nanoparticles……………………………..5
2.1 Comparison of Amino acid sequences in Gastrin releasing peptide and
Bombesin…………………………………………………………………………7 2.2 Synthesis protocol of BBN-AuNps…………………………………………….14
2.3 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Starch
Gold Nanoparticles ……………………………………………………………..15
2.4 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Bombesin Gold Nanoparticles…………………………………………………..16
2.5 Different stages of the cellular uptake process of BBN-AuNps. TEM images of
PC-3 cells depicting the arrival of a BBN-AuNps at the cell membrane, binding of the nanoparticles to surface receptors, membrane wrapping of the Nps, and finally internalization into the cell nucleus……………………………………...17
2.6 TEM images of MCF-7 cells showing the internalization of BBN-AuNps into
the nucleus………………………………………………………………………18
2.7 TEM images of PC-3 cells showing no uptake of BBN-AuNps when the GRP-receptors are blocked by excess free BBN (0.035 mg)………………………….19
2.8 TEM images of MCF-7 cells showing no uptake of BBN-AuNps when the GRP-
receptors are blocked by excess free BBN (0.035 mg)……………………….....20
2.9 TEM Images of three different PC-3 cells showing uptake of Unconjugated-AuNps in to the lysosomes……………………………………………………...21
2.10 TEM Images of three different MCF-7 cells showing uptake of
Unconjugated-AuNps in to the lysosomes…………………………....................22
3.1 Synthesis of Thioctic acid analogue of Annexin-V…………………………….27 3.2 Conjugation reaction of Annexin-V-TA with S-AuNps………………………..28
vii
3.3: a) UV-Visible absorption spectrum, b) TEM image of Annexin V-Gold Nanoparticles………………………………………………………………………..30 3.4: a, b, c, d, e, f: Effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells………………………………...32 3.5: Dose response curve for Anx V- AuNps in displacement of FITC-Anx from apoptotic Jurkat-T cells……………………………………………………………...34
3.7: Apoptotic Jurkat-T cell showing rough surface………………………………...35 3.8: a, b, c, d Back scatter SEM images showing surface binding of Anx V-AuNps to apoptotic Jurkat-T cells incubated with 278.4 µM Anx V-AuNps………………….36
4.1 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Tea gold nanoparticles…………………………………………………………………...41
4.2 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Soy gold nanoparticles…………………………………………………………………...42
4.3 a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Cinnamon Gold Nanoparticles………………………………………………………43
4.4 a, b, c: TEM Images of different MCF-7 cells showing uptake of Tea-AuNps in to the lysosomes……………………………………………………………………..45
4.5 a, b: TEM Images of different MCF-7 cells showing uptake of Soya -AuNps in to the lysosomes……………………………………………………………………..46
4.6 a, b: TEM images of MCF-7 cell showing uptake of Cin-AuNps in to the lysosomes……………………………………………………………………………47
4.7 a, b: Dose dependent cytotoxicity of Tea-AuNps in cultured PC-3 and MCF-7 cells after 24 hrs of exposure using MTT assay…………………………………….49
4.8 a, b: Dose dependent cytotoxicity of Soy-AuNps in cultured PC-3 and MCF-7 cells after 24 hrs of exposure using MTT assay…………………………………….50
4.9 a, b: Dose dependent cytotoxicity of Cin-AuNps in cultured PC-3 and MCF-7 cells after 24 hrs of exposure using MTT assay………….........................................51
4.10: Dose dependent cytotoxicity of NaAuCl4 in cultured PC-3 and MCF-cells after 24 hrs of exposure using MTT assay………………………………………….52
viii
LIST OF TABLES
3.6: Table showing the effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells…………………………………34
ix
LIST OF ABBREVATIONS
AuNps Gold nanoparticles
Anx V Annexin V
Anx V-AuNps Annexin V-Gold nanoparticles
BBN Bombesin
Cin-AuNps Cinnamon gold nanoparticles
DI De-Ionized Water
DMF Dimethylformamide
GA Gum Arabic
GRP Gastrin releasing peptide
HSA Human serum albumin
RS Raman Scattering
S-AuNps Starch stabilized Gold nanoparticles
SERS Surface Enhanced Raman Scattering
Soy-AuNps Soy Gold nanoparticles
TA Thioctic Acid
T A-BBN Thioctic acid-Bombesin conjugate
TEM Transmission Electron Microscopy
THPAL Tris hydroxyl phosphine alanine
T-AuNps Tea Gold nanoparticles
x
GOLD NANOPARTICLE–BIOMOLECULE CONJUGATES:
SYNTHESIS, PROPERTIES, CELLULAR INTERACTIONS
AND CYTOTOXICITY STUDIES
Swapna Mekapothula
Dr. Chada S. Reddy and Dr. Kattesh V. Katti, Thesis Supervisors
ABSTRACT
In recent years, Gold nanoparticles (AuNps) have been widely used in diverse biomedical
applications because of their efficient optical and electronic properties. Gold
nanoparticles also posses a strong surface chemistry which renders them suitable for
attachment with biomolecules. Researchers are considering the use of gold nanoparticles,
as potential contrast enhancement agents in X-ray Computed Tomography and Photo
Acoustic Tomography imaging techniques, useful in the early detection of specific
tumors. Fundamental to any further clinical developments in gold nanoparticulate based
imaging or drug design is the ability to synthesize gold nanoparticles conjugated with
proteins/biomolecules that impart high affinity to target various disease pathologies. This
research focuses on conjugation of two major bio-molecules i.e., Bombesin and Annexin
V, to gold nanoparticles and studying their target specificity. The target specificity of
gold nanoparticles coated with Bombesin, a GRP (Gastrin releasing peptide) receptor
specific protein was tested using two cancer cell lines (MCF-7, breast cancer cells; and
PC-3, prostate cancer cell line) that over-express GRP receptors. Both cell types exhibit
xi
significant uptake of Bombesin gold nanoparticles, internalizing them through a highly
specific receptor mediated endocytosis pathway.
Binding of AuNps coated with a phospholipid-binding protein Annexin V with
high affinity towards apoptotic cells was tested in Jurkat-T- lymphocytes. Annexin V-
gold nanoparticles showed excellent affinity towards the apoptotic Jurkat-T lymphocytes
binding to the cells in a manner similar to the biomolecule annexin V.
We further used environmentally benign so called green chemicals i.e.,
polyphenols, flavonoids, catechins, and various phytochemicals present in tea, soybean,
and cinnamon and their synergistic reduction potentials to reduce the gold salts into
AuNps. Such nanoparticles also showed excellent affinity toward epidermal growth
factor receptors (EGFR) on prostate and breast cancer cells and proved to be non-
cytotoxic at as high as 150µM. These studies showed that gold nanoparticles can be
coated not only to exert specific molecular interactions in specific cell types but also to
be devoid of adverse effects.
1
CHAPTER 1
Introduction:
Nanotechnology is a tremendously powerful technology, which holds a huge
promise for the design and development of many types of novel products with its
potential medical applications on early disease detection, treatment, and prevention. Gold
nanoparticles represent a new class of biocompatible vectors capable of fulfilling this
promise by selective cell and nuclear targeting of which will provide new means for the
site- specific diagnosis and treatment of medical conditions. This work outlines the
methodology for conjugation of AuNps with target specific biomolecules (Bombesin,
Annexin V) and details the results of studies assessing the target specificity and
Cytotoxicity effects of thus conjugated gold nanoparticles.
1.1 Gold Nanoparticles and their properties:
Gold nanoparticles are defined as stable colloid solutions of clusters of gold atoms
with sizes ranging from 1-100 nm (Figure 1.1).
Gold Nanoparticles Gold Nanoparticles are a cluster of gold atoms with
sizes in nanometer range.
Gold nanoparticle 1 nm – 100 nm Cluster of Gold atoms
Example: 0.65 nm Colloidal Gold-nanoparticle solution contains approximately 9 gold atoms
Figure 1.1: Gold Nanoparticles and their electronic properties
2
At this nanoscale, AuNps possess different physicochemical characteristics when
compared to the bulk gold [1, 2], most obvious example being the color change from
yellow to ruby red when bulk gold is converted into nanoparticulate gold. This ruby red
color of AuNps is explained by a theory called “surface plasmonics”.
According to this theory, when the clusters of gold atoms are hit by the
electromagnetic field of the incoming light, the surface free electrons (6 electrons in case
of AuNps) present in the conduction band of AuNps oscillate back and forth thus,
creating a plasmon band which has an absorption peak in the visible region at 530-540
nm (Figure 1.2) [3]. The surface plasmon band (SPB) of AuNps is used as an indicator for
formation during the synthesis of AuNps from their precursor salts. The sensitivity of
plasmon band absorptivity is the basic detection mechanism involved in the AuNps based
bio-sensors [4, 5].
Figure 1.2: Theory of Surface Plasmonics.
3
Physical properties of AuNps in turn depend on the size, shape, particle-particle distance
and the nature of the stabilizer used to prevent the agglomeration of nanoparticles [1].
According to Mie theory, Surface Plasmon Band (SPB) is absent for AuNps less than
2nm and greater than 500nm [2]. Gold nanorods have two SPB’s, one longitudinal-
wavelength band at 550-600nm and one transverse-wavelength band at 520nm [6, 7].The
longitudinal-wavelength band is very sensitive and changing the aspect ratio of Gold
nanorods changes the absorption region from visible to Near-infra red (NIR) [8]. This
unique optical property of Gold nanorods is used in Near-infra red ray therapy [9]; and
enhanced Raman scattering of adsorbed biomolecules [10]. Therefore, by changing the
size and shape of AuNps, the SPB and scattering may be tuned for application in cellular
imaging, drug delivery and therapy.
The six free electrons present in the conduction band of nanoparticulate gold
makes them potential candidates to bind with thiols and amines [11]. Therefore, AuNps
may be easily tagged with various proteins and bio-molecules rich in amino acids leading
to important biomedical applications including targeted drug delivery [12, 13], cellular
imaging [14], and biosensing [15]. Further, the free electrons also render AuNps useful as
contrast enhancement agents [16]. Imaging studies are based on comparisons of contrast
produced by the variations in the electron densities in different tissues. With their high
electron densities, AuNps serve as excellent contrast enhancement agents in the detection
of tumors.
4
1.2 Synthesis of Gold Nanoparticles:
Gold Nanoparticles are traditionally synthesized by reducing metallic gold in +3
state to nanoparticulate gold in +1 state. There are a number of reducing agents reported
in the literature for the synthesis of AuNps. Two most important ones are: Tri sodium
citrate (Citrate synthesis) discovered by Turkevitch in 1973 [17] and Sodium borate
(Borate synthesis) introduced by Brust-Schiffrin in 1994 [18]. These two synthesis
protocols pose potential problems in case of size control, stability and most importantly
toxicity [19]. Therefore, we followed a novel protocol reported by Katti et. al. in 2003
that uses a phosphino-amino acid based reducing agent, tris hydroxyl phosphine alanine
(THPAL) to synthesize AuNps (Figure 1.3).
THPAL is a water-soluble non-toxic reducing agent. It is reported that swine models can
withstand up to 100 mg/kg of body weight of THPAL with out showing toxicity, the most
important of criteria in the use of nanoparticles for bio medical applications.
Due to the strong surface reactivity of free electrons present on AuNps, they
easily tend to agglomerate posing stability problems. Naturally occurring, FDA approved
non-toxic compounds such as starch; gum arabic and gelatin were used to stabilize
AuNps immediately after they are formed [20]. These stabilizers form weak covalent
bonds with AuNps so that they easily shed off in the presence of biomolecules with
strong electronegative groups with which the AuNps can then react.
5
The Reaction:
Au+3(aq) + Reducing Agent (aq) + Stabilizer ----- Au0 (solid)/Au+1
[HAuCl4; NaAuCl4] THPAL [Starch; Gum Arabic; Gelatin]
Gold nano particles
5 minutes
NaAuCl4
THPALTHPAL
PNH
NH
NH
COOH COOH
COOH
Novel nontoxic phosphino- amino acid based reducing agent
Heat at 90-100C
+THPAL
(Katti et. al., J. Am. Chem. Soc. 2003, 125, 6955.)
STARCH +
Figure 1.3: General Synthesis protocol of Gold Nanoparticles.
1.3 Gold nanoparticle - based targeted delivery systems:
Gold nanoparticles; due to their ease of synthesis, uniform size distribution, rich
surface chemistry and a lack of toxicity; have been proven to be excellent candidates for
conjugation with numerous biomolecules for site-specific delivery. Selective cell and
receptor targeting of AuNps are likely to provide new pathways for the targeted delivery
of diagnostic/ therapeutic agents [21, 22]. Tkachenko et al. showed specific nuclear
targeting of AuNps by conjugating them with bovine serum albumin (BSA) using
differential contrast microscopy [23, 24]. Gold nanoparticles are used as targeted contrast
6
agents in detecting cervical cancer by tagging them with monoclonal antibodies and
oncoproteins associated with human papilomavirus [25]. Mercaptoalkyl-oligonucleotide-
conjugated AuNps have been used in the detection of polynucleotides by plasmon band
interactions with the surrounding environment [26]. In particular, Gold nanoshells, when
coated with breast tumor marker Her-2, effectively localized in the microscopic tumors
present in the breast tissue [27]. Most of the work in the area of nanoimaging is also
focusing on fabricating detectors that can detect efficiently cells undergoing apoptosis, an
effect common to many chemotherapeutic regimens. These examples of the use of
AuNPs in medical diagnosis although demonstrate the potential of AuNPs even in drug
delivery, their target specificity by conjugation with biomolecules has yet to attain the
desired refinement.
The overall goal of this research is to conjugate non-toxic AuNps with
biomolecules like Bombesin and Annexin V which posses’ high affinity towards specific
receptors over-expressed on cancer cells and phosphatidylserine (PS), a phospholipid
exposed on apoptotic cells respectively and studying their target specificity.
7
CHAPTER 2
Bombesin Conjugated Gold Nanoparticles for Targeted Delivery
2.1 Introduction:
Thirty years ago, Erspamer and Anastasi isolated Bombesin (BBN) from the skin of
amphibians Bombina variegata and Bombina bombina [29]. Bombesin is a
tetradecapeptide neurohormone with exocrine and endocrine effects. Its C-terminal amino
acid sequence is very similar to that of Gastrin releasing peptide (GRP) which has high
affinity towards G-protein coupled receptors (GPCRs) (Figure 2.1).
Gastrin-releasing peptide –Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2
Bombesin –Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2
Figure 2.1: Comparison of Amino acid sequences in Gastrin releasing peptide and Bombesin
Bombesin is proved to have numerous pharmacological effects, such as control of
gastrin release, modulation of gastrointestinal secretions, smooth muscle motility and the
amount of food intake [30-34]. Recently, it has been shown to play a major role in cell
proliferation, tumor growth and inflammation [35, 36]. There is substantial evidence
indicating increased BBN/GRP (Gastric releasing peptide) receptor expression in
prostate, small-cell lung, ovarian, breast, pancreatic, gastric, renal cell, and thyroid
8
cancers, suggesting their use as specific markers for targeting [37-45]. Since BBN and
GRP have high affinity towards GRP receptors, conjugating AuNps with these peptides
should allow for greater binding of these particles to the tumor sites, thus making them
suitable for early detection of tumors by imaging techniques.
By modifying the structure of BBN several derivatives of BBN with high affinity
towards GRP receptors (agonists) and some with low affinity towards receptors
(antagonists) have been synthesized [46, 47, 48]. In this research, 7-amino acid truncated
BBN (Figure 2.2) derivative was conjugated to AuNps that has been proven to have high
agonistic action towards prostate, small-cell lung and breast cancer cell lines. A Disulfide
bond (-S-S) present in thioctic acid (TA), an antioxidant, was used as a Linker to bind
truncated BBN with AuNps. Starch was used as a stabilizer.
2.2 Materials and Methods:
Sodium tetrachloroaurate (NaAuCl4) was obtained from Alfa-Aesar (Ward Hill,
MA). Tris hydroxyl phosphino alanine (THPAL) was synthesized according to the
standard protocols described in the literature [49, 50]. Sephadex G -100 gel was obtained
from Pharmcia (Newyork). 7-amino acid Bombesin (BBN) was obtained from Anaspec
(San Jose, CA). De-ionised water was used as a solvent to synthesize gold nanoparticles.
Minimum essential medium (MEM with nonessential amino acids, powdered),
HEPES, insulin, streptomycin sulfate, penicillin-G, were obtained from Sigma Chemical
Company (St. Louis, MO). Bovine calf serum, phenol red (sodium salt), and lyophilized
trypsin were obtained from Gibco BRL (Grand Island, NY).
9
Cell Culture:
MCF-7 breast cancer cells and PC-3 prostate cancer cells were obtained from V.
Craig Jordan (University of Wisconsin-Madison) and ATCC (Manassas, VA)
respectively. MCF-7 cells were maintained in MEM with nonessential amino acids, 10
pgml-1 phenol red, 10 mM HEPES, 6 ngml-1 insulin, 100 units ml-1 penicillin, 100 pgml-1
streptomycin, and 5% charcoal-stripped calf serum (maintenance medium) [51,52,53].
PC-3 cells were maintained in RPMI medium supplemented with 4.5 gL-1 D-glucose, 25
mM HEPES, 0.11 gL-1 sodium pyruvate, 1.5 gL-1 sodium bicarbonate, 2 mM L-glutamine
and 10 % FBS and100 units ml-1 penicillin and 100 pgml-1 streptomycin.
2.3 Synthesis of Bombesin conjugated Gold nanoparticles
(BBN-AuNps):
2.3.1. Step-1: Synthesis of Starch stabilized Gold Nanoparticles (S-AuNPs):
To a 10 ml vial, 0.0225 g of starch was added and dissolved in 6 ml of double-
distilled and deionized water. This starch solution was heated up to 900C -1000C and
stirred continuously until dissolved. To the dissolved starch solution, 100 μl of 0.1 M
NaAuCl4 solution was added resulting in a pale yellow color solution. To this solution, 20
μl of 0.1 M THPAL solution was added with continuous stirring. The resulting solution
slowly turned into purplish-wine color representing the formation AuNps. The mixture
was cooled to room temperature and S-AuNPs were characterized by UV-Vis absorption
Spectroscopy and Transmission Electron Microscopy (TEM) measurements [54].
10
2.3.2 STEP: 2 – Filtration:
Thus synthesized S-AuNps were filtered using Sephadex columns to remove any
unreacted starch from the mixture [54]. The absorbance peak of filtered S-AuNps was
adjusted to 0.7 by diluting the resulting solution to suit further conjugation with thioctic
acid-BBN conjugate.
2.3.3 STEP: 3-Conjugation of Thioctic acid-BBN (TA-BBN) conjugate to S-AuNps:
Based on the studies done by Kattumuri et. al [54] 0.885 μM of Thioctic acid-
BBN conjugate (1 mg in 1 ml of methanol) was added to 1 ml of filtered S-AuNps and
stirred for 60 hrs. The product, thus formed was further filtered and purified by washing
several times with mixture a of methanol and water to remove all traces of unconjugated
reactants. The purified Bombesin-conjugated gold nanoparticles (BBN-AuNPs) solution
was vacuum dried and re-dissolved in PBS (without Ca2+ and Mg2+). This conjugate was
used to study its target specificity towards GRP receptors present on PC-3 and MCF-7
cell lines.
2.4. Cell internalization procedure:
About 16,000 cells (PC-3/MCF-7) were plated into each well in a 6 well plate
were incubated at 370C for 20.0 hrs to allow the cells to recover. The medium from each
well was aspirated and 4 ml of fresh growth medium was added per each well. Cells were
allowed to grow until they reached confluence by changing the medium every alternate
day. To the confluent cell layer, 150 μl of BBN-AuNps solution made up in PBS was
added and further incubated for 4 hrs at 370C. The medium was then aspirated from each
11
well and the cell layer was rinsed 3 times with complete growth medium to remove any
traces of uninternalized BBN-AuNps. Then the cell layer was washed with CMFH-EDTA
(Calcium-Magnesium-Free-Hank’s +HEPES - EDTA) solution to remove all traces of
serum, a trypsin inhibitor. About 0.5 ml of 0.1 M Trypsin-EDTA solution was added to
each well to detach the cell layer from the plastic. Detached cells were dispersed in 4 ml
of complete growth medium and gently pipetted out of the well. The cell suspension was
transferred into a centrifuge tube and centrifuged at approximately 125 x g for 5 minutes.
Supernatant was discarded and cell pellet was fixed with 0.1 M Na-Cacodylate buffer
containing 2% glutaraldehyde and 2% paraformaldehyde. The pellets were post-fixed
with 1 % osmium tetraoxide, dehydrated and embedded in Epon/ Spurr’s resin and 80 nm
sections were collected and placed on TEM grids followed by sequential counterstaining
with uranyl acetate and lead citrate. TEM grids were observed under TEM (Joel 1400)
and images were recorded at different magnifications. As a control, similar incubations
were performed with either 25 μM unconjugated AuNps and/or with the same
concentration BBN-AuNps in the presence of 0.035mg of BBN, (since this is the amount
of BBN used to conjugate S-AuNps).
12
2.5 Results and Discussion:
2.5.1. Synthesis of Bombesin conjugated Gold nanoparticles (BBN-AuNps):
7-amino acid truncated BBN, a derivative of BBN, has been proven to have high
agonistic action towards GRP receptors [55, 56]. According to literature, AuNps show
high affinity towards sulfur [11]. Indeed, sulfur bonds present in various biomolecules
could be used as linkers to conjugate them with AuNps. In fact, disulfide bond (S-S)
present in thioctic acid serves as a linker to conjugate AuNps selectively with BBN even
in the presence of thiol groups in other biomolecules. Thioctic acid is conjugated to BBN
by first activating its carboxyl group using “2-(1H-benzotriazol-1-yl)-1, 1, 3, 3,-
tetramethyluronium hexafluorophosphate” (HBTU) followed by an addition reaction with
BBN (Figure 2.2). The product thus formed TA-BBN, was then conjugated with AuNps.
Gold Nanoparticles were synthesized using NaAuCl4 as a gold precursor, THPAL
as a reducing agent and starch solution as a stabilizer. Starch stabilizes AuNps by
forming weak coordination bonds between its –OH groups and gold atoms. These weak
coordination bonds are broken down in the presence of powerful -S-S- bond present in
Thioctic acid-BBN conjugates to remove starch coating on AuNps. Filtered S-AuNps
showed a uniform size distribution of 15±5 nm and an absorption peak at 530 nm.
Absorption studies and TEM analysis of the S-AuNps shows excellent uniformity and
desired size distribution of S-AuNps necessary for further conjugation with TA-BBN
(Figure 2.3).
For the synthesis of BBN-AuNps 1 ml of stable S-AuNps were stirred with 0.855
μM of TA-BBN for 60 hrs. The purified BBN-TA-AuNps when analyzed by UV-visible
spectroscopy and TEM showed a uniform size distribution of 20nm and has an absorption
13
peak at 530 nm (Figure 2.4). These values are similar to the values reported indicated by
Petersen.et.al for AuNps conjugated to CTAB and by Katti.et.al for AuNps conjugated to
cysteine [57].
14
Reaction steps in the synthesis of BBN-AuNps
Figure 2.2: Synthesis protocol of BBN-AuNps
15
a) b)
c)
Size distribution of Starch-AuNps
0
5
10
15
20
25
30
35
1 3 5 7 9 11 13 15 17 19 21 23 25
Size of Starch-AuNps
Num
ber o
f AuN
ps
Figure 2.3: a) TEM Image, b) UV-Visible absorption spectrum, and c) size distribution of Starch Gold Nanoparticles.
16
a) b)
c)
Size distribution of BBN-AuNps
0
5
10
15
20
25
30
35
1 3 5 7 9 11 13 15 17 19 21 23 25
Size of BBN-AuNps
Num
ber o
f AuN
ps
Figure 2.4: a) TEM Image, b) UV-Visible absorption spectrum, and c) size distribution of Bombesin-Gold Nanoparticles (BBN-AuNps).
17
a) b)
c) d)
Figure 2.5a, b, c, d: Different stages of the cellular uptake process of BBN-AuNps. TEM images of PC-3 depicting the arrival of a BBN-AuNps at the cell membrane, binding of the nanoparticles to surface receptors, membrane wrapping of the Nps, and finally internalization into the cell nucleus, respectively.
18
a)
b)
Figure 2.6: TEM images of MCF-7 cells showing the internalization of BBN-AuNps into the nucleus.
19
a)
b)
Figure 2.7 a, b: TEM images of PC-3 cells showing no uptake of BBN-AuNps when the GRP-receptors are blocked by excess free BBN (0.035 mg).
20
Figure 2.8: TEM images of MCF-7 cells showing no uptake of BBN-AuNps when the GRP-receptors are blocked by excess free BBN (0.035 mg).
21
a)
b)
c)
Figure 2.9 a, b, c: TEM Images of three different PC-3 cells showing uptake of Unconjugated-AuNps in to the lysosomes.
22
a)
b)
c)
Figure 2.10 a, b, c: TEM Images of three different MCF-7 cells showing uptake of Unconjugated-AuNps in to the lysosomes.
23
2.5.2. Cellular interactions of Bombesin conjugated Gold nanoparticles:
To determine whether BBN-AuNps can be used to label BBN/GRP receptors over
expressed in prostate and breast cancer cells, cultures of confluent PC-3 and MCF-7 were
exposed for 4 hrs to BBN-AuNps. These incubated samples are analyzed under TEM to
verify whether the BBN-AuNps are bound to the GRP receptors present in PC-3 and
MCF-7 cells. Figures 2.5a,b,c,d clearly show binding of BBN-AuNps to the plasma
membrane of PC-3 cells by forming clathrin pits and then those pits delivering the
nanoparticles into the nucleus via endosomes. Figures 2.6 show a similar uptake of BBN-
AuNps into the nucleus of MCF-7 cells. A control experiment, in which cells were
exposed to the same concentration of BBN-AuNps in the presence of 0.035 mg of BBN,
showed no uptake of BBN-AuNps Figures 2.7 a, b and 2.8. These results suggest that the
uptake of BBN-AuNps occurs likely by the same mechanism as that of Bombesin. These
results are also in agreement with the results of similar studies showing the uptake of
Bombesin coated Qdots in Swiss 3T3 cells, which over-express GRP receptors [58].
To further substantiate the results, we also incubated unconjugated gold
nanoparticles with PC-3 and MCF-7 cells. TEM analysis (Figures 2.9 a, b, c and 2.10 a,
b, c) demonstrated that unconjugated gold nanoparticles were packed inside intact single
membrane vesicles, similar to lysosomes or phagosomes, and showed no specific uptake
process. Therefore, it may be concluded that unconjugated AuNps were taken up by the
cells via non-specific phagocytosis pathway since they contained no targeting peptide and
BBN-AuNps likely being taken up by a GRP-receptor mediated endocytosis. Based on
these results, it appears feasible that Bombesin conjugated AuNps could be used to
24
specifically target and bind to the endogenously expressed GRP receptors on PC-3 and
MCF-7 cells and could be effectively used for targeted delivery and/or early detection of
tumors by various imaging techniques.
25
CHAPTER 3
Detection of Apoptosis using Annexin-Gold Nanoparticles
3.1 Introduction:
Apoptosis is an extremely organized, energy dependent form of programmed cell
death. It plays a crucial role in the maintenance of normal cellular homeostasis by
removing the damaged cells. Apoptosis is a fundamental feature reported in the
pathology of wide range of diseases including Alzheimer’s disease, various lung diseases,
in cardiac ischemia and in progressive heart failure [59, 60, 61]. The process of apoptosis
is characterized by distinct morphological features like loss of plasma membrane
integrity, nucleus and cytoplasm condensation, membrane blebbing, formation of
apoptotic bodies. Early apoptotic phase is marked by the exposure of a phospholipid
molecule, phosphatidylserine present on the cytoplasm side of the cell on to the outer cell
surface [62, 63]. This key feature may be effectively utilized in fabricating various
apoptosis detection probes. Therefore, Annexin V a 35 kDa phospholipid-binding protein
that has high affinity towards PS exposed on apoptotic cells has been chosen to
conjugated with gold nanoparticles to specifically target AuNps on to the apoptotic cells
[64, 65, 66]. Annexin V conjugated to various fluorescence and magnetic probes such as
FITC and Iron oxide are reported to show high affinity to apoptotic cells [67, 68, 69, 70].
26
3.2 Materials and Methods:
Annexin-V and an Annexin V-FITC Apoptosis Detection Kit was procured from
BioVision Research Products, (CA, USA). Thioctic acid, N-hydroxy succinimide (NHS),
dicylcohexyl carbodiamide (DCC), phosphate buffer (pH 7) and methanol are obtained
from Sigma Aldrich Chemicals, (St. Louis, MO).
Cell Culture:
Jurkat T cells obtained from ATCC (ATCC TIB-152) were used to induce apoptosis.
The cells were grown in ATCC complete growth medium, which contains RPMI 1640
medium, adjusted with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose,
10 mM HEPES, 1.0 mM sodium pyruvate, 90% and 10% FBS.
3.2.1 Synthesis of Annexin V-Gold Nanoparticles (Anx V-AuNps):
STEP: 1
3.2.1a Synthesis of Thioctic acid analogue of Annexin V:
Sixty micro grams of Annexin V was dissolved in 9 ml of 2:1 solution of
phosphate buffer (pH 7) and methanol by stirring continuously. To this solution, 400 µL
of Thioctic acid (26.89 mg of TA in 10 mL MeOH), 400 µL of H-hydroxy succinimide
(NHS) (15.1 mg of NHS in 10 mL MeOH) and 400 µL of dicyclohexyl carbodiamide
(DCC) (26.89 mg of DCC in 10 mL MeOH) were added. The reaction mixture was
stirred for 16 hrs and the small amount of urea (reaction by-product) that is formed at the
end of the reaction was filtered using 1.2 micron filter. The remaining Annexin V-
Thioctic acid analogue solution was used insitu further in the conjugation reaction with
S-AuNps.
27
Figure: 3.1 Synthesis of Thioctic acid analogue of Annexin-V
STEP: 2
3.2.1b Starch stabilized AuNps:
Starch stabilized AuNps are synthesized as described in the section 2.3.1.
STEP: 3
3.2.1c Conjugation reaction of Annexin V-TA with S-AuNps:
Annexin V-TA analogue and filtered S-AuNps obtained in steps 1 and 2 respectively are
used for further conjugation reactions. To 1 ml of Annexin V-TA in 2:1 mixture of
phosphate buffer/methanol 1 ml of S-AuNps solution was added drop-by-drop, stirring
continuously. This mixture was stirred for 16 hrs to obtain the gold conjugate of
Annexin-V. The Annexin V-AuNps conjugate thus obtained was purified to remove all
traces of unconjugated Annexin V-TA by repeated centrifugation and washing, with 2:1
phosphate buffer/ methanol solution. The pure Anx V-AuNps conjugate formed was
characterized using UV-Vis spectroscopy and TEM analysis.
28
Figure 3.2: Conjugation reaction of Annexin-V-TA with S-AuNps
3.2.2 Interaction of Annexin-V-AuNps with Apoptotic Cells:
To induce apoptosis, 7 µL of Camptothecin (1 mM in DMSO) per ml of culture
medium was added to approximately 2 million Jurkat T cells, followed by incubation at
370C for 7hrs [71] . Apoptosis induced by camptothecin was verified by staining the
samples with FITC-Annexin V and Propidium iodide (PI). PI stains the necrotic cells
(more specifically a DNA stain) and FITC-Annexin V stains the apoptotic cells present in
the sample. Increasing concentrations of Anx V-AuNps were added to a series of tubes
containing similar amounts of apoptotic Jurkat T cells in a binding buffer (from apoptosis
detection kit) followed by incubation for 30 min at room temperature in dark. Five micro
liters of FITC-Annexin V (fluorescent probe) and 5 µL of PI were added simultaneously
to each and every tube and allowed to incubate for 30 min. Then all the samples were
analyzed by FACS. As a reference, a positive control with no apoptosis (live cells) and a
negative control with 100% apoptosis were used.
29
3.2.3 Scanning Electron Microscopy:
Yuri/Jensen top-on SEM method:
Apoptotic Jurkat T cells were incubated for 3 hrs with 278.4 µM Anx V-AuNps, washed
3 times with chilled PBS buffer and fixed with 0.1 M Na-Cacodylate buffer containing
2% glutaraldehyde and 2% paraformaldehyde. A small amount of the fixed sample was
placed onto a 0.22 µM nucleopore membrane (shiny side up) sitting on top of a slightly
moistened piece of filter paper in a small petri dish. For each of the following steps listed
below enough buffer solution was added to the filter paper to keep the membrane
floating. As long as the whattman paper stays wet, it will pull ever-increasing amounts of
solution through the nucleopore filter.
After placing the fixed sample onto the membrane, series of five buffer rinses
with 0.1 M cacodylate buffer followed by ultra pure water were done. Samples were then
dehydrated with a series of 10%, 20%, 35%, & 50% ethanol dilutions. At this point
another 0.22 µM membrane was gently placed on top of the membrane with sample (top-
on). Dehydration was continued with 70%, 90%, 95% and 3x100% ethanol dilutions.
Following dehydration, samples were critical point dried immediately after which, the
sandwiched nucleopore membranes were separated and samples were mounted onto SEM
stubs coated with a thin carbon layer.
30
3.3 Results and Discussion:
3.3.1 Synthesis of Annexin V-Gold Nanoparticles (Anx V-AuNps):
We have successfully generated Annexin V-Thioctic acid analogue and
conjugated it with S-AuNps via activated carboxyl group present in Thioctic acid (TA)
and the amino groups on Annexin-V. N-hydroxy succinimide (NHS) was used to activate
the C-terminal carboxyl group in thioctic acid and dicylcohexyl carbodiamide (DCC), a
dehydrating agent was used to facilitate the coupling reaction between annexin V and
thioctic acid.
Figures 3.3 a and b show the results of UV-Vis spectroscopy and TEM images of
Anx V-AuNps respectively. Anx V-AuNps show a sharp absorption peak at 530 nm and
a uniform size distribution of 12±5 nm, which agree with the earlier reported values of
CLIO-nanoparticles conjugated to annexin V [72].
a) b)
Figure 3.3: a) UV-Visible absorption spectrum, b) TEM image of Annexin V-Gold Nanoparticles.
Annexin-V-TA-Au
0.09
0.1
0.11
0.12
0.13
0.14
0.15
0.16
400 450 500 550 600 650 700 750 800
Wavelength (nm)
Abs
31
3.3.2. Indirect Binding Assay:
Since, Anx V-AuNps do not posses any fluorescent probe on them, it makes it
difficult to analyze their binding capacity towards apoptotic cells using flowcytometry
(detects the fluorescence marker attached to apoptotic cells only). Therefore, an indirect
binding assay was designed to analyze the ability of Anx V-AuNps to detect the apoptotic
cells. According to this assay, increasing concentrations of Anx V-AuNps were added to
a series of tubes containing similar amounts of apoptotic Jurkat T cells in a binding buffer
followed by addition of equal amounts of FITC-Annexin V (fluorescent probe) and PI to
each and every tube. The strength of Anx V-Gold Nanoparticles to detect apoptotic Jurkat
–T cells was examined by using a dual wavelength FACS. As shown in Figures 3.4 a, b,
c, d, e, f as the concentration of Anx V –AuNps was increased from 58 µM to 278.4 µM;
signal given by FITC-Annexin V (fluorescent dye) was decreased from 99.13 to 5.55%
(Table 3.1). These results indicate that at higher concentrations Anx V-AuNps saturate all
the available apoptotic cells and are not competitively displaced by FITC–Annexin V.
Therefore, it may be concluded that Anx V-AuNps show high specificity to bind with
phosphatidylserine exposed on surface of the apoptotic Jurkat T cells.
32
3.4 a. Live Cells (No Apoptosis):
3.4 b. Anx-AuNps (0 µM) + FITC- Annexin V (5 µl) + PI (5 µl)
3.4 c. Anx-AuNps (58 µM) + FITC- Annexin V (5 µl) + PI (5ul)
33
3.4 d. Anx-AuNps (116 µM) + FITC- Annexin V (5 µl) + PI(5 µl)
3.4 e. Anx-AuNps (232 µM) + FITC- Annexin V (5 µl) + PI (5 µl)
3.4 f. Anx-AuNps (278.4 µM) + FITC- Annexin V (5 µl) + PI (5 µl)
Figures 3.4: a, b, c, d, e, f: Effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells.
34
Indirect Binding Assay:
Figure 3.5: Dose response curve for Anx V- AuNps in displacement of FITC-Anx from apoptotic Jurkat-T cells.
Treatment % of Apoptotic cells
detected in FACS
Live Cells (No Apoptosis) 0
Anx-AuNps (0 µM) + FITC-Annexin V (5 µl) + PI (5 µl) 75.89
Anx-AuNps (58 µM) + FITC- Annexin V (5 µl) + PI (5ul) 50.9
Anx-AuNps (116 µM) + FITC- Annexin V (5 µl) + PI(5 µl) 39.14
Anx-AuNps (232 µM) + FITC- Annexin V (5 µl) + PI (5 µl) 15.99
Anx-AuNps (278.4 µM) + FITC- Annexin V (5 µl) + PI (5 µl) 5.55
Figure 3.6: Table showing the effect of increasing concentrations of Anx V-AuNps on FITC-Anx fluorescence signal in apoptotic Jurkat T-cells.
In-direct Binding Assay
0
20
40
60
80
100
0 58 116 232 278.4
Concentration of Anx V-Gold nanoparicles (uM)
% o
f apo
ptot
ic c
ells
de
tect
ed in
FA
CS
35
3.3.3 Detection of Binding of Anx V-Gold Nanoparticles to Apoptotic Cells through
scanning electron microscopy:
To further validate our results we examined the ability of Anx V-AuNps to bind
with apoptotic cells via Scanning electron microscopy. Confluent cultures of Jurkat-T
cells are apoptocized using 7 µl of Camptothecin and then incubated with 278.4 µM Anx
V-AuNps, washed 3 times with chilled PBS buffer and fixed with 0.1 M Na-cacodylate
buffer containing 2% glutaraldehyde and 2% paraformaldehyde. Analysis of these
samples under dark field back scattering mode in SEM clearly show the Anx V-AuNps
attached on the rough surfaces of apoptotic Jurkat T cells as shinning little spots (Figures
3.8 a, b, c, d). As a control similar incubations are made with unconjugated AuNps
.These controls show absolutely no binding of unconjugated AuNps with the apoptotic
cells. Figure 3.7 shows an Apoptotic cell with rough, disrupted cell surface when viewed
under normal SEM mode (no back scattering applied).
Figure 3.7: Apoptotic Jurkat-T cell showing rough surface.
36
Figu
res 3
.8: a
, b, c
, d: B
ack
scat
ter S
EM im
ages
show
ing
surf
ace
bind
ing
of A
nx V
-AuN
ps to
apo
ptot
ic Ju
rkat
-T c
ells
incu
bate
d w
ith 2
78.4
µM
Anx
V-A
uNps
.
a b
c d
37
CHAPTER 4
Green Gold Nanoparticles
4.1 Introduction:
The use of phytochemicals in the synthesis of nanoparticles is an important
symbiosis between nanotechnology and green Chemistry [73, 74, 75]. As the
nanorevolution unfolds, it is imperative to develop ‘nano-naturo’ connections between
nanotechnology and green domains of the nature. Production of nanoparticles under
nontoxic green conditions is of vital importance to address growing concerns on the
overall toxicity of nanoparticles for medical and technological applications [76, 77, 78].
The power of phytochemicals, which initiate varieties of chemical transformations within
biological systems, is well known [77, 79, 80, 81]. For example, a high level of genistein
found in Soybeans is both a phytoestrogen and antioxidant, and has been extensively used
to treat conditions affected by estrogen levels in the body [82, 83]. Polyphenolic
flavonoids in tea, of which epigallocatechin gallate (EGCG) is the major constituent, has
anticarcinogenic activity [84, 85]. Cinnamon a common household spice is known to
have potential properties to treat diabetes mellitus [86, 87]. While the tremendous health
benefits of chemical cocktails present within tea, soya, cinnamon is beyond doubt, the
actual applications of the chemical reduction power of the myriad of chemicals present in
herbs and spices is still in infancy. Therefore we investigated the synergistic potentials of
polyphenols, flavonoids, catechins, and various phytochemicals present tea, soya,
cinnamon for the reduction reactions of gold salts to produce AuNps which have
potential applications in the diagnosis and therapy of various deadly diseases including
cancer.
38
4.2 Materials and Methods:
4.2.1 Synthesis of Soybean Gold Nanoparticles (Soy-AuNps):
Step 1: Soybean Extract Preparation
Intact soybeans (8 g) were washed with DI water to remove any traces of contaminants.
Soybeans were then soaked in 50 ml of DI water at room temperature for 72 hrs.The
supernatant was decanted, and centrifuged at 8000 rpm for 10 min at room temperature
and was stored at 40C and for use within 3 days.
Step 2: Four ml of soybean supernatant were diluted to 8 ml in DI water and was heated
to simmer for 1 min.
Step 3: To this solution, 100 µl of NaAuCl4 (0.1 M) were added and further heated to
simmering with constant stirring. Within 20minutes, the color of the solution turned to
ruby red indicating the formation of gold nanoparticles (Soy-AuNP).
4.2.2 Synthesis of Tea Gold Nanoparticles (T-AuNps):
Step 1: 100mg of Tea leaves (Darjeeling Tea) were added to 6 ml of DI water and the
reaction mixture was stirred continuously at 25 °C for 15 min.
Step 3: To the stirring mixture, 100 μl of 0.1 M NaAuCl4 solution (in DI water) were
added. The color of the mixture slowly turned purple-red from pale yellow within 10
minutes, which indicates the formation of gold nanoparticles (T-AuNps).
Step 4: The reaction mixture was stirred for an additional 15 minutes and the gold
nanoparticles thus formed were separated from residual tea leaves immediately using a
5µ filter and analyzed using UV-Visible spectroscopy and TEM.
39
4.2.3 Synthesis of Cinnamon gold nanoparticles (Cin-AuNps):
100 μl of 0.1 M NaAuCl4 was added to a stirring solution of 6ml DI water
containing 30 mg of cinnamon powder. Within 15 minutes, the color of the solution
turned to ruby red indicating the formation of gold nanoparticles (Cin-AuNps).
4.3 Cell Culture and Cellular Interactions:
MCF-7 breast cancer cells and PC-3 prostate cancer cells were cultured and the cellular
interactions were studied as described earlier for AuNps in Chapter 2.
4.4 Cytotoxicity Studies (MTT assay):
Cytotoxicity evaluation of soy-, tea- and Cin-AuNps was performed using MTT
assay as described by Mosman [88]. Approximately 1 × 105 ml-1 cells (MCF-7 and PC-3)
in their exponential growth phase were seeded in a flat-bottomed 96-well polystyrene-
coated plate and were incubated for 24 hrs at 37 °C in a 5% CO2 incubator. Series of
dilutions (10, 30, 50, 70, 90, 110, and 150 µM) of AuNps in the medium was added to the
plate in hexaplets. After 24 hrs of incubation, 10 μl of MTT reagent was added to each
well and was further incubated for 4 hrs. Formazan crystals formed after 4 hrs in each
well were dissolved in 150 μl of detergent and the plates were read immediately in a
microplate reader (Spectramex, 190 Molecular Devices Inc., USA) at 570 nm. Wells with
complete medium, nanoparticles, and MTT reagent, without cells were used as blanks. A
control experiment with series of dilutions of NaAuCl4 was performed using the same
MTT kit to validate the assay.
40
4.5 Results and Discussion:
4.5.1 Synthesis of Green gold Nanoparticles:
Our new green process for the production of gold nanoparticles uses direct
interaction of sodium tetrachlroaurate (NaAuCl4) with black Darjeeling tea, soyabean
supernatant and cinnamon powder in the absence of man-made chemicals and thus,
satisfies all the principles of a 100% green chemical process. Various phytochemicals
present in tea, soy and cinnamon are presumably responsible for making a robust coating
on gold nanoparticles and thus, rendering stability against agglomerations. Absorption
measurements indicated that the plasmon resonance wavelength, λmax of T-AuNPs, Soya-
AuNps and Cin-AuNps are 535 nm, 540 nm and 540 nm respectively. The sizes of T-
AuNPs, Soya-AuNps and Cin-AuNps are in the range of 12±4 nm; 16±5 nm and 15±5
nm respectively as measured from TEM techniques (Figures 4.1, 4.2, 4.3).
41
a) b)
c)
Size Distribution of TGAAuNP-H
0
5
10
15
20
25
6 8 10 12 14 16
Size (nm)
No.
of P
artic
les
Figure 4.1: a) UV-Visible absorption spectrum, b) TEM Image c) size distribution of Tea Gold Nanoparticles.
42
a) b)
c)
Figure 4.2: a) UV-Visible absorption spectrum, b) TEM Image, c) size distribution of Soy Gold Nanoparticles
0
2
4
6
8
10
12
6 8 10 12 14 16 18 20 22More
S ize (n m )
No.
of P
artic
les
43
a) b)
c)
Figure 4.3: a) TEM Image, b) UV-Visible absorption spectrum, c) size distribution of Cinnamon Gold Nanoparticles
Size distribution of Cinnamon stabilized AuNP
05
1015202530
7 9 11 13 15 17 19
Size (nm)
No.
of P
artic
les
44
4.5.2 Cellular Internalization studies:
Results of cellular internalization studies of AuNps solutions are key to providing
insights into their use in biomedicine. Their selective cell and nuclear targeting will
provide new pathways for their site-specific delivery as diagnostic/ therapeutic agents. A
number of studies have demonstrated that phytochemicals in tea, soya and cinnamon
have the ability to penetrate the cell membrane and internalize within the cellular matrix
[89, 90]. Cancer cells are highly metabolic and porous in nature and are known to
internalize solutes rapidly compared to normal cells [90]. Therefore, we hypothesized
that tea, soya and cinnamon derived phytochemicals, if coated on AuNps, will show
internalization within cancer cells.
TEM images of prostate (PC-3) and breast tumor (MCF-7) cells treated with
AuNPs unequivocally validated our hypothesis. Significant internalization of
nanoparticles via endocytosis within the MCF-7 and PC-3 cells was observed (Figures
4.4a, b, c; 4.5a, b, c; 4.6a, b, c). The internalization of nanoparticles within cells could
occur via processes including phagocytosis, fluid-phase endocytosis, and receptor-
mediated endocytosis. The viability of both PC-3 and MCF-7 cells post-internalization
suggests that the phytochemical coating renders the nanoparticles non-toxic to cells. Such
a harmless internalization of AuNps will provide new opportunities for probing cellular
processes via nanoparticulate-mediated imaging.
45
a) b)
c)
Figures 4.4a, b, c: TEM Images of different MCF-7 cells showing uptake of Tea-AuNps in to the lysosomes.
46
a)
b)
Figures 4.5a, b: TEM Images of different MCF-7 cells showing uptake of Soya-AuNps in to the lysosomes.
47
a)
b)
Figures 4.6 a, b: TEM images of MCF-7 cell showing uptake of Cin-AuNps in to the lysosomes.
48
4.5.3 Cytotoxicity Studies:
Untreated PC-3 and MCF-7 cells as well as cells treated with 10, 30, 50, 70, 90,
110 and 150 μM concentrations of various AuNps for 24 hrs were subjected to the MTT
assay for cell-viability determination. In this assay, only cells that are viable after 24 h
exposure to the sample are capable of metabolizing a dye (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) efficiently and produce a purple colored precipitate
which is dissolved in a detergent and analyzed sphectrophotometrically. After 24 hrs
post-treatment, PC-3, MCF-7 cells showed excellent viability even up to 150 μM
concentrations of tea, soy and cinnamon-AuNps (Figures 4.7 a, b; 4.8 a, b;4.9 a, b). These
results clearly demonstrate that the phytochemicals within these herbs provide a non-
toxic coating on AuNps and corroborate the results of the internalization studies
discussed above. It is also important to recognize that a vast majority of Gold (I) and
Gold (III) compounds exhibit varying degrees of cytotoxicity to a variety of cells (Figure
4.10). The lack of any noticeable toxicity of tea, soy and cinnamon-AuNps provides new
opportunities for the safe application in molecular imaging and therapy.
49
a)
b)
4.7 a, b: Dose dependent cytotoxicity of Tea-AuNPs in cultured PC-3 and MCF-cells after 24 hrs of
exposure using MTT assay.
MTT Assay of Tea-AuNps in PC-3 cell line
0
20
40
60
80
100
120
140
160
10 30 50 70 90 110 150Concentration (um )
% C
ell V
iabi
lity
PC-3 cells-Tea AuNps
MTT Assay of Tea-AuNps in MCF-7 cell line
0
20
40
60
80
100
120
140
10 30 50 70 90 110 150Concentration (um )
% C
ell V
iabi
lity
MCF-7cells-Tea-AuNps
50
4.8 a, b: Dose dependent cytotoxicity of Soy-AuNPs in cultured PC-3 and MCF-cells after 24 hrs of
exposure using MTT assay.
MTT Assay of Soy-AuNps in PC-3 cell line
0
20
40
60
80
100
120
140
160
10 30 50 70 90 110 150Concentration (um )
% C
ell V
iabi
lity
PC-3 cells-Soy-AuNps
M TT Assay of Soy-AuNps in M CF-7 ce ll line
0
20
40
60
80
100
120
140
10 30 50 70 90 110 150Concentration (um )
% C
ell V
iabi
lity
MCF-7 cells-Soy-AuNps
51
a)
b)
4.9 a, b: Dose dependent cytotoxicity of Cin-AuNPs in cultured PC-3 and MCF-cells after 24 hrs of
exposure using MTT assay.
M TT Assay of Cin-AuNps in PC-3 ce lls
0
20
40
60
80
100
120
140
10 30 50 70 90 110 150Concentration (um )
% C
ell V
iabi
lity
PC-3 cells-Cin-AuNps
M TT Assay of Cin-AuNps in M CF-7 ce ll line
0
20
40
60
80
100
120
140
10 30 50 70 90 110 150Concentration (uM )
% C
ell V
iabi
lity
MCF-7 cells-Cin-AuNps
52
MTT Assay of natural constructs in MCF-7and PC-3 cell lines.
0
20
40
60
80
100
120
0.01 0.1 1 10 10
01,0
00
Concentration (M)
% C
ell V
iabi
lity NaAuCl4 in MCF-
7 cellsNaAuCl4 in PC-3cells
4.10: Dose dependent cytotoxicity of NaAuCl4 in cultured PC-3 and MCF-cells after 24 hrs of exposure
using MTT assay
53
CHAPTER 5
SUMMARY:
Functionalized and biomolecule conjugated gold nanoparticles will play an
indispensable role in the overall design and development of AuNps-based nano
pharmaceuticals. For molecular imaging and therapy, it will be of pivotal importance to
generate nanoparticles in bio-friendly media including, in the presence of chemically
sensitive proteins, peptides and receptor specific bio vectors. This research resulted in the
synthesis of non-toxic, biologically friendly gold nanoparticles that are conjugated with
highly specific biomolecules such as Bombesin and Annexin V. In addition, the
conjugated AuNps were tested for their target specificity towards cancerous cells and
apoptotic cells.
Our results show that Bombesin conjugated gold nanoparticles can enter prostate
and breast cancer cells via BBN/GRP receptor-mediated mechanism. These results show
a proof of concept for the in vivo delivery of AuNps to the tumor regions. Their use as
contrast enhancement agents to visualize tumors in vivo, using Computer Tomography
(CT) imaging technique depends on whether their uptake is selective in such cells as
opposed to surrounding non-tumerous tissue at non-toxic doses.
Conjugates of AuNps with Annexin V to detect apoptosis may be of use: in the
detection of apoptosis by CT-imaging, for cell sorting, and for better appreciation of
interactions between various chemotherapeutic agents and cells. Therefore, the synthesis
of biomolecule/protein conjugated gold nanoparticles outlined opens the way for the
54
fabrication of a wide range of biological probes, which can specifically target wide range
of cells.
Future Work
The present work may be continued by working towards the following directions
1. Evaluating the in vivo uptake profile of Bombesin gold nanoparticles and
Annexin V gold nanoparticles in tumors and apoptotic cells.
2. Determining the apt amount of conjugated AuNps to use them as contrast
enhancement agents.
3. Investigating the target specificity of Anx V-AuNps by inducing apoptosis via
different mechanisms.
4. Fabricating gold nanoparticles with more than one targeting agent such as
conjugating a receptor specific biomolecule and a florescent probe.
55
BIBLIOGRAPHY
1. Alivisatos AP. Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science
271: 933-937, 1996.
2. Feldheim DL, Colby AF, Marcel D. Metal Nanoparticles: Synthesis,
Characterization and Applications, J. Phys. Chem 300: 11202-11208, 2002.
3. Kreibig U, Vollmer M. Optical Properties of Metal Clusters. Springer 25: 532-538,
1995.
4. Chandrasekharan N, Kamat PV, Hu J, Jones G. Dye- Capped Gold Nanoclusters:
Photoinduced Morphological Changes in Gold/Rhodamine 6G Nanoassemblies. J.
Phys. Chem 104:11103-11109, 2000.
5. Nath N, and Chilkoti A. Interfacial Phase Transition of an Environmentally
Responsive Elastin Biopolymer Adsorbed on Functionalized Gold Nanoparticles
Studied by Colloidal Surface Plasmon Resonance. J. Am. Chem. Soc 123:8197-8202,
2001.
6. Link S, El-Sayed MA. Shape and size dependence of radiative, non-radiative and
photothermal properties of gold nanocrystals. Int. ReV. Phys. Chem 19 (3): 409-453,
2000.
7. Link S, El-Sayed MA. Optical properties and ultrafast dynamics of metallic
nanocrystals. Annu. ReV. Phys. Chem 54:331-366, 2003.
8. Link S, Mohamed MB, El-Sayed MA. Simulation of the optical absorption spectra
of gold nanorods as a function of their aspect ratio and the effect of the medium
dielectric constant. J. Phys. Chem. B. 103:3073-3077.
9. Link S, El-Sayed MA. Additions and corrections to “Simulation of the optical
absorption spectra of gold nanorods as a function of their aspect ratio and the effect of
the medium dielectric constant”. J. Phys. Chem. B 109:10531-10532, 2005.
10. Nikoobakht B, Wang J, El-Sayed MA. Surface enhanced Raman scattering of
molecules adsorbed on gold nanorods: off-surface plasmon resonance condition.
Chem. Phys. Lett 366: 17-23, 2002.
56
11. Leff DV, Brandt L, and Heath JR. Synthesis and Characterization of Hydrophobic,
Organically-Soluble Gold Nanocrystals Functionalized with Primary Amines.
Langmuir 12:4723, 1996.
12. Niemeyer CM, Angew. Nanoparticles, Proteins, and Nucleic Acids: Biotechnology
Meets Materials. Science Chem. Int. Ed 40:4128-41582001, 2001.
13. Sanford JC, Smith FD, and Russell JA. Optimizing the biolistic process for
different biological applications. Methods Enzymol 217: 483-509, 1993.
14. Bielinska A, Eichman JD, Lee I, Baker JR, Balogh LJ. Imaging {Au0-PAMAM}
Gold-dendrimer Nanocomposites in Cells. Nanopart. Res 4: 395-403, 2002.
15. Olofsson L, Rindzevicius T, Pfeiffer I, Kall M, and Hook F. Surface-Based Gold-
Nanoparticle Sensor for Specific and Quantitative DNA Hybridization Detection.
Langmuir 19: 10414-10419, 2003.
16. Anderson SA, Rader RK, Westlin WF, Null C, Jackson D, Lanza GM, Wickline
SA, Kotyk JJ. Magnetic resonance contrast enhancement of neovasculature with
alpha(v)beta(3)-targeted nanoparticles. Magn Reson Med 44:433–439, 2000.
17. Turkevitch J, Stevenson PC, Hillier J. Nucleation and Growth Process in the
Synthesis of Colloidal Gold. Faraday Soc 11:55-75, 1951.
18. Brust M, Walker M, Bethell D, Schiffrin DJ, and Whyman RJ. Synthesis of
Thiol-Derivatized Gold Nanoparticles in a Twophase Liquid-Liquid System. J. Chem.
Soc., Chem. Commun 801-802, 1994.
19. Guo S, Wang E. Synthesis and electrochemical applications of gold
nanoparticles. Analytica Chimica Acta 598: 181-192, 2007.
20. Kattumuri V, M Chandrasekhar, S Guha, K Raghuraman, KV Katti, K Ghosh,
and RJ Patel. Agarose-stabilized gold nanoparticles for surfaceenhanced Raman
spectroscopic detection of DNA nucleosides. Applied Physics Letters 88(15), 2006.
21. Yang PH, Sun XS, Chiu JF, Sun HZ, He QY. Functionalized gold nanoparticles for
drug delivery. Bioconj. Chem. 16:494-496, 2005.
22. Chithrani BD, Chan WCW. Elucidating the Mechanism of Cellular Uptake and
Removal of Protein-Coated Gold Nanoparticles of Different Sizes and Shapes. Nano
Lett. 7:1542-1550, 2007.
57
23. Hong R, Han G, Fernandez JM, Kim BJ, Forbes NS, and Rotello VMJ.
Glutathione-Mediated Delivery and Release Using Monolayer Protected Nanoparticle
Carriers J. Am. Chem. Soc. 128:1078-1079, 2006.
24. Tkachenko AG, Xie H, Coleman D, Glomm W, Ryan J, Anderson MF, Franzen
S, Feldheim DL. Multifunctional gold nanoparticle-peptide complexes for nuclear
targeting. J. Am. Chem. Soc. 125:4700-4701, 2003.
25. Tkachenko AG, Xie H, Liu Y, Coleman D, Ryan J, Glomm WR, Shipton MK,
Franzen S, Feldheim DL. Cellular trajectories of peptide-modified gold particle
complexes: comparison of nuclear localization signals and peptide transduction
domains. Bioconjugate Chem. 15:482-490, 2004.
26. Sokolov K, Aaron J, Hsu B, Nida D, Gillenwater A, Follen M, MacAulay C,
Adler-Storthz K, Korgel B, Descour M, Pasqualini R, Arap W, Lam W,
Richards-Kortum R. Optical systems for in vivo molecular imaging of cancer.
Technol Cancer Res Treat. 2:491– 504, 2003.
27. Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA. Selective
colorimetric detection of polynucleotides based on the distance-dependent optical
properties of gold nanoparticles. Science 277:1078–81, 1997.
28. Hirsch LR, Stafford RJ, Bankankson JA, Sershen SR, Rivera B, Price RE, Hazle
JD, Halas NJ, West JL. Nanoshell-mediated nearinfrared thermal therapy of tumors
under magnetic resonance guidance. Proc Natl Acad Sci 100:13549–13554, 2003.
29. Anastasi A, Erspamer V, Bucci M. Isolation and structure of bombesin and alytesin
2 analogous active peptides from the skin of the European amphibians Bombina and
Alytes. Experientia 27: 166–167, 1971.
30. Hampton LL, Ladenheim EE, Akeson M, Way JM, Weber HC, Sutliff VE,
Jensen RT, Wine LJ, Arnheiter H, Battey JF. Loss of bombesin-induced feeding
suppression in gastrin-releasing peptidereceptor-deficient mice. PNAS 95: 3188-3192,
1998.
31. Ladenheim EE, Emond M, Moran TH. Leptin enhances feeding suppression and
neural activation produced by systemically administered bombesin. Am J Physiol
Regulatory Integrative Comp Physiol 289: R473-R477, 2005.
58
32. Michaud D, Anisman H Merali Z. Capsaicin-sensitive fibers are required for the
anorexic action of systemic but not central bombesin. Am J Physiol Regulatory
Integrative Comp Physiol 276: R1617-R1622, 1999.
33. Stephen CW, Randy JS, Daniel P, Michael WS. Signals That Regulate Food
Intake and Energy Homeostasis. Science 280: 1378-1383, 1998.
34. McDonald TJ, Jornvall H, Nilsson J, Vagne M, Ghatei M, Bloom SR, Mutt V.
Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue.
Biochem Biophys Res Commun 90: p. 227, 1979.
35. Dembinski A, Konturek PK, Konturek SJ. Role of gastrin and cholecystokinin in
the growth-promoting action of bombesin on the gastroduodenal mucosa and the
pancreas. Regul Pept 27: p. 343, 1990.
36. Dembinski A, Warzecha Z, Konturek SJ, Banas M, Cai RZ, Schally AV. The
effect of antagonist of receptors for gastrin, cholecystokinin and bombesin on
growth of gastroduodenal mucosa and pancreas. J Physiol Pharmacol 42: 263-65,
1991.
37. Cuttitta F, Caney DN, Mulshine J, Moody TW, Fedorko J, Fishler A, Minna
JD. Bombesin-like peptides can function as autocrine growth factors in human
small-cell lung cancer. Nature 316: 823 – 826, 1985.
38. Reubi JC, Wenger S, Schmuckli-Maurer J, Schaer J.C, Gugger M. Bombesin
receptor subtypes in human cancers: detection with the universal radioligand (125)I-
[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)] bombesin(6-14). Clin Cancer Res,
8:1139-1143, 2002.
39. Carroll RE, Matkowskyj KA, Chakrabarti S, McDonald TJ, Benya RV. Aberrant expression of gastrin-releasing peptide and its receptor by well-
differentiated colon cancers in humans. Am J Physiol 276: G655-65, 1999.
40. Siegfried JM, DeMichele MA, Hunt JD, Davis AG, Vohra KP, Pilewski JM. Expression of mRNA for gastrin-releasing peptide receptor by human bronchial
epithelial cells. Association with prolonged tobacco exposure and responsiveness to
bombesin-like peptides. Am J Respir Crit Care Med 156: 358-66, 1997.
41. Pansky A, De Weerth A, Fasler-Kan E, Boulay JL, Schulz M, Ketterer S, Selck
C, Beglinger C, Von Schrenck T, Hildebrand P. Gastrin releasing peptide-
59
preferring bombesin receptors mediates growth of human renal cell carcinoma. J Am
Soc Nephro 11:1409-1418, 2000.
42. Smith CJ, Volkert WA, Hoffman TJ. Gastrin releasing peptide (GRP) receptor
targeted radiopharmaceuticals: a concise update. Nucl Med Biol 30:861-868, 2003.
43. Sun B, Schally AV, Halmos G. The presence of receptors for bombesin/GRP and
mRNA for three receptor subtypes in human ovarian epithelial cancers. Regul Pept
30: 77-84, 2000
44. Burghardt B, Wenger C, Barabás K, Rácz G, Oláh A, Flautner L, Coy DH,
Gress TM, Varga G. GRP-receptor-mediated signal transduction, gene expression
and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF.
Peptides 22: 1119-1128, 2001.
45. Bartholdi MF, Wu JM, Pu H, Troncoso P, Eden PA, Feldman RI. In situ
hybridization for gastrin-releasing peptide receptor (GRP receptor) expression in
prostatic carcinoma. Int J Cancer 20:82-90, 1998.
46. Lin KS, Luu A, Baidoo KE, Hashemzadeh-Gargari H, Chen MK, Brenneman
K, Pili R, Pomper M, Carducci MA, Wagner HN Jr. A new high affinity
technetium-99m-bombesin analogue with low abdominal accumulation. Bioconjug
Chem 16: 43-50, 2005.
47. Kunstler JU, Veerendra B, Figueroa SD, Sieckman GL, Rold TL, Hoffman TJ,
Smith CJ, Pietzsch HJ. Organometallic 99mTc(III) '4 + 1' bombesin(7-14)
conjugates: synthesis, radiolabeling, and in vitro/in vivo studies. Bioconjug Chem
18: 1651-1661, 2007.
48. Hoffman TJ, Quinn TP, Volkert WA. Radiometallated receptor-avid peptide
conjugates for specific in vivo targeting of cancer cells. Nucl Med Biol 28:527-532,
2001.
49. Raghuraman K, Katti KK, Barbour LJ, Pillarsetty N, Barnes CL, Katt KV.
Characterization of supramolecular (H2O)(18) water morphology and water-
methanol (H2O)(15)(CH3OH)(3) clusters in a novel phosphorus functionalized
trimeric amino acid host. Journal of the American Chemical Society 125: 6955-6960,
2003.
60
50. Neves M, Gano L, Pereira N, Costa MC, Costa MR, Chandia M, Rosado M,
Fausto R. Synthesis, characterization and biodistribution of bisphosphonates Sm-
153 complexes: correlation with molecular modeling interaction studies. Nuclear
Medicine and Biology 29:329-335, 2002.
51. Grady LH, Nonneman DJ, Rottinghaus GE,Welshons WV. PH-Dependent
cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells.
Endocrinology 129:3321-3330, 1991.
52. Welshons WV, Grady LH, Engler KS, Judy BM. Control of proliferation of
MCF-7 breast cancer cells in a commercial preparation of charcoalstripped adult
bovine serum. Breast Cancer Res Treat 23:97-104, 1992.
53. Read LD, Greene GL, Katzenellenbogen BS. Regulation of estrogen receptor
messenger ribonucleic acid and protein levels in human breast cancer cell lines by
sex steroid hormones, their antagonists, and growth factors. Mol Endocrinol 3:295-
304, 1989.
54. Kattumuri V. Gold Nanoparticles for Bio-medical applications: Synthesis,
Characterization, In Vitro and In Vivo studies. Thesis, Dept of Physics and
Astronomy, UMC.
55. Hoffman TJ, Sieckman GL, Volkert WA, Truman HS. Iodinated bombesin
analogues: Effect of N-terminal vs side chain iodine attachment on BBN/GRP
receptor binding. Journal of Nuclear Medicine 37: 850-855, 1996.
56. Hoffman TJ, Quinn TP, Volkert WA. Radiometallated receptor-avid peptide
conjugates for specific in vivo targeting of cancer cells. Nucl Med Biol 28 :527-532,
2001.
57. Narayanan R, Lipert RJ, Porter MD. Cetyltrimethylammonium bromide-modified
spherical and cube-like gold nanoparticles as extrinsic Raman labels in surface-
enhanced Raman spectroscopy based heterogeneous immunoassays. Anal Chem 80:
2265-71, 2008.
58. Steven H Young and Enrique Rozengurt. Qdot Nanocrystal Conjugates
conjugated to bombesin or ANG II label the cognate G protein-coupled receptor in
living cells. Am J Physiol Cell Physiol 290:C728–C732, 2006.
61
59. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science
267:1456-1462, 1995.
60. Peter ME, Heufelder AE, Hengartner MO. Advances in apoptosis research. Proc.
Natl. Acad. Sci. U.S.A. 94:12736-12737, 1997.
61. Bursch W, Oberhammer F, Schulte-Hermann R. Cell death by apoptosis and its
protective role against disease. Trends Pharmacol. Sci. 13:245-251, 1992.
62. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, Schie RC, LaFace
DM. Early redistribution of plasma membrane phosphatidylserine is a general
feature of apoptosis regardless of the initiating stimulus: inhibition by
overexpression of Bcl-2 and Abl. J. Exp. Med. 182:1545-1556, 1995.
63. Schlegel RA, Williamson P. Phosphatidylserine, a death knell. Cell Death Differ
8:551-563, 2001.
64. Funakoshi T, Heimark RL, Hendrickson LE, McMullen BA, Fujikawa K.
Human placental anticoagulant protein: isolation and characterization. Biochemistry,
26:5572-5578, 1987.
65. Van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, and Reutelingsperger
CP. Annexin V-affinity assay: a review on an apoptosis detection system based on
phosphatidylserine exposure. Cytometry 31:1-9, 1998.
66. Hammill AK, Uhr JW, Scheuermann RH. Annexin V staining due to loss of
membrane asymmetry can be reversible and precede commitment to apoptotic death.
Exp. Cell Res 251:16-21, 1999.
67. Bossy-Wetzel E, Green DR. Detection of apoptosis by annexin V labeling. Methods
Enzymol 322:15-18, 2000.
68. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for
apoptosis. Flow cytometric detection of phosphatidylserine expression on early
apoptotic cells using fluorescein labeled Annexin V. J Immunol. Methods 184:39-51,
1995.
69. Schellenberger E, Bogdanov AJ, Petrovsky A, Ntziachristos V, Weissleder R,
and Josephson L. Optical imaging of apoptosis as a biomarker of tumor response to
chemotherapy. Neoplasia 5:187-192, 2003.
62
70. Petrovsky A, Schellenberger E, Josephson L, Weissleder R, and Bogdanov A Jr.
Near-infrared fluorescent imaging of tumor apoptosis. Cancer Res. 63:1936-42,
2003.
71. Sestier C, Da-Silva MF, Sabolovic D, Roger J, and Pons JN. Surface modification
of superparamagnetic nanoparticles (Ferrofluid) studied with particle
electrophoresis: application to the specific targeting of cells. Electrophoresis 19:
1220-1226, 1998.
72. Eyk A Schellenberger, David Sosnovik, Ralph Weissleder, and Lee Josephson.
Magneto/Optical Annexin V, a Multimodal Protein. Bioconjugate Chem. 15 (5),
1062 -1067, 2004.
73. Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, Wang H, Wang Y, Shao W, Hong
NJ, Chen C. Biosynthesis of silver and gold nanoparticles by novel sundried
Cinnamomum camphora leaf. Nanotechnol. 18:105104-105115, 2007.
74. Jorge L, Torresdey G, Gomez E, Peralta-Videa JR, Parsons JG, Troiani H,
Yacaman MJ. Phytoremediation of heavy metals and study of the metal
coordination by X-ray absorption spectroscopy. Langmuir 19: p.p1357, 2003.
75. Gardea-Torresdey JL, Tiemann KJ, Parsons JG, Gamez G, Herrera I, Jose
Yacaman M. Investigation into the Mechanism(s) of Au (III) Binding and
Reduction by Alfalfa Biomass. Microchemical Journal. 71:193-204, 2002.
76. Hardman R. Toxicologic Review of Quantum Dots: Toxicity Depends on
Physicochemical and Environmental Factors. Environ. Health. Perspect.114: p.p165,
2006.
77. Curtis J, Greenberg M, Kester J,Phillips S, Krieger G. Nanotechnology and
Nanotoxicology: A Primer for Clinicians. Toxicol. Rev. 25: p.p 245, 2006.
78. Lewinski N, Colvin V, Drezek R. Cytotoxicity of nanoparticles. Small 4:26-49,
2008.
79. Espín JC, García-Conesa MT, Tomás-Barberán FA. Nutraceuticals: Facts and
fiction. Phytochemistry 68: p.p 2986, 2007.
80. Rochfort S, Panozzo J. Class targeted metabolomics: ESI ion trap screening
methods for glucosinolates based on MSn fragmentation. J. Agric. Food. Chem. 55:
p.p7981 2007.
63
81. Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear
WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of pure isoflavones in
healthy humans and analysis of commercial soy isoflavone supplements. J Nutr.
131:1362S-75S, 2001.
82. Magee PJ, Rowland IR. Phyto-oestrogens, their mechanism of action: current
evidence for a role in breast and prostate cancer. Br. J. Nutr 91:513-520, 2004.
83. Limer JL, Speirs V. Phyto-oestrogens and breast cancer chemoprevention.Breast
Cancer Res 6: 119-127, 2004.
84. Bandele OJ, Osheroff N. (-)-Epigallocatechin Gallate, A Major Constituent of
Green Tea, Poisons Human Type II Topoisomerases. Chem Res Toxicol. 21:936-43,
2008.
85. Shankar S, Ganapathy S, Srivastava RK. Green tea polyphenols: biology and
therapeutic implications in cancer. Front Biosci 12:4881-99, 2007.
86. Dannemann K, Hecker W, Haberland H, Herbst A, Galler A, Schäfer T,
Brähler E, Kiess W, Kapellen TM. Use of complementary and alternative
medicine in children with type 1 diabetes mellitus - prevalence, patterns of use, and
costs. Pediatr Diabetes, 2008.
87. Suppapitiporn S, Kanpaksi N. The effect of cinnamon cassia powder in type 2
diabetes mellitus. J Med Assoc Thai 89 Suppl 3:S200-5, 2006.
88. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J Immunol Methods 65:55-63, 1983.
89. Mizuno H, Cho YY, Zhu F, Ma WY, Bode AM,Yang CS,Ho CT, Dong ZG.
Theaflavin-3, 3′-Digallate Induces Epidermal Growth Factor Receptor Down-
Regulation Mol. Carcinog 45: 204-212, 2007.
90. Sun DJ, Liu Y, Lu DC, Kim W, Lee JH, Maynard J, Deisseroth A. Endothelin-3
growth factor levels decreased in cervical cancer compared with normal cervical
epithelial cells Human Pathology 38: 1047-1056, 2007.