Yunsuk Jo

Magnetic Nanoparticles for Biomedical Applications

Mamoun MuhammedRoyal Institute of Technology, (KTH)

Kista –Campus, Stockholm, Sweden.mamoun@kth.se

Advanced Magnetic MaterialsPori, October 9-11, 2007

Nano Medicine

Biomedical Applications of Magnetic Nanoparticles

Hyperthermia

RF-Genarator410kHz3.5kW 2 loop

coil sample

Thermocouple

Ice water 0oCreference

Dataacquisition

Digital Voltmeter Tissue Engineering

Targeted Drug deliveryMR imaging

Diagnostics

Cell Separation

Biomedical Applications of SPION

In-vivo Applications In-vitro Applications

250mlN S1ml

100ul

Pre-enrichment Immunocapture SeparationWashing &

Concentration Detection

Plating

ELISA

DNA/RNA

MP

N

S

N

S

Assay A :

biospecific surface analyte

1st reaction

magnetic conjugate

2nd reaction

Assay B :isolation of complexes by High GradientMagnetic Separation

Assay C :

magnet

Immunomagnetic assays (IMA)

Cell separation

Tissue Engineering MR imaging

Hyperthermia

RF-Genarator410kHz3.5kW 2 loop

coil sample

Thermocouple

Ice water 0oCreference

Dataacquisition

Digital Voltmeter

Drug delivery and release

Antibody

++ Alexa Fluor

As a Nano-carriers

1. Design target specific tracers by using Fe3O4 as exogenous contrast media

2. Develop methods to monitor extracellular macromolecules both at a single cell level (genes and proteins) and at a network level (intercellular communication)

Outline

• Magnetic nanoparticles– Superparamagnetic nanoparticles for MRI

– Magnetic relaxation of Thermally blocked nanoparticles

• Controlled Drug Release systems– Multifunctional PLA and PEG Nanoparticles

– Temperature sensitive polymeric nanospheres

• Summary

Superparamagnetic and Thermally blockednanoparticles with strong magnetic response

Design of tailored Magnetic Nanoparticles

• Magnetite (Fe3O4)• Maghemite (γFe2O3)• Ferrites (CoFe2O4, ZnFe2O4, MnFe2O4, …) • Iron Platinum (FePt) & CoPt

Bio-compatibility and surface functionalisation

• Inorganic: Gold, Silica, hydroxyappatite, ...• Organic: Dextran, PVA, PEG, mPEG, …

Synthesis Techniques

Magnetite by co-precipitation (90o)

Magnetite by sol-gel

Maghemite by oxidation (RT)

Cobalt Ferrite by co-precipitation (90o)

( ) 43C90,NO

22 OFeOHFeOH2Fe 3 ⎯⎯⎯ →⎯→+ °−+ −

[ ] 3243 OFe3OOFe2 γ→+

OH4OFeOH8Fe2Fe 24332 +→++ −++

−

−−++

++→

→+++

2242

322

NOOH3OCoFe

NOOH6Fe2Co

A)

B)

2 3 4 5 6 7 8 90

10

20

30

40

50

60

<D> = 5.7 nm

Freq

uenc

y (%

)

Diameter (nm)

6 8 10 12 14 16 180

5

10

15

20

25

<D> = 12nm

Freq

uenc

y (%

)

Diameter (nm)

TEM images (left) and the corresponding particle size histograms (right) of magnetite nanoparticles prepared by controlled coprecipitation. (A) without heat treatment and (B) after heat treatment (80ºC for 1hrs)

Magnetite

Magnetite by sol-gelLarge particle size

Superparamagnetic iron oxide nanoparticles

• Average particle size=12 nm• XAS shows nonstochiometric phase Fe3O4-δ, the curve shifts to Fe2+.

After one year shelf storage

Silica Coated Magnetic Nanoparticles

Control of thickness, porosity of coating layer

Silica layer

Magnetic core

Magnetic characterisation

VSM measurement for SiO2 coated Fe3O4

by co-precipitation

Magnetic characterisation

VSM measurement for SiO2 coated Fe3O4

by sol-gel

Development of Superparamagnetic Nanoparticles

Fe48Pt52

*Monodispersed Fe-Pt nanoparticles for biomedical applications. D.K. Kim, et al. 2004, Nanotechnology

FePt and CoPtAdvatages

Narrow particle size distribution -60000 -40000 -20000 0 20000 40000 60000

-8

-6

-4

-2

0

2

4

6

8

x = 0.70 x = 0.52 x = 0.48

Mas

s M

agne

tizat

ion

(em

u/g)

Applied field (Oe)

Superparamagentic NP

Low toxicity- Fe48Pt52nanoparticles in brain endothelial cells*

Exosomes (biological vesicles exerted by dendritic cells) used a coating layer for the reduction of toxicity, recognition tumor cells and also as a cage for delivering drug to the inflammation site.

Magnetic Nanoparticles• Oxide : magnetite, ferrite • Metal : Fe, Co, PtFe, CoPt

COATING• Gold• Silica• Hydroxyapatite• Dextran• Starch• Albumin• Sodium Oleate• Folic acid• L-aspartic acid• PVA • PEG• mPEG• PLLA (PDLLA)• PCL• PGA

Surface Functionalization of Magnetic Nanoparticles

2 3 4 5 6 7 8 9 10 11-0.05

-0.04

-0.03

-0.02

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

ESA

(mPa

*M/V

)

pH

Au@SPION SPION

ESA measurement of SPION and Au@SPION prepared by μE system

100 200 300 400 500 600

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

(b)

(a)

Heat

(mW

/s)

Temp (oC)

DSC analysis of bare and coated nanoparticles. (a) Magnetite, and (b) Au coated SPION.

Effect of surface modificationAu Coating

Nano Medicine

Immunoassay formats for selective Multi-component detection

direct sandwich competition

γ-, , )

, )α ß ß

nanoparticles

antibodiesantigenes = analytes

Rutheniumchemiluminescentlabel

Magneto Diagnostics

Examples of assays:

Cytokines (IL-2, IL-4, IL-1ra, IL-1ß, IL-6, IL-10, γ-interferon)Hormones (FSH, LH, PTH, prolactin, TSH, insulin, testosterone, progesterone, estradiol)Peptides and Proteins (calcitonin, troponin, ICAM, IgG, IgE, Aß1-40, Aß1-42, APP)Growth Factors (NGF, TNF-α, TNF-ß, TGFß1, TGF-α)Signal transduction cAMPDrugsEnzymatic activity assays

Néel relaxation

Brownian relaxation

Externalapplied field

Externalapplied field

kTKV

eN 0ττ =

τN = Nèel rel. timeτ0 = characteristic rel. timek = Boltzmann constantT = temperatureK = magnetic anisotropyV = single domain volume

kTVH

Bητ 3

=τB = Brownian rel. timeVH = Hydrodynamic particle

volumeη = viscosity

Relaxation of magnetic nanoparticles

Brownian relaxation process can be detected in the frequency domain

M = magnetisationH = alternating external

magnetic fieldχ = complex magnetic

susceptibility

H)i(HM ''' χχχ −==

Bio-diagnostics based on Magnetic Relaxation

IMEGO AB

Detect specific biomolecules by measuring changes in Brownian relaxation of thermally blocked magnetic nanoparticles.

Biosensor principleMagnetic relaxation

Kindly provided by IMEGO Institute (Göteborg - Sweden)IMEGO AB

Detection of PSA by using magnetic nanoparticles

Kindly provided by IMEGO Institute (Göteborg - Sweden)

Brownian relaxation frequency decreaseswhen PSA binds to the particles

1000100

Nano Medicine

• SPION: – mono-dispersed, Very narrow particle size distribution– highly crystalline high magnetization

• Synthesis in organic media and phase transfer to water phase

• High r2/r1 ratio good T2 contrast agent

Magnetic Nanoparticles for MRI

Dose response of Fe3O4 nanoparticles in MRI

Kim, Do-Kyung, Thesis, KTH, 2001

A)

B)

2 3 4 5 6 7 8 90

10

20

30

40

50

60

<D> = 5.7 nm

Freq

uenc

y (%

)

Diameter (nm)

6 8 10 12 14 16 180

5

10

15

20

25

<D> = 12nm

Freq

uenc

y (%

)

Diameter (nm)

TEM images (left) and the corresponding particle size histograms (right) of magnetite nanoparticles prepared by controlled coprecipitation. (A) without heat treatment and (B) after heat treatment (80ºC for 1hrs)

Preparation of SuperparamagneticIron Oxide Nanoparticles (SPION)

Fe2+ + 2 Fe3+ + 8 OH-

Co-precipitation

Fe3O4 + 4 H2O

Pyrolysis of Fe salt of fatty acids

Fe oleate complex

Fe3O4 and/or γ-Fe2O3300 OC

Widely used to prepare currentcommercial T2 contrast agents

Pros & Cons of Two Major Methods

Pros Cons

Co-precipitation 1. Neither organic solvents nor capping agents are used, water-based synthesis, “clean” for biomedical applications;

2. Low temperature (< 100 oC)

1. Broad size distribution;

2. Aggregation;

3. Formation of antiferromagnetic phase,

i.e. γ-FeOOH

Pyrolysis 1. Narrow size distribution;

2. Identical morphology;

3. Highly crystalline

Organic solvents (dioctyl ether) and capping agents (oleic acid) are used,

Not suitable for most biomedical applications

Phase transfer from organic to aqueous solution is required

Hydrophobic-Hydrophilic Phase Transfer

Hydrophobic Hydrophilic

Organic coating molecules

Amphiphilic macromolecules with PEG section

Phase transfer

Water

Hexane

Water

Hexane

SPION

SPION: Suiperparamagnetic iron oxide nanoparticlesPEG: Poly(ethylene glycol)

Jian Qin et al, Advanced Material

(in press)

Mechanism of phase transfer through Surface Coating

Hydrophobic poly(propylene oxide)

Hydrophilic poly(ethylene oxide)

ABA type triblock copolymer: Pluronic® F127 (PF127)

Amphiphilic coating layer

PF127/Oleic acid (POA)

Superparamagnetism Retained

Magnetization curve of (a) as-synthesized SPION and (b) POA@SPION

Cytotoxicity Tests

Viability of HeLa and MCF-12A cells exposed to POA@SPION at various iron concentrations.

Compare with Conventional Iron OxideNanoparticle Based Contrast Agents

Particle name Surface polymer r2/r1 ratio

(0.47 T, 310 K)

Mean hydrodynamic

diameter (nm)

POA@SPION

AMI-25 (Feridex; Advanced Magnetics, Cambridge, Mass)

AMI-227 (Combidex; Advanced Magnetics, Cambridge, Mass)

MION-37 (R. Weissleder, Massachusetts General Hospital, Boston, Mass)

MION-37 (R. Weissleder, Massachusetts General Hospital, Boston, Mass)

NC100150 (Clariscan, Nycomed, Amersham, Oslo, Norway)

SH U 555 A (Schering AG, Berlin, Germany)

USPIO S (Schering AG, Berlin, Germany)

11641.5Pluronic F127 + Oleic acid

Dextran

Dextran

Dextran T10

Dextran T10

Oxidized Starch

72

Carboxydextran

4.0

2.2

2.2

2.2

1.6

7.1

19

16-28

18-24

11.9

Carboxydextran 2.3

65

21

MRI Studies

Dextrane

MagneticCore

Blodkärl

fMRI

MagneticTargeting

Nanoparticles

Neuron

B. B. BjelkeBjelke

Use of SPION in Brain MRI Study Use of SPION in Brain MRI Study

T2 values as a function of unit pixel along the x-axis and y-axis. As indicated, a concentration gradient is observed with declining values towards the periphery of the injection.

100 120 140 160 180 2002x106

3x106

4x106

5x106

6x106

7x106

8x106

9x106

1x107

Inte

nsity

(T2)

Distance(pu)30 40 50 60 70 80

3x104

4x104

5x104

6x104

7x104

8x104

9x104

1x105

Inte

nsity

(T2)

D istance(pu)

24h 0 ug Fe 24h 10 ug Fe

48h 10 ug Fe

Labelling stem cells from neonatal mouse cerebellum(C17-2 cell lines) with Au-SPION.

STEM CELLS

1 month after transplantation

Neuronal stem cells labelled with Au-SPIONand transplanted into the rat spinal cord

2668 labelled cells, 4µl (4 x 667 cells/µl)

Nano Medicine

Design of MultifunctionalNanoparticles for DDS

• Magnetic: Imaging – markers – thermal• Carrier for a certain functional compound, e.g. drug, genes,

DNA, etc• Formation of stable suspension - physiologically

compatible• Controlled targeting of location, an organ or tissue, within

the living body• Keeping the particles localized in a given location for

desired period of time• Senstive to external stimulii, Temp, pH, bio-molecules, etc. • Controlled release of drugs through the pores of the shell

according to the pre-defined conditions • Bio-degradable and bio-resorable

Fe2+ +Fe3+ + OH-

Etching

1 2

3

45

Fabrication of inorganic NP with loading capacity

SiO2

Au

Core-shell Processing

Core-shell ProcessingTemplated Gold-Magetite Nanoparticle

Filling sequence into gold hollow shell

Etched Filled Refilled Final coated

Multifunctional NP for smart drug delivery system

Biocompatible polymers

* O *

O

Poly ¥å-caprolactone

*O

*

O

Poly lactide

*O

*

O

Poly glycolide

*NH

*

O

CH2

CH2

CH2

CH2

NH2

Poly L-lysine

* CH2 *

N

O

O

Poly(ethyl-2-cyanoacrylate)

Synthesis of PLA-mPEG amphiphilic diblock copolymer

hydrophilic hydrophobic

‘amphiphilic’ diblock copolymer

Procedures for preparation of drug-loaded nanospheres

Drug and Fe3O4

loaded in the cavity

TEM images of BSA-loaded nanospheresBSA-loaded PLA-mPEG nanospheres

BSA and Fe3O4-loaded PLA-mPEG nanospheres

Drug

Drug Fe3O4

‘Smart’ polymeric nanomaterials

Poly(N-isoprorylacrylamide)

T<LCST T>LCST

Drug Drug

LCST : Lower critical solution temperature

Under the condition that temperature exceeds the LCST, amphiphlic micelles are collapsed so as to start to release the entrapped drug.

Thermosensitive polymer

Jo, Y. S., Muhammed, et al., Macromolecular Rapid Communications 2003, 24, 957.

Qin et al (2006)

Schematic View of the “Shell-in-Shell” Structure

PNIPAAm: hydrophilic, stable PNIPAAm: hydrophobic, unstable

LCST: Lower critical solubility temperature

Below LCST

Drug release

Above LCST

DSC curves

-2

-1.5

-1

-0.5

0

20 70 120 170 220 270 320

Temperature [˚C]

Hea

t flo

w [W

/g]

LL-SH DL-SH LL-FL

Endo

DL-SHTg: 43 C̊

LL-SHTc: 99 C̊

LL-SHTm: 162 C̊

DL-SHTd: 257 C̊

LL-SHTd: 293 C̊

LL-FLTc: 101 C̊

LL-FLTm: 171 C̊

LL-FLTd: 290 C̊

TEM images

Toxicity AssayAu@PLLA-PEG@PNIPAAm-PDLAand PLLA-PEG@PNIPAAm-PDLA

(a) (c)(b)

1000 μg/mL uncoated1000 μg/mL Au control

(a) (c)(b)

1000 μg/mL uncoated1000 μg/mL Au control

TCCV assay

MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

TCCV: Two-color cell fluorenscence viability

Green spots = living cellsRed spots = dead cells

Mathematical modelingof controlled release rate

• Diffusion model • Dissolution model

⎟⎟⎠

⎞⎜⎜⎝

⎛∂∂

+∂∂

=∂∂

rc

rrcD

tc 2

2

2

⎟⎟⎠

⎞⎜⎜⎝

⎛∂∂

+∂∂

=∂∂

rc

rrcD

tc 2

2

2

( )∑∞

−

−

∞ +++

−=1

221

1 2

2

)39(161

n

DtRq

n

n

eqc

cαα

αα

)( 1cKckdtdcr pd −=−=

⎥⎦⎤

⎢⎣⎡ +

−−+

= )1exp(1)1(

01 kt

Kcc

p αα

α

Diffusion

Diffusion

Dissolution

Time

Jo. Y. S, Muhammed, M. et al, Nanotechnology 2004, 15 (9),1186-1194.

Self-assembly of gold nanoparticleson the surface of PLA-mPEG nanospheresSilanization

Gold nanoparticle self-assembly

Y. S. Jo, M. Muhammed, et al., J. Materials Science: Materials in Medicine 2005,

TEM images of ‘shell-in-shell’ structure nanoparticles

a) PLLA-PEG micelleb) ‘shell-in-shell’ structures covered with Au nanoparticles in partc) - d) ‘shell-in-shell’ structures fully covered with Au nanoparticles

Releasing profile – DL1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20

Time [hrs]

c1∞

Vr/

c0

Diffusion modelDissolution modelDL1

Releasing profile – LL2

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20

Time [hrs]

c1∞

Vr/

c0

Diffusion modelDissolution modelLL2

Application for controlled-release of estrogen

OH

HO

17β -estradiol

Hormone replacement therapy (HRP)

Estrogen or estrogen/progestin medication

Relieving menopausal symptoms

Reducing risk of osteoporosis, cardiovascular diseases, Alzheimer’s diseases, colon cancer, etc.

Target to evaluate estrogen effect

in vivo test: estrogen release by PLA-PEO DDS

Group D

PLA-PEO DDS (loading 17¥â-estradiol) injected Dosage;(39.6 ¥ìg / 39.6 ¥ìg / 550 ¥ìL = 17¥â-estradiol / PLA-PEO DDS / PBS buffer solution)

D-IID-I

Group N

550 ¥ìL of PBS buffer solution injected

N-IIN-I

Group P

P-I; 550 ¥ìL of positive control solution (17¥â-estradiol dissolved in olive oil) injectedP-II; 17¥â-estradiol tablet implanted

P-IIP-I

Aromatase P450 (P450arom) Knocked-Out (ArKO) mice used in this work

Androgen precursor steroids

Estrogens

Aromatase P450

Hearing ResearchHearing ResearchNanNanO O earear

Prof I. PykkProf I. Pykköö, Tampere Univ, Tampere Univ

B Bjelke

Summary

• Magnetic relaxation using NP for multi-compound detection

• Magnetic nanoparticles useful for functional MRI studies• Construction of ‘smart’ materials responsive to external

stimuli (from surroundings) such as temperature, pressure, pH, etc– Use of Poly(N-isopropylacrylamide) (PNIPAAm) as Thermosensitive

polymeric materials. Tunable lower critical solution temperature(LCST)

• Applications– Can be cosntructed to sense changes in body temperture– Can be applied for external heating– Can be used in Nanosphere, Hydrogel, …

Acknowledgement

Thanks for your Attention

For contacts

mamoun@kth.se