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Synthesis, Modification, and

Bioapplications of Magnetic Nanoparticles

By

Jin Xie

B.S., Nanjing University, 2003

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

IN THE DEPARTMENT OF HISTORY AT BROWN UNIVERSITY

Provedience, Rhode Island

May, 2009

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Copyright 2009 by Jin Xie

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iii

This dissertation by Jin Xie is accepted in it present form

by the department of Chemistry as satisfying the

dissertation requirement for the degree of Doctor of Philosophy

Date __________________ _________________________

Shouheng Sun

Recommended to the Graduate Council

Date __________________ _________________________

Dwight Sweigart

Date __________________ _________________________

William Risen

Approved by the Graduate Council

Date __________________ _________________________

Shelia Bonde

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iv

VITA

Jin Xie was born on August 16, 1980, in Jiangsu, China. He went to Nanjing

University for undergraduate study since 1999 and graduated with a B.Sc. in

Chemistry in 2003. Jin joined Brown University in August, 2004 and worked in Prof.

Shouheng Suns research group for his Ph.D. in Chemistry. His research interest is

magnetic nanoparticles synthesis, surface modification and bioapplications and have

published over 15 peer-reviewed papers.

Publications

1. Xie, J.; Chen, K.; Lee, H. Y.; Xu, C.; A., H. R.; Peng, S.; Chen, X. Y.; Sun, S.

Ultra-Small c(RGDyK)-Coated Fe3O4 Nanoparticles and Their Specific

Targeting to Integrin v3-rich Tumor Cells. Journal of American Chemical

Society, 2008, Online publication.

2. Xie, J.; Xu, C.; Kohler, N.; Hou, Y.; Sun, S. Controlled PEGylation of

monodisperse Fe3O4 nanoparticles for reduced non-specific uptake by macrophage

cells,Advanced Materials, 2007, 19, 3163-+.

3. Xie, J.; Xu, C. J.; Xu, Z. C.; Hou, Y. L.; Young, K. L.; Wang, S. X.; Pourmond,

N.; Sun, S. H. Linking hydrophilic macromolecules to monodisperse magnetite

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v

(Fe3O4) nanoparticles via trichloro-s-triazine, Chemistry of Materials, 2005, 18,

5401-5403.

4. Xie, J.; Huang, J.; Li, X.; Sun, S. H.; Chen, X. Y.; Iron oxide nanoparticle

platform for biomecial applications, Current Medicinal Chemistry, 2008,

Accepted.

5. Xie, J.; Peng, S.; Brower, N.; Pourmand, N.; Wang, S. X.; Sun, S. H. One-pot

synthesis of monodisperse iron oxide nanoparticles for potential biomedical

applications,Pure and Applied Chemistry, 2006, 78, 1003-1014.

6. Xie, J.; Sun, S. H. Chemical synthesis and surface modification of monodisperse

magnetic nanoparticles in Nanomaterials: Inorganic and Bioinorganic

Perspectives - Encyclopedia in Inorganic Chemistry, eds. C. M. Lukehart & R. A.

Scott, John Wiley & Sons, Ltd., 2008

7. Xu, C.; Xie, J.; Ho, D.; Wang, C.; Kohler, N.; Walsh, E. G.; Morgan, J. R.; Chin,

Y. E.; Sun, S. H. Au-Fe3O4 dumbbell nanoparticles as dual-functional probes,

Angewandte Chemie-International Edition, 2008, 47, 173-176.

8. Peng, S.; Wang, C.; Xie, J.; Sun, S. H. Synthesis and stabilization of

monodisperse Fe nanoparticles,Journal of the American Chemical Society, 2006

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vi

128, 10676-10677.

9. Peng, S.; Xie, J.; Sun, S. H. Synthesis of Co/MFe2O4 (M = Fe, Mn) Core/Shell

Nanocomposite Particles,Journal of Solid State Chemistry, 2008, In press.

10.Xu, C. J.; Xie, J.; Kohler, N.; Walsh, E. G.; Chin, Y. E.; Sun, S. H.

Monodisperse magnetite nanoparticles coupled with nuclear localization signal

peptide for cell-nucleus targeting, Chemistry-an Asian Journal, 2008, 3, 548-552.

11.Lee, H.Y.; Lee, S.H.; Xu, C. J.; Xie, J.; Lee, J. H.; Wu, B.; Koh, A. L.; Wang, X.

Y.; Sinclair, R.; Wang, S. X., et al. Synthesis and characterization of PVP-coated

large core iron oxide nanoparticles as an MRI contrast agent, Nanotechnology,

2008, 19, 165101.

12.Lee, H. Y.; L., S. H.; Xu, C. J.; Xie, J.; Lee, J. H.; Wu, B.; Koh, A.L.; Wang, X.Y.;

Sinclair, R.; Wang, S. X., et al. PET/MRI Dual Modality Tumor Imaging Using

Conjugated Radio-labeled Iron Oxide Nanoparticles, Journal of Nuclear

Medicine, 2008, In press.

13.Chung, S. H.; Grimsditch, M.; Hoffmann, A.; Bader, S.D.; Xie, J.; Peng, S.; Sun,

S.H., Magneto-optic measurement of Brownian relaxation of magnetic

nanoparticles,Journal of Magnetism and Magnetic Materials, 2008, 320, 91-95.

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14.Shen, W. F.; Schrag, B. D.; Carter, M. J.; Xie, J.; Xu, C. J.; Sun, S. H.; Xiao, G.,

Detection of DNA labeled with magnetic nanoparticles using MgO-based

magnetic tunnel junction sensors, Journal of Applied Physics, 2008, 103,

07A306.

15.Xie, J.; Xu, C. J.; Shen, W. F.; Xiao, G.; Sun, S. H. PEGlyated Fe3O4

nanoparticles for DNA detection in MagArray, Manuscript in preparation.

16.Young, K.; Xie, J.; Xu, C. J.; Sun, S. H. Conjugating methotrexate to magnetic

magnetite (Fe3O4) nanoparticles via trichloro-s-triazine, Manuscript in

preparation.

17.Li, M.X., Cai, P., Duan, C.Y., Lu, F., Xie, J., and Meng, Q.J. (2004). Octanuclear

metallocyclic Ni(4)Fc(4) compound: Synthesis, crystal structure, and

electrochemical sensing for Mg2+. Inorganic Chemistry 43, 5174-5176.

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ACKNOWLEDGEMENTS

There are many people I would like to thank for their support and help during my 4

year Ph.D. study. First of all, I would like to thank my advisor, Prof. Shengsun. It is

him who led me into the kingdom of nanoscience and taught me how to think, talk,

write and perform experiments like a scientist. Actually, he himself is the best

interpretation of a successful scientist.

Thanks Prof. William Risen and Prof. Dwight Sweigart. Your classes did help me a

lot in my research. And thank you for being in my committee for RPD and the final

defense. And thanks Prof. Robert Hurt for letting me use the facilities. Also thanks

Prof. Gang Xiao from engineering department. We had a great cooperation. Thanks

Prof. Y. Eugene Chin from Rhode Island Hospital, for giving me suggestions on

research and letting me use biology facilities.

Thanks Prof. Xiaoyuan Chen from Stanford University, I was benefited a lot from

the discussions with you. And also thanks for your great group members, for helping

me do the animal studies. Without you, I can not finish the project alone. Thanks Prof.

Shan X. Wang, also from Stanford. It was such a pleasure to work with you and your

group and may I have chances to continue the cooperation when I move to Stanford.

Thanks my group members. Thanks Chenjie Xu, for all your help, and I really

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learned a lot from you, in and out of science. Thanks Sheng Peng, Chao Wang and

Jaemin Kim, it has been great four years working with you. Thanks Kaylie Young, and

wish you have a great time in Northwestern. And thanks Dr. Nathan Kohler, for the

great advices you gave. And thanks for all the people who have worked in this group,

you are the best.

Thanks also for the department staff: Tun-Li Shen, Gen Goditt, Lynn Rossi,

Virginia McGee and Stacey Lee Calis. You are always available whenever I need help.

Thanks Mon and Dad for your everlasting love and support, which helped me

surpass all the difficulties. Like I always said, you are the best parents in the world, I

love you. And thanks Lin Xin. You are always by my side, whenever I was depressed

or was happy. It was you who taught me the importance of sharing and forgiving. And

only with your involvement and support, then this moment is meaningful.

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To Mom and Dad

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Table of Contents

Chapter 1: Introduction: iron oxide nanoparticle for biomedical applications........ 1

1.1 Introduction.. 2

1.2 IONP synthesis and surface modification 3

a. Introduction.. 3

b. IONP synthesis. 5

c. Surface modification of IONPs 8

d. Bioconjugation. 12

1.3 Pharmacokinetics and biodistribution.. 16

1.4 IONPs as T2-weighted MR contrast agent... 18

a. Passive targeting... 19

b. Specific targeting.. 22

c. Labeled cells for imaging. 30

1.5 IONPs for therapy 32

a. Drug delivery.... 32

b. Hyperthemia. 36

1.6 IONPs as nanosensor.. 39

1.7 Multifunctional IONPs for multimodality imaging 41

1.8 Conclusions and perspectives. 48

References 50

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Chapter 2: One step synthesis of monodisperse MFe2O4 (M=Fe, Mn, Co)

nanoparticles... 54


2.2 Synthesis of 6 nm Fe3O4 nanoparticles 57

2.3 5 nm CoFe2O4 nanoparticle synthesis.. 58

2.4 7 nm MnFe2O4 nanoparticle synthesis. 58

2.5 Size and shaper control of the nanoparticles 59

2.6 Magnetic properties of the nanoparticles. 65

2.7 Conclusions.. 66

Methods.. 68

References.. 71

Chapter 3: Synthesis and protection of Co and Fe nanoparticles. 72


3.2 Synthesis and protection of Co nanoparticles.. 73

3.3 Synthesis and protection of Fe nanoparticles. 81

Methods 82

References 98

Chapter 4: Surface modification of iron oxide nanoparticles. 99

4.1 Introduction..... 100

4.2 Ligand addition to make the particles water

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soluble and functionable... 101

4.3 Ligand exchange to make the particles water

soluble and functionable... 112


Methods.. 131

References.. 137

Chapter 5: Biovector conjugated iron oxide nanoparticles

for specific targeting... 138


5.2 Coupled with NLS peptide for cell-nucleus targeting.. 139

5.3 Au-Fe3O4 dumbbell nanoparticles as dual functional probes... 148

Methods.. 160

References.. 168

Chapter 6: Ultra-small c(RGDyK)-Fe3O4 nanoparticles

and their specific targeting to integrin v3-rich tumour cells... 170


6.2 Synthesis and characterizations of 4-MC IONPs. 172

6.3 Specific targeting study in vitro and in vivo. 178


Methods.. 185

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References.. 190

Chapter 7. Detection of magnetic nanoparticle labeled DNA

with magnetic tunnel junction sensors. 192

7.1 Introduction 193

7.2 Single strand DNA detection.. 195

7.3 DNA hybridization detection. 198

7.4 Conclusions 206

Methods 207

References 210

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List of Figures

1.1 .. 4

1.2 .. 7

1.3 .. 8

1.4 .. 9

1.5 .. 11

1.6 .. 15

1.7 .. 15

1.8 .. 22

1.9 .. 25

1.10 27

1.11 29

1.12 31

1.13 33

1.14 34

1.15 36

1.16 38

1.17 40

1.18 43

1.19 45

1.20 47

2.1 .. 60

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2.2 .. 62

2.3 .. 65

2.4 .. 67

3.1 .. 75

3.2 .. 77

3.3 .. 78

3.4 .. 79

3.5 .. 81

3.6 .. 84

3.7 .. 84

3.8 .. 86

3.9 .. 87

3.10 89

3.11 90

4.1 103

4.2 104

4.3 106

4.4 109

4.5 110

4.6 112

4.7 115

4.8 116

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4.9 116

4.10 118

4.11 120

4.12 122

4.13 123

4.14 124

4.15 125

4.16 126

4.17 127

5.1 141

5.2 144

5.3 146

5.4 146

5.5 147

5.6 148

5.7 150

5.8 150

5.9 152

5.10 152

5.11 153

5.12 154

5.13 156

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5.14 158

5.15 159

6.1 174

6.2 174

6.3 175

6.4 176

6.5 177

6.6 179

6.7 180

6.8 182

6.9 183

6.10 184

7.1 196

7.2 200

7.3 201

7.4 202

7.5 203

7.6 205

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1

Chapter 1

Introduction: iron oxide nanoparticle for

biomedical applications

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1.1. Introduction

The special and superior properties demonstrated by nanomaterials have attracted a

lot of attention in the past two decades. Many breakthroughs have been witnessed

since their emergence, and with the current progress, their continued interactions with

the traditional disciplines are highly expected. One of the most promising fields is the

integration of nanoscience and biomedicine, which might revolutionize the current

diagnosis and therapeutics techniques.

Among all the nanomaterials, IONPs might be one of the first and most studied,

which is attributed to their superior magnetic property, inexpensiveness,

biocompatibility and biodegradability. While the in vivo applications of a lot of other

materials are hurdled for the toxicity concern, several kinds of IONPs have already

been approved by the Federal Drug Administration (FDA) and are now widely used in

clinic for MR imaging and for hyperthermia therapy1-4

. This further motivates the

research interests in improving the quality of the IO materials. Owing to the

continuous efforts in synthetic method development, the precise control over the size,

size distribution, shape, crystallinity and magnetic property of the IONPs, is now

possible5-7. Meanwhile, the advances in the surface modification methods and

bioconjugation techniques have gifted the IONPs with different functions and

bioactivities8-10. Moreover, the recent exploits in trying to combine IONPs with other

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nanometerials have further expanded their capabilities10-12

. All these efforts have

endowed the old materials with whole new elements and stimulated the long-lasting

research interests in their applications in biomedicine.

This chapter will summarize some of the great works that have been done. Not only

that we will focus on the recent advances in IONPs applications in MR imaging,

hyperthermia and drug delivery, but also we will stress on the materials design,

synthesis, surface modification, and functionlization. And in the end, a current

tendency of combining IONPs with other nanostructures to form multimodality

nanocomposites will be discussed.

1.2. IONP synthesis and surface modification

1.2.a. Introduction

There are two stable constitutions of iron oxides at room temperature (r.t.), which is

magnetite (Fe3O4) and maghemite (-Fe2O3). The former has a so-called cubic inverse

spinel structure with oxygen forming an fcc closed packing while Fe cations

occupying the interstitial tetrahedral and octahedral sites. More specifically, half of

Fe(III) cations occupy the tetrahedral sites, while the other half occupy the octahedral

sites along with Fe(II) cations13. The standard formula for magnetite is AB2O4, where

A represents Fe(II) and B represents Fe(III). Other kinds of metal ferrites, such as

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MnFe2O4, CoFe2O4, NiFe2O4, can be formed by substituting Fe(II) with the

corresponding metal cations. In ambient environment, magnetite will be gradually

oxidized to maghemite. But in biomedical applications, we dont usually distinguish

these two but generally call them iron oxide.

Iron oxide is a good magnetic material, with magnetic saturation (Ms) moments of

92 emu/g (127 emu/g Fe) and 78 emu/g (111 emu/g Fe) for bulk magnetite and

maghemite, respectively. Those values are found to be smaller for the corresponding

particles, which has been attributed to the surface effects (spin surface disorders, spin

canting)14. However, with the recent advances in better controlling the crystallinity,

some IONPs were reported to have Ms values close to the bulk ones8, 15

(Figure 1.1).

Figure 1.1 A) energy diagram of magnetic nanoparticles with different magnetic spin alignment

showing ferromagnetism in a large particle (top) and superparamagentism in a small nanoparticle

(bottom)16; B) Hysteresis loop of superparamagnetic nanoparticles17.

A B

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IONPs with sizes ranging from 2 to 20 nm in diameter represent an important class

of artificial nanomaterials. At this scale regime, the thermal energy is sufficient to

overcome the anisotropy energy, therefore the magnetic moments begin to fluctuate

randomly. More specifically, the relaxation of the magnetization orientation of each

particle is determined by = 0KV/2kT, in which is the relaxation time at one

orientation, K is the particles anisotropy constant, V is the particle volume, k is

Boltzmanns constant, and T is temperature. As the size of the particle decreases to a

level where KV (free-energy barriers) becomes comparable to kT (thermal energy), its

magnetization starts to fluctuate from one orientation to another, leaving no coercivity

and net magnetic moment. Such phenomenon is called superparamagnetism16, 17.

IONPs at this scale respond quickly to an external magnetic field to reach Ms, while

withdrawing back to zero when the external field is removed. Such feature ensures the

particles long-term stability, which, together with their extreme small sizes hence

lower nonspecific in vivo uptake, explains why they are highly preferred in MRI and

drug delivery. Larger IONPs, on the other hand, were found to be more efficient in

hyperthermia and cellular labeling.

1.2.b. IONP synthesis

The synthesis of IONPs has been well documented. The classic way is to

co-precipitate Fe(II) and Fe(III) salts in aqueous solution, by addition of some bases,

usually NH4OH or NaOH (Figure 1.2). In order to control the IONPs growth and

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stabilize the particles from agglomeration, many kinds of polymers were added during

the synthesis, such as dextran, dendrimer, poly(aniline)18, 19

, which were coated onto

the particle surface to create steric or statistic repulsion hence balancing the attraction

forces among the particles. This method is simple and high-throughput, therefore is

still widely used today (for example dextran coated IONPs). However, the major

drawback of the co-precipitation method is the limited control over the NPs shape,

size, crystallinity and magnetization. In order to solve these problems, an organic

phase synthesis, which relies on pyrolysis of iron precursors with the presence of

surfactants, was later developed.

Two most widely used iron precursors in organic phase IONP synthesis are

Fe(acac)3 and Fe(CO)5. In the former case, one heats the mixture of Fe(acac)3,

1,2-hexadecanediol (reducing agent), oleyamine and oleic acid (surfactants) in benzyl

ether (high boiling solvent) stepwise up to the refluxing point7. During this process,

the salt forms nuclei and further grows to desired sizes through Ostwald ripening

process (the process when the small particles shrink while the big particles grow

larger20). One advantage of this method is that, it can be easily extended to make other

kinds of metal ferrites by simply mixing corresponding metal salt into the precursors.

For example, by mixing M(acac)2 (M = Mn, Co, Ni, etc.) and Fe(acac)3 in an 2:1 ratio

with the other starting materials, following the same treatment, MFe2O4 (M = Mn, Co,

Ni, etc.) were yielded7. Related work please refers to chapter 2. Similarly, IONPs can

be synthesized by decomposing Fe(CO)5 in refluxing octyl ether with oleic acid as the

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surfactant5.

Figure 1.2. Schematic illustration of IONPs synthesized through coprecipitation and pyrolysis

methods.

Compared to the co-precipitation method, the organic phase synthesis allows better

control over the size and monodispersity of IONPs. For example, Hyeon et al.

reported the synthesis of IONPs ranging from 4-15 nm in one-nanometer precise

control. Briefly, 4, 8 and 11 nm iron nanoparticles can be synthesized from thermal

decomposition of Fe(CO)5 with oleic acid at 1:1, 1:2 and 1:3 molar ratios, respectively.

Other sizes of IONPs were made by using these particles as the starting seeds, while

using Fe oleate (pre-made by reacting Fe(CO)5 with oleic acid in this case) as the iron

sources to go through a seed mediated growing process. By changing the

combinations between the seeds and Fe precursors, monodisperse IONPs with sizes of

6, 7, 9, 10, 12, 13 and 15 nm can be yielded (Figure 1.3)21. This procedure was further

extended for large-scale IONP synthesis. In this case, a common iron salt, iron

chloride, which is economically available and non-toxic, was reacted with sodium

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oleate first to generate iron-oleate complex. Afterwards, similar to the previously

described process but without presence of seed IONPs, this complex was decomposed

in 1-octadecene, yielding tens of grams monodisperse IONPs in a single pot reaction6.

Figure 1.3. TEM images of iron oxide nanoparticles with particle diameters of 6, 7, 8, 9, 10, 11, 12,

and 13 nm (clockwise from bottom right with 6 nm; scale bar: 20 nm); the diameter is controllable to

one nanometer accuracy. 21

1.2.c. Surface modification of IONPs

Surface coating is of great importance in determining the NPs stability under

physiological conditions. Due to the strong magnetic dipole-dipole interactions, the

IONPs tend to agglomerate without a hydrophilic coating layer. For those IONPs

made by co-precipitation in water using hydrophilic polymer (like dextran, dendrimer,

PASP) as the capping agents, this might be a minor issue. But for those made from

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high temperature decomposition in organic solvent, a surface modification step is

necessary to render the particles water soluble, biocompatible and functionalizable.

Various methods have been developed, which can be roughly divided into three

categories, i.e. 1) ligand exchange; 2) ligand addition; and 3) inorganic coating

(Figure 1.4).

Figure 1.4: (A) ligand exchange (B) ligand addition and (C) inorganic coating. F represents functional

chemical group that can be used for further conjugation.

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Ligand exchange refers to the approach of replacing the original hydrophobic

surfactants (usually alkylamine or alkylacid), with new, hydrophilic ones. This

happens when the new ligands have higher affinity to the particle surface than the

original capping ligands. One such example is the dopamine based ligands. Studies

have shown that, the bidentate enediol ligands such as dopamine could convert the

under-coordinated Fe surface sites back to a bulk-like lattice structure with an

octahedral geometry for oxygen-coordinated iron, which result in strong binding

between dopamine moiety and the surface of the IONPs. This was first reported by the

Xu group, who successfully anchored nickel nitrilotriacetic acid (Ni-NTA) onto the

SmCo5 core iron oxide shell nanoparticles with the help of dopamine22, and later

extended by the our group by introducing PEGylated dopamine onto the nanoparticle

surface, yielding particles with great stability against agglomeration in physiological

environment (Figure 1.5)23

. Please refer to chapter 4 for the details.

Another example is to use dimercaptosuccinic acid (DMSA) to replace the original

organic coating. The DMSA first forms a stable coating onto the NP surface through

its carboxylic chelate bonding and further stabilizing the ligand shell by forming

intermolecular disulfide cross-linkages among the ligands9 (Figure. 1.5).

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NH

HO

HO

OO

n

HN

OHNH

O

O

O

O

O

SH OH

OH SH

O

NH

O

O

OO

n

HN

OHNH

O

O

O

O

OOC

OOC

S

S

SH

S

S

SS

S

OOC

OOC

S

HS

OOC

OOC

HS

S

SSH

Figure 1.5. Ligand exchange by A) Dop-PEG-COOH and B) DMSA. Note that, in B, the ligands are

crosslinked by forming intermolecular disulfide bonds.

An alternative way of modifying hydrophobic IONPs is by ligand addition. In this

case, the newly added ligand needs to be amphiphilic, with one end being

hydrophobic and interacting with the inner hydrophobic IONPs core, while sticking

the hydrophilic tail into aqueous solution, offering the NPs hydrophilicity and stability.

Different kinds of block co-polymers, lipids, etc. are proved to be sufficient to render

the NPs water soluble in this way. For example, PEGylated phospholipids with

different kinds of terminal functional groups are commercially available and are

widely used in functionalizing IONPs. In a typical such conversion, phospholipids are

mixed with hydrophobic IONPs in chloroform to form a homogeneous solution.

Afterwards, the chloroform is evaporated and the IONPs can be redispersed in

water/PBS with the help of sonication or vortexing24. Our related work can be seen in

chapter 3 and 4.

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12

Thick and dense inorganic coating was also exploited as coating materials.

Benefiting from its rigid structure, the inorganic coating can prevent the IONPs from

contacting with each other or even the environment, so as to disperse and protect the

IONPs. The most commonly used materials for coating IONPs is silica. It is usually

done in a reverse microemulsion containing hydrophobic IONPs and appropriate

silane precursors25, 26. For example, polyoxyethylene(5)nonylphenyl ether (also

known as IGEPAL CO-520), which is a surfactant, can be dispersed in cyclohexane

by sonication, followed by addition of IONPs in cyclohexanes and ammonium

hydroxide to form a transparent, reverse microemulsion. Next, tetraethyl orthosilicate

(TEOS) is added, and the solution is stirred overnight. During this process, TEOS will

hydrolyze and subsequently condense to form a silica coating onto the NPs25.

1.2.d. Bioconjugation

Besides making the IONPs hydrophilic and stable against agglomeration, making

the IONPs functionalizable after modification is another concern. This requires the

new ligand/coating to have some chemically active groups on the end, such as

carboxyl, thiol, amine, etc. (Figure 1.6). These groups will react with some active

groups on the biovectors with the presence of some catalyst23, 27; or, alternatively, a

bifunctional linker will be adopted, which can react with the chemical groups on both

the particle surface and biovectors, to facilitate the coupling8. Either way, the reaction

condition needs to be mild, so as not to destroy the IONPs and the biovectors.

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13

For silica coating, it is very common to have APS co-polymerized with TEOS.

Therefore, after coating, there are multiple amine groups on the surface, which can be

used for further conjugation with amine or NHS terminated species12

. As for DMSA

coating, after crosslink, the remaining free thiol groups of DMSA ligand can be used

for the attachment of bioactive molecules. The coupling with amine terminated

species, such as proteins was achieved by using sulfo-SMCC as the linker8, 9. For

dop-PEG coated IONPs, the PEG is terminated with carboxyl group, which by

employing EDC/NHS as the catalyst, will form covalent bond with amine terminated

compounds23.

As mentioned above, synthesis of IONPs can be generally divided into two

categories. One is co-precipitation, with the advantage of high throughput,

comparatively better water solubility but are usually wider size distributed,

polycrystalline, and less magnetic. There is a trend to replace this method with high

temperature decomposing iron precursors with the presence of surfactant. This

approach offers better control over the particles size, shape, crystallinity and

magnetization, however, at the cost of an extra modification step to make the particles

water soluble and functionable.

Recently, there have been some efforts to mend the original recipes from both

approaches; or alternatively, to explore the third route. For example, PASP coated

IONPs was reported by co-precipiation of FeCl3 and FeCl2 in basic aqueous solution

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but with a heating treatment at 100 C. The yielded 5 nm Fe3O4 nanoparticles are

PASP coated and with a saturation moment as high as 116.9 emu/g Fe, compared to 70

emu/g of dextran coated nanoparticles with similar core size. The carboxyl groups and

amine groups on the PASP can be used for conjugation with other bimolecules28.

Another trial is recently done by our group. In this system, the Fe 3O4 NPs with 4.5

nm core size were synthesized by thermal decomposition of Fe(CO)5 in benzyl ether,

followed by air oxidation. Different from any of the previous synthesis methods, the

current preparation used a new ligand 4-methylcatechol (4-MC) as the surfactant,

which was inspired by the acknowledgement that, catechol based derivatives, such as

dopamine, have high affinity to the IONP surface. This design allows the particles to

be synthesized at high temperature in organic solutions. However, unlike their

alkylacid or alkylamine coated counterparts, the surface modification step can be

avoided, because the 4-MC coated IONPs, can be directly conjugated with amine

terminated species, such as a peptide, c(RGDyK), via a simple one step Mannich

reaction29 (Figure 1.7). Please refer to chapter 6 for details.

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Figure 1.6. Surface modification of IONPS: the inner magnetic core (black) combined with the outer

coating through chemical or physical interactions, ending with the active groups for further

modification.

Figure 1.7. Schematic illustration of coupling c(RGDyK) peptide to Fe3O4 NPs.29

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1.3. Pharmacokinetics and biodistribution

One key point regarding the in vivo applications of IONPs is to understand their

pharmacokinetics, and further control the in vivo behavior by modifying size (both

core size and hydrodynamic particle size) and the coating of the nanoparticles. In most

cases, IONPs, whether for drug delivery purpose or as MRI contrast agents, are

subject to intravenous administration, and reach the targeted tissues through blood

circulation. This naturally raises the questions like how long will the particles stay in

the blood circulation and where they are going to end up with? Both are crucial

concerns that directly determine the diagnosis and therapeutics efficacy.

Recognizing the particles as invaders, the body will try to eliminate them out of the

circulation, but amid different manners depending on the particles hydrodynamic

sizes. For example, since the smallest capillaries in body is 4 m30

, particles larger

than 4 m will be most likely captured in the lungs31

. Particles smaller than that, will

be usually eliminated by mononuclear phagocytes system (MPS), part of bodys

immune system also known as reticuloendothelial system (RES). It refers to the

family of cells, primarily monocytes and macrophages, that are extensively distributed

in the liver (Kupffer cells), spleen, bone marrow and lymph nodes, etc. Due to the

easier accessibility, most of the particles will be taken by the macrophages in the liver

and spleen. Between these two organs, although liver is the predominant terminus,

there is a tendency that particles larger than 200 nm may have better chances to be

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taken up by the spleen31, 32

. On the other hand, IONPs smaller than 7 nm are easily

subjected to extravasation and renal clearance and can be removed from the

circulation quickly33

. These facts narrow the window down to from about 10 nm to

200 nm, which is thought to be optimal for intravenous injection. For the ones out of

this range, the IONPs dont have enough circulation time to reach the desired sites.

And within this range, generally smaller particles are usually with longer circulation

time34.

Features of the coating on the IONPs surface also play important roles in the

IONPs biodistribution and elimination. Once injected intravenously, the IONPs are

exposed to the plasma proteins where many of them are adsorbed onto the particle

surface, a process called opsonization. The adsorbed proteins, including opsonins such

as immunoglobulins, complement proteins and fibronectin32

, together with the so

caused increased size, will enhance the particles chances of being recognized by the

macrophage cells therefore accelerating the clearance through RES. It is in this aspect

that some amphiphilic coating materials, like PEG, are important, for their capability

of resisting opsonization, hence elongating the circulation time35. Surface charge also

greatly influences the MPS uptake. It was found that negatively charged

carboxydextran-coated IONPs have an enhanced macrophage uptake compared to

neutral dextran coated IONPs1. On the other hand, positively charged particles are

known to cause nonspecific sticking to arbitrary cell membranes due to the electronic

interaction. Altogether, a neutral surface is favored to increase the circulation time.

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As pharmaceutical substances, IONPs biodegradability and biocompatibility is of

great concern and was also intensely studied. For the IONPs taken up by the cells,

either through phagocytosis by macrophage cells, or through receptor-mediated

endocytosis by targeting cells, they will be trapped in endosomes first which will later

fuse into lysosomes, where pH is low (4.8) and full of digestive enzymes, thereafter

progressively decomposed into iron cations1, 35. In one previous research, in which

radiolabeled IONPs were injected into mice and dogs and tracked with MRI, it was

found that most of the IONPs went to liver and spleen and slowly degraded there, with

plasma half-lives being 3 days in liver and 4 days in spleen, respectively. The released

iron ions were found to be subsequently incorporated into hemoglobin erythrocytes 31.

It is known that, chronic iron toxicity develops only after the liver iron concentration

exceeds 4 mg Fe/gram, 20 times that of the normal liver. However, in diagnosis, the

employed amount of IONPs is only 50 to 200 mg of Fe, comparing to 3500 mg of the

total human iron stores. Injection at this small scale is proved to be safe and nontoxic1.

1.4 IONPs as T2-weighted MR contrast agent

The biocompatibility and the superior magnetic property make IONPs important

contrast agent in magnetic resonance imaging (MRI). MRI is a noninvasive technique

which is now becoming a powerful tool in clinic for visualizing the structure and

function of body. It relies on the variations in local proton environment, which are

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reflected in the image as variable intensities, to depict organs or tissues in depth.

Introduction of appropriate contrasts will help accelerate the relaxation time of T1

(spin-lattice or longitudinal) and/or T2 (spin-spin or transverse) therefore further

enhance the local contrast. IONPs attracted interests because of their superior negative

enhancement effect on T2 and T2*-weighted sequences, creating dark or negative

contrast effects. IONPs can also accelerate T1 relaxation time, and have been reported

as T1 contrast agent for blood tool imaging36-38.

1.4.a. passive targeting

As discussed earlier, the in vivo pharmacokinetics of IONPs greatly relies on the

overall sizes. By controlling the sizes of the particles, IONPs can be specifically or at

least predominantly taken up by some of the tissue cells instead of the others. This

difference in uptake will subsequently change the local proton environment, therefore

achieving improved contrast and highlighting the pathologic sites. This is the basic

mechanism of passive targeting.

For example, IONPs larger than 100 nm will have greater chances to be recognized

by RES and subject to macrophage uptake compared to the smaller ones. This feature

was widely used for liver imaging, where Kupffer cells are exclusively located, and

are more accessible than other organs. Once administrated intravenously, most of the

IONPs will be phagocytosed by those macrophage cells, causing signal decrease of

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normal liver areas on the T2 weighted maps. On the other hand, pathologic tissues,

like liver tumors or metastases, which devoid of macrophages, show unaltered signals

after IONP injection. This difference increases the contrast between the diseased

tissues and healthy tissues hence increasing lesion conspicuousness1. Ferumoxides,

T10-dextran coated IONPs with a hydrodynamic size of 120-180 nm, is among one of

the first tested for liver imaging, and has been marketed and widely used in clinic.

Decrease in overall size can usually cause less macrophage uptake therefore longer

circulation time. It allows the IONPs to evade from the major RES uptake, thus

having enough time to slide into areas where are otherwise not accessible. For

example, Ferumoxtran-10, another marketed dextran coated IONPs, which are similar

in core size with Ferumoxides but with thinner coating, are proved to compensate

Ferumoxides application by its differed biodistribution. With a hydrodynamic size of

30 nm, following administration, Ferumoxtran-10 can reach normal lymph nodes1.

Similarly, this uptake can improve the contrast by darkening normal lymph node areas

on T2 weighted images, while the intensities from metastatic nodes remain unchanged.

Many other research using Ferumoxtran-10 as the contrast agents have also been

reported, including inflammatory and degenerative diseases detection, blood tool

agents for MR angiography, and perfusion imaging of tumor, kidneys, brain and heart,

all by taking advantage of their smaller size and therefore longer plasma half-life1, 35,

39.

Considering the fact that the RES uptake is also greatly dependent on the coating

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materials, many other materials were tried to coat the IONP surface as well to change

the targeting feature. This exploit becomes more intense with the recent advances in

organic phase based NP synthesis as well as the development in surface modification

techniques, which allows different kinds of polymers, molecules and peptides to be

decorated onto the IONPs.

For example, in a recent report, poly(vinyl pyrolidine) (PVP), a water-soluble,

non-charged and non-toxic polymer, was used as the capping ligand for the IONP

preparation. In this study, PVP-coated IONPs were produced in a one-step synthesis

by thermal decomposition of Fe(CO)5 with PVP in DMF at 160C. The final product

are 8-10 nm single crystallized IONP cores with an overall size around 40-50 nm.

These IONPs showed better magnetic moment (110 emu/g Fe) than Ferumoxide (70

emu/g Fe) and higher macrophage uptake in vitro and in vivo than Ferumoxide,

potentially being a better contrast agent for inflammation disease detection15

(Figure

1.8).

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Figure 1.8 a) T2*-weighted MR images before, 3, 7, and 20 min after intravenous injection of Feridex

(up) and PVP-coated iron oxide nanoparticles (down). b) Both Feridex and PVP-IO were able to reduce

the intensity in the rabbit liver parenchyma but the T2* effect of PVP-IO is more prominent than

Feridex. C) HR-TEM image of the PVP-IONPs. 15

1.4.b. Specific targeting

A more promising approach is to make the IONPs targetable, therefore able to reach

the pathological sites specifically. In order to achieve this goal, biovectors must be

first loaded onto the IONPs before administration, which will render the particles

targeting ability toward some specific tissues. Taking tumor targeting for example, the

biovectors such as antibodies or peptides, will be recognized and captured by the

corresponding biomarkers that are exclusively expressed or overexpressed on the

surface of tumor cell membranes. Then through the specific interaction between the

biovector on IONP surface and the receptors on cell membranes, IONPs can be

accumulated on the cell membranes or even internalized through receptor-mediated

5 nm c

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endocytosis. The normal cells, on the other hand, will not be labeled. Therefore, on a

T2 weighted map, the tumors can be distinguished from the normal cells with deceased

signal intensities.

Regarding active targeting, the nanoparticles are expected to stay in the circulation

long enough before targeting to the aiming sites, therefore smaller nanoparticles are

preferred for decreasing nonspecific macrophage uptake. Another request is that, it is

necessary to have multiple reactive groups, such as amines, carboxyls or thiols, etc. on

the IONP surface, for exerting bionconjugation with the targeting agents.

One of such platforms that has been extensively studied is the so-called

amine-terminated cross-linked iron oxide (CLIO) nanoparticle. It is produced by

cross-linking dextran coated IONP surface with epichlorohydrin and ammonia. Many

types of targeting agents, such as transferrin, annexin V, anti-VCAM mAb,

anti-E-Selectin mAb, oligonucleotides, and TAT peptides have been successfully

grafted on to CLIO NPs and have shown expected targeting ability40-44

.

However, as a dextran coated IONP derivative, which is synthesized from low

temperature co-precipitation method, CLIO shows a relatively low saturation moment

(about 70 emu/g)8, 15, therefore less prominent T2 contrast effect compared with their

single crystalline counterparts8. Additionally, substantial amount of the particles are

found trapped in liver and spleen for CLIO, partially due to its incapability of

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precisely controlling the core and hydrodynamic sizes. More importantly, the

cross-linking agent, epichlorohydrin is classified as a carcinogenic, mutagenic and

reprotoxic substance1, which limit their clinical potential. Therefore, the efforts for

searching new IONP platforms have never been stopped.

One promising approach is to graft the IONP surface with PEG, which is, efficient

in reducing non-specific adsorption and enhance IONP stability in plasma. One of the

efforts was done by PEGylating IONPs with a home-made ligand

dopamine-PEG-COOH. In this case, IONPs were synthesized first in organic medium.

Afterwards, this dopamine-PEG-COOH ligand was introduced to replace of the

original organic coating and render the IONPs water soluble and functionable. While

keeping the cores the same, the overall sizes of the NPs can be modulated by PEG

lengths. For example, 9 nm Fe3O4 core PEG 3000 coated IONPs have an overall

diameter around 50 nm, whereas the PEG 20000 coated IONPs have a diameter of 90

nm in aqueous solution. More importantly, in an in vitro uptake study with RAW

264.7 macrophage cells, all the four tested PEGylated NPs (PEG 600, 3000, 6000 and

20000) showed much lower uptake compared with dextran coated IONPs23. This

feature makes it a good platform for specific targeting purpose. Related work please

refer to chapter 4 and 5. Once conjugated with targeting agents such as RGD peptide

from the terminal carboxyl group and injected intravenously, those particles were

successfully concentrated in tumor area, which was tracked by MRI (Figure 1.9)

(unpublished data).

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Figure 1.9 MR images of mice with A) Dop-PEG-RGD NPs and B) Dop-PEG-RGD NPs plus block

dosage of free RGD peptide.

Another important work was done by Cheon group8. In their system, the IONPs

were nonhydrolytically synthesized by high-temperature decomposition of iron

precursors in the presence of oleic acid and oleylamine, followed by a surface

modification step with 2,3-dimercaptosuccinic acid (DMSA) to render them water

soluble and functionable. The magnetization value for these DMSA modified 12 nm

IONPs is 101 emu/g Fe. Doping the oxides with different kinds of metal cations was

found to influence the magnetization. For 12 nm MnFe2O4, CoFe2O4 and NiFe2O4

nanoparticles, the magnetization values are 110, 99 and 85 emu/g magnetic atoms,

respectively. The correspondingR2 (1/T2) values of these particles at 1.5 tesla (T) are

358, 218, 172 and 152 l/mmol/s for MnFe2O4, Fe3O4, CoFe2O4 and NiFe2O4,

respectively All of them are much higher than that of CLIO NPs (62 l/mmol/s) under

A B

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the same condition. Next, herceptin, which specifically binds to HER-2/neu marker

overexpressed in breast and ovarian cancers were conjugated onto IONPs by using

sulfosuccinimidyl-(4-N-maleimidomethyl) cyclohexane-1-carboxyl (sulfo-SMCC) as

the crosslinker. After administrating intravenously at a scale of 20 mg/kg, these IONPs

were found to be successfully accumulated in tumor site, as proved by MRI by giving

a 31% R2 enhancement (R2/R2control). As a comparison, herceptin conjugated CLIO

NPs at the same condition only give very marginal (5%) signal change. The author

attributed this improvement to the superior magnetic property of these IONPs8

(Figure 1.10).

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Figure 1.10 In vivo MR detection of cancer using magnetic nanoparticleHerceptin conjugates. (af)

Color maps ofT2-weighted MR images of a mouse implanted with the cancer cell line NIH3T6.7, at

different time points after injection of MnFe2O4 (MnMEIO)-Herceptin conjugates or CLIO-Herceptin

conjugates (preinjection (a,d); and 1 h (b,e) or 2 h (c,f) after injection). In ac, gradual color changes at

the tumor site, from red (that is, low R2) to blue (that is, high R2), indicate progressive targeting by

MnMEIO-Herceptin conjugates. In contrast, almost no change was seen in the mouse treated with

CLIO-Herceptin conjugate (df). (g) Plot of R2 change versus time. In the mouse treated with

MnMEIO-Herceptin conjugate (squares), significantR2 changes (up to 34%) were observed with time

after treatment. In contrast,R2 changed by

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With the era of using targeting IONPs as MR imaging agents getting closer and

closer, price becomes an important issue. While there exist a long list of biovectors

which have been tried to conjugate onto IONPs, many of them might be crossed out

simply because they are too expensive, which might include most of the antibodies.

Currently research adopts a dosage scale about 10-20 mg Fe/kg for mice. As for

human, this means that, antibody in tens of mg scale might be needed to functionalize

IONPs for a single injection, which is economically unrealistic for early stage cancer

detection. Some small peptides, however, are favored for their inexpensiveness and

being unlikely to denature during coupling and storage. Such an example that has

been actively studied is arginineglycineaspartic acid (RGD) containing peptides,

which can interact with integrin v3 overexpressed on many kinds of solid tumor

cells and tumor vasculature. For example, as mentioned already, it was used as the

biovector to conjugate onto Dop-PEG-COOH NPs (Figure 1.9). CLIO can as well be

conjugated with RGD peptide, and these particles could target predominately to BT-20

tumor cells in vitro and in vivo45, 46

. The recent developed 4-MC IONPs have also

been tried to coat with RGD peptide (Figure 1.7) through one step Mannich reaction.

After intravenous injection, these particles were found to concentrate on xenoplanted

U87MG tumor cells 29 (Figure 1.11). Please refer to chapter 6 for details.

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Figure 1.11 MRI of the cross section of the U87MG tumors implanted in mice: (A) without NPs, (B)

with the injection of 300 g of c(RGDyK)-MC-Fe3O4 NPs, and (C) with the injection of

c(RGDyK)-MC-Fe3O4 NPs and blocking dose of c(RGDyK); and Prussian staining of U87MG tumorsin the presence of (D) c(RGDyK)-MC-Fe3O4 NPs and (E) c(RGDyK)-MC-Fe3O4 NPs plus blocking

dose of c(RGDyK).29

It is worth noting that, direct targeting the pathological tissue is not the only way to

increase the MR contrast. Some other approaches have also been tried with success.

One example is to target normal cells instead of the diseased cells, which has similar

mechanism to the passive targeting approach, but was done by active targeting. For

example, asialoglycoprotein (ASG) receptors are expressed on normal hepatocytes but

not on metastatic tumors. After coupling IONPs with arabinogalactan (AG), a ligand

of ASG receptor, and intravenous injection of such IONP-AG conjugate into

orthotopic hepatocarcinoma xenograft model in rats, MR intensities from the normal

liver cells were decreased on the T2 weighted map but not the liver cancer, thus the

tumor can be detected by MRI as pseudo positive contrast4. Other designs include a

two-step amplification approach, in which a biotinylated antibody was administrated

and accumulated on pathological sites first, followed by a second step

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IONP-streptavidin injection. Through the strong streptavidin-biotin interaction, IONPs

can be localized onto the diseased sites to improve the contrast4.

1.4.c Labeled cells for imaging

Other than being used as targeting probes, IONPs have also been used to label cells

ex vivo and MRI has been used to track the migration of the injected cells. This

technique is of special importance regarding the recent progress in stem cells and

progenitor cells based therapies, which are anticipated to ultimately change the

treatment of disease1.

One of the key issues in IONP-based cell tracking is to load adequate amount of

IONPs into cells. Nonspecific strategies have been tried to enhance the internalization

efficacy, such as simply adopting high concentration47, 48

. More effectively, a variety

of transfection agents were employed to enhance the uptake through the electrostatic

interactions, like protamine sulfate, polylysine, cationic liposome, cationic dexdrimer1.

For example, mesenchymal stem cells (MSCs) were magnetically labeled with

ferumoxides in culture medium for 24 to 48 hours with 375 ng/mL poly-L-lysine

(PLL; average MW 275 kDa). After washing, these Feridex-labeled MSCs

(MR-MSCs) were suspended in PBS, and their viability was studied right before the

injection, which was >95%. The MR-MSCs were then intramyocardially injected, and

their migration was successfully tracked by MRI49.

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IONPs have also been coupled with HIV-TAT peptides to facilitate internalization.

IONP-TAT conjugate can be internalized into hematopoietic CD34+

cells in quantities

up to 10-30 pg Fe per cell, with unharmed cell viability. Following intravenous

injection, these IONP-labeled cells were found to home to bone marrow and were

detected by MRI successfully 50 (Figure 12).

Fig. 1.12. MR imaging. Axial MR images of bone marrow samples obtained from mouse femurs. (A)

NOD/SCID mouse had been injected intravenously with TAT-IONPs labeled CD34+ cells (4 106

cells/animal). Single cells are detectable by MR imaging as dark signal voids (arrows). (B) Control

animal.50

Instead of trying to put millions of particles into cells to make them detectable, an

alternative way is to simply use large particles. It was found that micro-sized IONPs

can be detected by MRI in vitro in agarose samples, in cultured cells, and further in

mouse embryos51. According to the report, this method can help accommodate cells

with ~100 pg Fe. This micro-sized IONP labeling was later extended to monitor

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hepatocytes transplantation with success52

.

1.5. IONPs for therapy

1.5.a. Drug delivery

IONPs can also be employed as the carrier for drug delivery. This topic is very

important in cancer treatment, where traditional chemotherapy and radiotherapy faces

serious side effect problem. The idea is to load drugs onto the IONPs, chemically or

physically, and then taking advantage of the modified IONPs targeting ability to

concentrate them to areas of interest, where the drugs will be released in a controlled

manner. By this approach, the dosage of drug can be dramatically reduced and side

effects can be greatly eliminated.

However, the realization of this specific targeting idea is not easy and most of the

work is still at the proof-of-concept stage. Several critical factors need to be

considered: such as how to load the drugs, how to specifically target the particles to

the tumor cells, and how to release the drugs?

As demonstrated above, with the advancement of surface chemistry, the IONPs can

be modified with a variety of functional groups, which can be used for conjugation

with the drugs through mild chemistry and/or appropriate crosslinkers. For example,

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IONPs can be modified with an amine terminated PEGylated saline first, and an

anti-cancer drug Methotrexate (MTX) can be loaded onto the particles through

EDC/NHS mediated coupling. This IONP-MTX conjugate was proved to enhance the

glioma cell uptake. And after taking into the cells, the MTX was cleaved from the

IONP surface in the lysosome due to the low pH and presence of proteases27, 53, 54

(Figure 1.13). This method can be potentially extended to load other drugs, however

an obvious premise is that these drugs must have some chemically active groups for

coupling. There has been no in vivo study of such drug-loaded IONPs so far.

Figure 1.13. Schematic representation of the intracellular uptake of MTX modified nanoparticles and

the following drug release.54

Alternatively, the drugs can be loaded onto IONPs through physical absorption.

This approach seems more universal considering that many drugs are lack of

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functional groups for coupling or will lose their activity after coupling. A porous

structure is known to favor the drug loading, like silica NP based drug delivery. But

such structure was rarely reported for IONPs until recently solved by Hyeon et al.

They started with spindle-shaped -FeOOH NPs, yielding through hydrolysis of FeCl3

in aqueous solution, and subsequently applied a so-called wrapbakepeel process, a

three-step treatment composing of silica coating, heat treatment and removal of silica,

to get hollow IONPs (Figure 1.14). Afterwards, they loaded doxorubicin (DOX) into

these hollow particles, and tested their cytotoxicity against SKBR-3 cancer cells.

Unlike DOX alone, the DOX loaded IONPs seem to exert a more controlled and

sustained release manner55.

Figure 1.14. TEM images of A) -FeOOH nanoparticles B) silica coated -FeOOH nanoparticles C)

iron oxide nanocapsules after wrapbakepeel process treatment.55

The intrinsic superior magnetic property gifts IONPs the ability to respond and

accumulate upon the applied the external field. This feature was employed for

transporting drug loaded IONPs to areas of interests, in which case strong magnetic

A B C

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field, large IONPs as well as high concentration seem to be necessary to achieve

adequate localization. Nevertheless, many trials have been done by employing

external field to concentrate drug loaded IONPs in animals2, 3

. More promisingly,

some clinical trials in humans were also reported. In one of the cases, seven patients

with metastatic breast cancer were fused with epirubicin loaded IONPs (100 nm, 0.5%

of the estimated blood volume). Afterwards, a magnetic field was built up over the

tumor. It was found that, for about one half of the patients, ferrofluid were

successfully directed to the tumors, where tumor regression was found31, 56. Although

in both animal studies and human trials some discoloration of the skin was observed,

no other obvious clinical symptoms were noted31.

IONPs are also actively involved in particle assisted gene delivery.

Polyethyleneimine (PEI) coated Fe3O4 nanoparticles, with an external magnetic field

applied in a vertical direction, were proved to greatly enhance the transfection

efficiency and even worked well with some cell lines that are otherwise hard to be

transfected19, 31

. The developers attributed this phenomenon to the enhanced

permeability given by the added magnetic field, and named this IONP assisted gene

transfection magnotransfection19, 57, 58. Alternatively, IONPs can be associated with

virus to work as a transfection agent meanwhile serving as an effective probe for MRI.

In one of the trials, MnFe2O4 nanoparticles were conjugated onto adenoviruses. These

hybrid particles were demonstrated as being able to efficiently deliver eGFP to U251N

cells in vitro, the process of which was successfully monitored by MRI and

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fluorescent tracking of the expressed eGFP59

(Figure 1.15).

Figure 1.15. a) Schematic of the formation of adenovirus-MnFe2O4(MnMEIO) hybrid nanoparticles. b)

TEM image of adenovirus-MnMEIO hybrid nanoparticles. c)-e), Determination of eGFP expression of

adenovirus-MnMEIO treated cell lines. c) MnMEIO-treated U251N cells (control), d)

adenovirus-MnMEIO treated U251N cell lines, e) adenovirus-MnMEIO treated CHO-1 cell lines.59

1.5.b. Hyperthermia

Instead of being a carrier and killing tumor cells by the loaded drugs, the IONPs

can serve as mediators and help induce heat to the local tumors to make damage,

which is called hyperthermia. It is based on the theory that, when exposing to an

external alternating magnetic field (AMF), the mediators magnetic moments

oscillates, during which process the electromagnetic energy is converted into heat

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(magnetic entropy change)2, 3

. Hyperthermia also takes advantage of the fact that,

tumor cells are more susceptible to elevated temperatures in the range of 42-45C than

the normal cells, making it possible to deliver magnetic materials specifically to tumor

cells, and generate heat locally to damage them, without influencing the normal

tissues2, 3, 19.

One of the most crucial parameters of such mediator is its specific absorption rate

(SAR), which indicates the heat evolution rate in hyperthermia. SAR values depend

on a large number of parameters (e.g. size, size distribution, shape, surface chemical

compositions, frequency and amplitude of the magnetic field viscosity of the

surrounding medium) and vary from a few tenths to a few hundreds of Watt per gram

of magnetic materials60

. Nevertheless, IONPs appear to be the best compromise

choice between biocompatibility and adequate SAR values, and have thus been

intensively studied.

Again, the specificity of targeting onto tumor cells is of great concern, which is

greatly influenced by the surface nature. In one study, IONPs coated with a liposome

layer (phospholipids) were incubated with the T-9 glioma cells. It was found that, the

positively charged surface facilitated the IONP uptake, and increased the uptake by 10

times compared to their neutral counterpart. These particles were then injected into

solid tumor formed subcutaneously in F344 rats, which were subsequently exposed to

AMF. For the rats without IONP injection, the temperature in tumor cells remained

unaltered. Oppositely, the temperature of tumor with IONP injection quickly exceeded

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43C. Meanwhile, prominent tumor regression was observed with repeated AMF

exposures2.

Antibody directed IONP targeting was also tried in hyperthermia. In one study, Fab

fragment of anti-human MN antigen-specific antibody was chemically anchored onto

the liposome surface, and their targeting towards MN antigen overexpressing

carcinoma cells was studied in vitro and in vivo (Figure 1.16) . Over 50% of the total

IONPs were found to be accumulated in the tumor cells, also leading to regression

after exposure to AMF2, 61.

Figure 1.16. Magnetite uptake of IO NPs for carcinomas and various organs 48 h after the intraarterial

injection (0.4 ml, net magnetite: 3 mg). Animals in group I (open columns), i.e., the control group, were

transplanted with MN antigen-overexpressing mouse renal cell carcinoma (MN-mRCC) and injected

with unmodified IONPs. Group II animals (closed columns) were transplanted with MN-mRCC and the

antibody-conjugated IONPs were injected.61

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1.6. IONPs as nanosensor

When the IONPs change from dispersed state to clustered state, the spin-spin

relaxation time (T2) of the water protons will concomitantly vary. If the IONPs are

modified with some probe molecule, and the crosslinking is caused by the existence of

a targeting molecule, then the interaction between the two molecules can be studied

by monitoring the T2 change. These particles are referred to as magnetic relaxation

switches (MRS), and they are proved as promising materials in the detection of

enzyme, antibody bacteria and virus62-66.

For example, a bi-biotinylated peptide (BBP) can cause agglomerates among avidin

coated CLIO NPs (CLIO-A), and give reduced T2. However, upon the addition of

protease (such as trypsin), if the peptide is a specific enzyme substrate, the clusters

will be redispersed, and the T2 loss will be regained (Figure 1.17). This phenomenon

was used for quantitative protease assay by monitoring the T2 change. It is worth

mentioning that although this assay was done in a 96 well plate, it is possible to

upgrade it to a high throughput fashion, such as simultaneous monitoring of stacked

microtiter plates with 1536 wells per plate and 10 L solution per well64. Compared to

conventional FRET method, this MRS protease assay can tolerate extreme pH and

temperatures and is free from light interferences. Antibody, bacteria and virus

detections were also reported with similar mechanism but modified design62, 63, 65, 66.

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Figure 1.17. A) Schematic view of the BBP-MRS assay. B) Plot of the increase in the average cluster

size and the increase in relaxivity R2 among CLIO-A nanoparticles, in the presence of BBP. c) T2

mapping of 96-well plate samples measuring trypsin activity. The upper row contains CLIO-A and

trypsin from 0 to 3.2 mg/mL. In the lower row are reference wells with CLIO-A only. T2 values are

coded according to the bar on the right.64

IONPs have also been tried as tags in DNA microarray to take place of the

traditional organic dyes. In one of the demonstrations, a probe DNA was immobilized

onto the surface of the biochips. A second biotinylated DNA, was then applied, which

would be immobilized onto the surface through hybridization if it was complementary

with the first one. The neutravidin coated Fe3O4 nanoparticles were added on

A B

C

C

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afterwards, and were immobilized through biotin-avidin interaction. The magnetic

signals generated from the IONPs can then be detected by either TMR or GMR sensor

underneath67, 68

. Please refer to our related work in chapter 7.

Recent research found that IONPs are also good enzyme mimetics. With the

presence of H2O2, they are capable of catalyzing reacting peroxidase substrates such

as 3,3,5,5-tetramethylbenzidine (TMB), di-azo-aminobenzene (DAB),

o-phenylenediamine (OPD) to their colorful oxidized states. The advantage is that,

IONPs can sustain a broad pH or high temperature, i.e. are able to work at harsh

environments, whereas peroxidase denatures and loses its activity quickly in such

circumstances. Actually, even at normal environment, their catalytic activity is 40-fold

higher than that of horseradish. Another benefit is that, IONPs can be recollected due

to their magnetic property, hence allowing recycling and repeated uses69

.

1.7 Multifunctional IONPs for multimodality imaging

The idea of integrating two or more modalities in one system is not new. It is

initiated by the fact that, the imaging modalities that are well suited in some

applications might be poorly suited in other circumstances70. For example, MRI

enables high-resolution anatomical and functional imaging at the cellular level but it

suffers from the low sensitivity. Radionuclide imaging is highly sensitive and allows

tomographic display but with relatively poor spatial resolution. Near-infrared

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fluorescence (NIRF) imaging has high sensitivity and low background but its usage is

restricted by the limited penetration depth of light and therefore poor anatomical

resolution. These facts lead to the exploits of building these modalities together and

therefore simultaneously collecting information from different aspects to achieve

better understandings of the tissues or organs under study. It is obvious that challenges

lie in both the instrumentation and the probe parts. The concern of hybrid

instrumentation is beyond the scope of this review. We will go over some of the

efforts made in developing multifunctional imaging probes in this section.

The most common way to add another function onto the magnetic IONPs is to

conjugate onto them some organic dyes by taking advantage of the chemical groups

on the surface of the IONPs71, 72

. This combination will allow one to get not only

macroscopic information about which tissue the IONPs are concentrated in, but also

telling at cellular and subcellular level where do the particles go. However, the

practical usage of such optical-magneto probe is restricted due to the fact that most of

the organic dyes have limited tissue penetration. On the other hand, IONPs have been

labeled with a variety of radioisotopes for PET/MRI and SPECT/MR imaging studies.

PET/MRI in particular, could be a prospective integration for complementing the

MIRs high resolution with PETs high sensitivity. One such example was given by

attaching 64Cu-DOTA chelats onto PASP coated IONPs, together with cyclic RGD

peptide28 (Figure 1.18). Such particles could specifically bind to tumor cells when

administrated intravenously, and was traced successfully by both MRI and small

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animal PET.

Figure 1.18. A) The synthesis of the PET/MRI dual functional probe DOTA-IO-RGD. DOTA-IO was

prepared similarly except that no RGD peptide was used. B) PET study of64Cu-DOTA-IO-RGD C-J)

MRI of IO-RGD injected mice.28

Recent progresses have allowed us to add one extra modality to the IONPs directly

through particle synthesis. For example, Au-Fe3O4 NPs can be synthesized by

growing Fe3O4 composite onto the pre-made Au seeds, forming a dumbbell like

nanostructure with two nanocomposites attaching with each other side-by-side

(Figure 1.19). The novel structure have two main advantages. First, this dumbbell

particle offers two different surface platforms, which allow selectively conjugating

B

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onto each moiety different species. Second, it may serve as both a magnetic (Fe3O4)

and optical (Au) probe. It is worth mentioning that, although Au nanoparticles are

well known for their surface plasma absorption, they are also demonstrated to have

superior reflection ability, i.e. reflects the incidence light back at the same wavelength,

which can be captured by fluorescence microscope for imaging purpose. The

advantage of utilizing gold particles as probe is that this reflectance effect will never

decay, which is a common problem for molecular based fluorescence. As a

proof-of-concept demonstration, Au-Fe3O4 NPs were modified with EGFR antibody

and selectively targeted onto A431 cells, which overexpress EGFR on the membrane.

Both MRI and fluorescence microscopy were employed and confirmed the

dual-modalities of these particles10. In a similar scenario, synthesis of CdSe-Fe3O4

NPs was recently reported which might be more promising for combining both

magnetic and fluorescent properties11

. Please read chapter 6 for details.

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Figure 1.19. a) T2-weighted MRI images of i) 20-nm Fe3O4, ii) 320-nm AuFe3O4, iii) 820-nm

AuFe3O4 nanoparticles, and iv) A431 cells labeled with 820-nm AuFe3O4 nanoparticles. b)

Reflection images of the A431 cells labeled with 820-nm AuFe3O4 nanoparticles. c, d) Images of

A431 cells labeled with 820-nm dumbbell particles, floating in the medium before (c) and after (d) an

external magnetic field was applied (field gradient in the sample area was in 500100 G). The dashed

circles denote individual cells; the numbers label the same cells in (d) and (d); the arrow and H indicate

the direction of the applied magnetic field.10

Instead of forming a side-by-side structure, the second moiety can also be

introduced in a core-shell manner, usually having Fe3O4 NPs as the core. For example,

there was a recent report on synthesizing magnetic core/shell Fe3O4/Au and

Fe3O4/Au/Ag nanoparticles. Briefly, the pre-made Fe3O4 nanoparticles were mixed

with a mixture solution of HAuCl4 and oleylamine, where HAuCl4 was reduced and

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formed a thin layer of Au shell over the Fe3O4 surface. More coating is achieved by

mixing these seeds with HAuCl4 or AgNO3 and ascorbic acid in the presence of

cetyltrimethylammonium bromide (CTAB), and incubating the mixture at 30C.

Interestingly, by controlling the shell thickness, the plasmonic properties could be

influenced, either red shift or blue shift73.

More commonly, as previously mentioned, silica was chosen as the coating

materials, where TEOS and APS mixture are hydrolyzed to form a uniform silica

coating onto the IONP surface. Part of the APS can be pre-conjugated with some

common dyes, so the formed silica nanoparticles will be both fluorescent and

magnetic. At the same time, the free amine groups from the unconjugated APS will

allow further conjugation. Alternatively, the silica coating can capsulate both Fe3O4

nanoparticles and fluorescent quantum dots together to make dual-functional probes26

(Fig. 1.20).

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Figure 1.20. Magnetic-optical dual-mode detection of polysialic acids (PSAs) expressed from

neuroblastoma cells by using dye-doped silica (DySiO2) core and multiple Fe3O4 satellite hybrid

nanoparticles. (a) Schematic and (b) TEM image of DySiO2(Fe3O4) coresatellite nanoparticles. (c)

Schematic of molecular recognition of nanoparticleantibody conjugates with PSAs. (d) MR and (e)

optical detections of neuroblastoma.74

A recent report about a core-satellite nanostructure with core being dye-doped silica

nanoparticles (fluorescent) and satellites being Fe3O4 nanoparticles (magnetic)

introduces another interesting model of multimodality probe. It was made by

cross-linking the DMSA coated IONPs and amine rich silica surface with sulfo-SMCC.

Interestingly, a remarkable increase in r2 was observed (397 mM-1 s-1 for core-satellite

particles versus 116 mM-1 s-1 for that of IONPs alone). As a demonstration, NPs were

conjugated with HmenB1 antibodies and specifically bound to polysialic acids that are

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expressed on CHP-134 cells, which was detected by both fluorescence microscope

and MRI74, 75

.

1.8. Conclusions and perspectives

In this chapter, weve gone through some important work regarding IONPs

utilizations in nanomedicine. As discussed above, the progresses in the synthesis and

modification methods have now allowed us to finely tailor almost all the aspects of

IONPs properties, such as size, size distribution, crystallinity, structure, magnetism,

physiology stability, hydrodynamic size and conjugation ability, etc. These advances

in materials have further stimulated the proceedings of IONPs bio-related

applications in diagnosis, drug delivery, gene delivery, hyperthermia, etc.

However, challenges still remain. On one hand, although we are now able to

conjugate onto IONPs different bio-species, their in vivo behaviors at this stage are

still too complex to be controlled. Most of the IONPs after administration would still

end up in liver and spleen, which hurdles their clinical applications in site specific

cancer diagnosis. And another concern is the inability of IONPs to extravsate the

blood vessels and penetrate into the interstitial space. The diffusion of particles in the

interstitial space is also rather ineffective so tumor cell targeting is very poor. This

targeting inefficiency limits most of the drug delivery and gene delivery studies at in

vitro level, which may otherwise overwhelmingly change our therapy techniques.

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Therefore, on one hand, keeping improving the IONP synthesis and modification is

necessary; on the other hand, there is urgent need in catching up the fast progress in

materials advances by putting more efforts on testing their in vivo performances.

These results might in turn direct the materials development and help screen the

optimal coating materials and biovectors. On the ether hand, the efforts of integrating

more modalities onto IONPs should be strengthened to further expand the systems

capability.

In the following chapters, I will talk about some of the related work Ive got

involved with. Chapter two will be mainly focused on IONP synthesis and quality

control. Chapter three will mainly talk about Co and Fe nanoparticle synthesis and

stabilization. In chapter four, I will introduces some of the methods weve developed

for IONP surface modification. Chapter five will talk about some specific targeting

examples, at in vitro level, based on the synthesis and modification methods discussed

in chapter two and four. In chapter six, I will introduce our recent work in making

ultrasmall IONPs for specific in vivo targeting and MR imaging. In chapter seven, I

will talk about our IONPs application in DNA biochips.

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