Jian Qin 秦健11985/FULLTEXT01.pdf · 2007. 9. 18. · Postal address Materials Chemistry Division Department of Materials Science and Engineering Royal Institute of Technology Brinellvägen

Nanoparticles for Multifunctional Drug Delivery Systems

Jian Qin

秦健

Licentiate Thesis

Stockholm 2007

Materials Chemistry Division

Department of Materials Science and Engineering

Royal Institute of Technology

Postal address Materials Chemistry Division

Department of Materials Science

and Engineering

Royal Institute of Technology

Brinellvägen 23, 2tr

SE 100 44, Stockholm, Sweden

Phone: +46-8-7908148

Fax: +46-8-7909072

Supervisor

Mamoun Muhammed, Prof.

E-mail: [email protected]

Phone: +46-8-7908158

Tutor

Maria Mikhaylova Dr.

E-mail:[email protected]

ISBN 978-91-7178-659-3 ISRN KTH/MSE--07/21--SE+CHEM/AVH © JIAN QIN, 2007

Universitetsservice US AB, Stockholm 2007

Materials Chemistry Division, KTH, 2007 i

Abstract Multifunctional drug delivery systems incorporated with stimuli-sensitive drug release,

magnetic nanoparticles and magnetic resonance (MR) T2 contrast agents is attracting

increasing attention recently. In this thesis, works on polymer nanospheres response to

temperature change, superparamagnetic iron oxide nanoparticles (SPION)/polymeric

composite materials for MR imaging contrast agents are summarized.

A “shell-in-shell” polymeric structure has been constructed through a “modified

double-emulsion method”. Thermosensitive inner shell is comprised of poly(N-

isopropylacrylamide) which undergoes phase transition at body temperature. Such a

feature could facilitate drug release at an elevated temperature upon administration.

Furthermore, the dual-shell structure is covered by a layer of gold nanoparticles.

According to the cytotoxicity tests, the biocompatibility is shown to be enhanced due to

the layer of gold.

SPION have been prepared using a high temperature decomposition method. Particle

growth of SPION is monitored by transmission electron microscope and synchrotron X-

ray diffraction. Poly(L,L-lactide)@SPION (PLLA@SPION) composite particles have

been prepared through surface-initiated ring-opening polymerization which has been

developed in our lab. For biomedical applications, it is essential to transfer the particles to

physiological solutions from organic solutions. Phase transfer of SPION has been carried

out by utilizing small molecules. Stability at the neutral pH is of large concern for such

transfer systems. A novel phase transfer agent, Pluronic F127 (PF127), a triblock

copolymer has been applied and the stability of the aqueous PF127@oleic acid

(OA)@SPION solution has been greatly enhanced over a broad pH range. Most

interestingly, PF127@OA@SPION show remarkable efficacy as T2 contrast agents as

indicated by relaxometric measurements compared with commercially available products.

Keywords: drug delivery, stimuli-sensitive, SPION, PLLA, PNIPAAm, gold, MRI,

cytotoxicity, Pluronic, phase transfer

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Materials Chemistry Division, KTH, 2007 iii

LIST OF PAPERS This thesis is based on following publications: 1. Qin, Jian; Jo, Yun Suk; Ihm, Jong Eun; Kim, Do Kyung; Muhammed, Mamoun

"Thermosensitive Nanospheres with a Gold Layer Revealed as Low-Cytotoxic Drug Vehicles" Langmuir 2005, 21, 9346-9351

2. Qin, Jian; Laurent, Sophie; Jo, Yun Suk; Roch, Alain; Mikhaylova, Maria; Muller,

Robert N.; Muhammed, Mamoun "A High-Performance Magnetic Resonance Imaging T2 Contrast Agent", accepted by Advanced Materials

3. Vallhov, Helen; Qin, Jian; Johansson, Sara M.; Ahlborg, Niklas; Muhammed,

Mamoun A.; Scheynius, Annika; Gabrielsson, Susanne "The Importance of an Endotoxin-Free Environment during the Production of Nanoparticles Used in Medical Applications" Nano Letters 2006, 6, 1682-1686

Other work not included: 1. Qin, Jian; Nogues, Josep; Mikhaylova, Maria; Roig, Anna; Muñoz, Juan S.;

Muhammed, Mamoun "Differences in the Magnetic Properties of Co, Fe, and Ni 250-300 nm Wide Nanowires Electrodeposited in Amorphous Anodized Alumina Templates" Chemistry of Materials 2005, 17, 1829-1834

Conference presentations 1. Jo, Yun Suk; Kim, Do Kyung; Qin, Jian; Muhammed, Mamoun "Temperature-Triggered

Release of Bovine Serum Albumin from Dual-Shell Drug Delivery Systems (oral)" 7th International Conference on Nanostructured Materials, 20-24 June 2004, Wiesbaden/Germany

2. Gras, Raphäel; Qin, Jian; Mikhaylova, Maria; Muhammed, Mamoun "Fabrication and

Assembly of Metal Nanowires (poster)" 7th International Conference on Nanostructured Materials, 20-24 June 2004, Wiesbaden/Germany

3. Qin, Jian; Jo, Yun Suk; Ihm, Jong Eun; Kim, Do Kyung; Muhammed, Mamoun

"Environmentally Responsive Drug Delivery System (speaker)" Medicinteknik dagarna, Sep 27-28, 2005, Södertälje/Sweden

4. Qin, Jian; Jo, Yun Suk; Ihm, Jong Eun; Kim, Do Kyung; Muhammed, Mamoun "Thermally

Responsive Low-Cytotoxic Drug Delivery System (poster)" Life is Matter – Life Matters: Conversations in Bionanotechnology, Oct 25-27, 2005, Göteborg/Sweden

iv

5. Vallhov, Helen; Qin, Jian; Muhammed, Mamoun; Gabrielsson, Susanne; Scheynius, Annika

"Gold Nanoparticles as Potential Medical Carriers-The Effect on Human Dendritic Cells (poster)" Life is Matter – Life Matters: Conversations in Bionanotechnology, Oct 25-27, 2005, Göteborg/Sweden

6. Qin, Jian; Jo, Yun Suk; Ihm, Jong Eun; Kim, Do Kyung; Muhammed, Mamoun "Thermally

Responsive Low-Cytotoxic Drug Delivery System (poster)" BioNanoMaT, Bioinspired Nanomaterials for Medicine and Technologies, Nov 23-24, 2005, Marl/Germany

7. Vallhov, Helen; Qin, Jian; Johansson, Sara; Muhammed, Mamoun; Gabrielsson, Susanne;

Scheynius, Annika "The Effect of Gold Nanoparticles on Dendritic Cells (poster)" NSTI Nanotech 2006 9th Annual, May 7-11, 2006, Boston/US

8. Qin, Jian; Laurent, Sophie; Roch, Alain; Muller, Robert N.; Muhammed, Mamoun

"Superparamagnetic Iron Oxide Micelles Revealed as High-Performance T2 Contrast Agents (oral)" 10th Special Topic Conference on Molecular Imaging, Magnetic Resonance and Intermodality Contrast Agent Research, June 14-16, 2006, Vilnius/Lithuania

9. Qin, Jian; Laurent, Sophie; Roch, Alain; Muller, Robert; Muhammed, Mamoun

"Fe2O3@Poly(L,L-lactide) Core-Shell Nanoparticles Synthesized by Surface-Initiated In Situ Polymerization (poster)" 6th International Conference on the Scientif and Clinical Applications of Magnetic Carriers, May 17-20, 2006, Krems/Austria

10. Fornara, Andrea; Qin, Jian; Muhammed, Mamoun "PLLA-PEG Nanospheres Encapsulating

Monodisperse Iron Oxide Nanoparticles for Simultaneous Drug Delivery and MRI Visualization (poster)" 6th International Conference on the Scientif and Clinical Applications of Magnetic Carriers, May 17-20, 2006, Krems/Austria

11. Qin, Jian; Laurent, Sophie; Muller, Robert N.; Muhammed, Mamoun "A Facile Approach to

Prepare Water-Soluble Monodisperse Superparamagnetic Iron Oxide Nanoparticles (poster)" 8th International Conference on Nanostructured Materials, Aug 20-25, 2006, Bangalore/India

12. Fornara, Andrea; Qin, Jian; Muhammed, Mamoun "3-G Multifunctional Nanoparticles for

Simultaneous Drug Delivery and MRI Visualization (poster)" 8th International Conference on Nanostructured Materials, Aug 20-25, 2006, Bangalore/India

13. Marian, Carmen M.; Fornara, Andrea; Qin, Jian; Muhammed, Mamoun "Optimized

Magnetic Nanoparticle for High Resolution Contrast in MRI (poster)" 8th International Conference on Nanostructured Materials, Aug 20-25, 2006, Bangalore/India

Materials Chemistry Division, KTH, 2007 v

Contributions of the author Paper 1. Performing experiments, characterization of the samples, evaluation of the

results and writing main parts of the article.

Paper 2. Planning of experiments, performing experiments, characterization of the

samples, evaluation of the results and writing.

Paper 3. Participate in initiating the idea, preparing the particles with LPS, developing

the method of preparing particles without LPS contamination, writing part of the

manuscript.

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Materials Chemistry Division, KTH, 2007 vii

Abbreviations and symbols

1H NMR proton nuclear magnetic resonance 13C NMR carbon 13 nuclear magnetic resonance AAS atomic absorption spectroscopy AOT aerosol OT, sodium dioctylsulphosuccinate APTMS 3-aminopropyltrimethoxysilane ATRP atom transfer radical polymerization BCA bicinchoninic acid BPO benzoyl peroxide BSA bovine serum albumin CMC critical micelle concentration [mol/L] Cos-7 African green monkey kidney cell line CT computed tomography CTAB cetyltrimethylammonium bromide Dc superparamagnetic critical size [m] DEM double-emulsion method DMEM Dulbecco's Modified Eagle's Medium DMSA meso-2,3-dimercaptosuccinic acid DLS dynamic light scattering DSC differential scanning calorimetry EA anisotropy energy [eV] EO ethylene oxide FBS fetal bovine serum FC field cooling FT-IR Fourier transform infrared GPC gel permeation chromatography H magnetic field [T] HeLa human cervical epithelial carcinoma cell line HLB hydrophilic-lipophilic balance HRTEM high-resolution transmission electron microscopy kB Boltzmann constant [m2kgs-2K-1]LA L-lactic acid LBL layer-by-layer LCST lower critical solution temperature M magnetization [Am2kg-1] MDEM modified double-emulsion method MEM-α modified eagle's medium alpha MRI magnetic resonance imaging MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfop henyl)-2H-tetrazolium

Mw molecular weight [Dalton] NP nanoparticle

viii

NIPAAm N-isopropylacrylamide NIR near infrared o/w oil-in-water OA oleic acid PALA N-(phosphonacetyl)-L-aspartate PBS phosphate buffer saline PDLA poly(D,D-lactide) PEG poly(ethylene glycol) PEO poly(ethylene oxide) PF127 Pluronic F127 PLLA poly(L,L-lactide) PNIPAAm poly(N-isopropylacrylamide) PO propylene oxide PPO poly(propylene oxide) PS polystyrene PtBA poly(tert-butyl acrylate) PVA poly(vinyl alcohol) PVP polyvinylpyrrolidone RES reticuloendothelial system ROP ring-opening polymerization SDS sodium dodecylsulphate SEC size exclusion chromatography SIROP surface-initiated ring-opening polymerization SPIO superparamagnetic iron oxide SPION superparamagnetic iron oxide nanoparticle SQUID superconducting quantum interference device Tc crystalline temperature [°C] TCCV two-color cell fluorescence viability THF tetrahydrofuran TEM transmission electron microscopy Tg glass transition temperature [°C] TGA thermogravimetric analysis Tm melting temperature [°C] TMAOH tetramethylammonium hydroxide TMA-POSS trimethylammonium-polyhedral oligomeric silsesquioxanes TOP trioctylphosphine TOPO trioctylphosphine oxide TSC trisodium citrate UV-Vis ultraviolet-visible VSM vibrating sample magnetometer w/o/w water-in-oil-in-water XRD X-ray diffraction ZFC zero-field cooling σ standard deviation τ relaxation time of magnetic particle [s] χm magnetic susceptibility [cm3g-1]

Materials Chemistry Division, KTH, 2007 ix

Table of contents

ABSTRACT ................................................................................................................................................... I

LIST OF PAPERS......................................................................................................................................III

ABBREVIATIONS AND SYMBOLS......................................................................................................VII

TABLE OF CONTENTS ........................................................................................................................... IX

1 INTRODUCTION................................................................................................................................1

1.1 OBJECTIVES .......................................................................................................................................1 1.2 OUTLINE ............................................................................................................................................2 1.3 DRUG DELIVERY SYSTEMS .................................................................................................................4

1.3.1 Polymeric spheres....................................................................................................................5 1.3.2 Liposomes ................................................................................................................................6 1.3.3 Magnetic NPs ..........................................................................................................................7 1.3.4 Gold and silica NPs .................................................................................................................7 1.3.5 Mesoporous materials .............................................................................................................8

1.4 MULTIFUNCTIONAL DDS...................................................................................................................8 1.4.1 Functionalization with targeted ligands ..................................................................................8 1.4.2 Visualization ............................................................................................................................9 1.4.3 Stimuli-responsive....................................................................................................................9

1.5 MRI APPLICATION ...........................................................................................................................11 1.5.1 Physical background .............................................................................................................11 1.5.2 Contrast agents......................................................................................................................12 1.5.3 Superparamagnetic iron oxide NPs.......................................................................................13

1.5.3.1 Superparamagnetism ....................................................................................................................... 13 1.5.3.2 Preparation methods of magnetic NPs............................................................................................. 14 1.5.3.3 Surface modification of SPION....................................................................................................... 17

1.5.3.3.1 Phase transfer........................................................................................................................ 17 1.5.3.3.2 Pluronic copolymers ............................................................................................................. 18 1.5.3.3.3 Biocompatible surface .......................................................................................................... 19

2 EXPERIMENTAL.............................................................................................................................21

2.1 THERMOSENSITIVE DRUG CARRIERS.................................................................................................21 2.1.1 Synthesis of PNIPAAm-PDLA and PLLA-PEG copolymers..................................................21 2.1.2 Fabrication of PLLA-PEG@PNIPAAm-PDLA and Au@PLLA-PEG@PNIPAAm-PDLA nanospheres .........................................................................................................................................22

2.2 MAGNETIC NPS ...............................................................................................................................22 2.2.1 Preparation of SPION ...........................................................................................................22 2.2.2 Evolution study of SPION in a hot organic solution..............................................................22 2.2.3 Surface modification of SPION..............................................................................................23

2.2.3.1 Preparation of PLLA@SPION ........................................................................................................ 23 2.2.3.2 Exchange with OA .......................................................................................................................... 23 2.2.3.3 Coating with PF127......................................................................................................................... 24

2.3 CHARACTERIZATIONS ......................................................................................................................24 2.4 DRUG RELEASE ................................................................................................................................25 2.5 CYTOTOXICITY TESTS ......................................................................................................................25

2.5.1 Thermosensitive DDS ............................................................................................................25 2.5.2 POA@SPION ........................................................................................................................26

3 RESULTS AND DISCUSSIONS ......................................................................................................27

3.1 THERMOSENSITIVE DDS..................................................................................................................27 3.1.1 Microscopic studies ...............................................................................................................27 3.1.2 Determination of LCST..........................................................................................................28

x

3.1.3 Drug release ..........................................................................................................................30 3.1.4 Cytotoxicity evaluation of polymeric nanospheres ................................................................31

3.2 MAGNETIC NPS ...............................................................................................................................33 3.2.1 Morphology and structure studies .........................................................................................33 3.2.2 Evolution of SPION ...............................................................................................................36 3.2.3 Surface modification of SPION..............................................................................................37

3.2.3.1 PLLA@SPION................................................................................................................................ 37 3.2.3.1.1 Microscopic observation....................................................................................................... 37 3.2.3.1.2 Characterization of surface coatings ..................................................................................... 39 3.2.3.1.3 Magnetic properties .............................................................................................................. 41

3.2.3.2 Phase transfer of SPION.................................................................................................................. 41 3.2.3.2.1 Microscopic observation....................................................................................................... 41 3.2.3.2.2 Magnetic properties .............................................................................................................. 44 3.2.3.2.3 Cytotoxicity test of POA@SPION........................................................................................ 45

3.3 MR STUDIES OF POA@SPION........................................................................................................46

4 CONCLUSIONS ................................................................................................................................50

ACKNOWLEDGEMENTS ........................................................................................................................52

REFERENCES ............................................................................................................................................53

Materials Chemistry Division, KTH, 2007 1

1 Introduction Nanotechnology is a broad and interdisciplinary area of research that has been growing

explosively worldwide in the past few years. Manufacturing nanomaterials such as clusters,

nanoparticles, nanorods, nanowires, nanotubes and thin films is the key component for

successful development of nanotechnology due to their unusual and extraordinary physical

and chemical properties result from the nanosize effect.1 Fueled by flourish development on

preparation methodologies of nanomaterials, a number of applications in biomedical field

have been proposed and some of them are close enough to successful development such as

DNA sensors, controlled drug delivery, tumor therapy etc.2 Nanotechnology has

demonstrated a tremendous potential in improving both biomedical research and clinical

applications because nanoobjects are generally in the similar size range with biological

entities, e.g. cells, organelles, DNA, proteins and so on. Benefiting from various properties

of nanostructures, one can greatly increase the efficiency and accuracy of medical diagnosis,

monitoring and therapy at the level of single molecules or molecular assemblies.

1.1 Objectives In this thesis, we are aiming to develop NPs used for multifunctional DDS associated

with the imaging functionality.

Biodegradable and biocompatible PLLA is employed as the main frame to construct the

drug carriers. Thermosensitive PNIPAAm is used as a trigger to achieve temperature-

programmed drug release due to their unique phase transition behavior upon temperature

changes. Gold NPs are used to increase the biocompatibility of drug carriers.

SPION are prepared through high temperature decomposition methods and developed as

markers for MRI after transferred to the aqueous solution. We have been seeking different

ways to transfer the NPs from organic solution to aqueous solution. The obtained solution

should be stable over broad pH range and surface coating materials should be non-toxic for

future in vivo administration. MR relaxometric analysis has shown that they are high-

performance T2 contrast agents.

2

1.2 Outline This thesis deals with development of multifunctional drug delivery system

incorporating stimuli-sensitive drug release, magnetic drug targeting and MRI contrast

agent.

Chapter 1.3 briefly introduces basic concepts on drug delivery systems in terms of

different categories e.g. biodegradable polymeric spheres, liposomes, magnetic NPs,

gold/silica NPs, and mesoporous materials. Chapter 1.4 introduced multifunctional DDS

including three categories which are targeting, visualization and stimuli-responsive.

Chapter 1.5 gives a brief introduction on the magnetic resonance phenomena together with

the major basics on the proton relaxation and contrast agents for MRI. Brief concepts of

superparamagnetic NPs are introduced from physical background to chemical preparation.

The preparation methodologies of magnetic NPs have been sketched in terms of

precipitation, microemulsion, sol-gel and pyrolysis of organometallic compounds. For

particles synthesized in organic solvent through pyrolysis, phase transfer from organic to

water solution are required especially for biomedical applications and different approached

reported by other research groups are overviewed. As a new phase transfer agent, pluronic

copolymers and their ability to solubilize hydrophobic substances in the micelles are

presented. Various kinds of biocompatible surface for safe administration of DDS are also

introduced.

Chapter 2.1 starts with the preparation of a novel temperature-responsive DDS.

Amphiphilic copolymers PLLA-PEG, PNIPAAm-PDLA, and Au NPs have been used to

engineer the DDS. A dual-shell structure has been constructed by an MDEM developed in

our group. In Chapter 2.2, as a state-of-the-art method of producing monodisperse and

highly crystalline SPION, the decomposition of the fatty acid salt is carried out. To further

understand the nucleation and growth of the particles, the growing process has been

monitored both with TEM and synchrotron XRD. To modify the surface of SPION, a layer

of L-lactic acid is used to replace the surface capping molecules of as-synthesized SPION

and serves as an active site for subsequent polymerization of L,L-lactide. Phase transfer of

SPION from organic to aqueous phase has been performed. Conventional methods using

ligand exchange with TMAOH, TSC, HNO3 and DMSA are presented first. PF127

copolymers, as a new candidate to couple with surface capping molecules, are used to


transfer SPION to aqueous phase. Followed by various means of characterizations,

performances such as drug release, MR relaxivities and cytotoxicity tests are carried out.

In Chapter 3.1, the LCST of thermally responsive nanospheres is manipulated to the

human body temperature (36.7°C) which has been confirmed by UV-Vis spectroscopy and

DSC. BSA is entrapped in the structure as a model drug and the thermosensitive release

behavior is monitored. Interestingly, cytotoxicity of such polymeric DDS has been

decreased after coating a layer of Au NPs onto the surface of polymeric spheres as

evidenced by tests on both HeLa and Cos-7 cell lines. In Chapter 3.2, monodisperse

SPION have been studied by XRD, TEM and HRTEM. The fine control of the particle

diameter is studied. A general method of controlling the size of SPION synthesized through

high temperature decomposition method is thus proposed based on the TEM and XRD

results. In the next section, surface of as-synthesized SPION are modified with PLLA for

further use as drug carriers, and the obtained particles have been characterized with TEM,

TGA and FTIR to confirm the coating structure. The PLLA layer has been separated from

the composite materials by dissolving the SPION with HCl and characterized by SEC, DSC, 1H NMR and 13C NMR. Results of phase transfer of SPION from organic to aqueous with

assistance of small molecules are presented. PF127 is used to form a stable aqueous

solution of SPION over a broad range of pH. The surface coating was characterized with

FTIR, TGA and DSC to confirm the presence of PF127 layer. The magnetic properties

before and after the phase transfer are not altered much upon the transfer. The obtained

particles show no significant cytotoxicity seen from MTS and MTT assays after incubated

with HeLa and MCF 12A cell lines, indicating that they are potentially suitable for both in

vitro and in vivo biomedical applications. Chapter 3.3 reveals that particles coated with

PF127 show incredible performance as a T2-weighed contrast agent. The ratios of

relaxivities (r2/r1) are much higher than those of commercially available T2-weighed

contrast agents. Mechanism of such enhancement are proposed and verified by comparing

with TMAOH and DMSA coated SPION.

4

1.3 Drug delivery systems

One of the most focused areas in the engineering of medical devices is DDS prepared

through the combination of biodegradable and biocompatible materials. DDS can be

specifically designed to control the release rate and/or amount of therapeutic agents that

will allow desired effects on target sites. Controlled drug release can be achieved by

combination of carrier materials and active agents. The carrier matrices are usually formed

by biocompatible materials such as solid lipid NPs,4-6 inorganic materials7, 8 and spheres

fabricated from biodegradable polymers.9, 10 Specifically designed DDS can effectively

reduce the chance of both underdosing and overdosing, thus provide better use of the active

agents. It can also alleviate disadvantages from allergy of some patients.11

In drug release, most important function of drug carriers is to regulate the release rate of

the drug. Reducing the frequency of drug release enables the patients to comply with

dosing instructions. Conventional dosage forms often lead to wide swings in serum-drug

concentration. Most of the drug contents tend to be released rapidly after the administration,

which may cause rapid increase of the drug concentration in the body. The vibration of

administered drug might cause an alternating period of ineffectiveness and toxicity (Figure

1.1). For instance, the controlled DDS can eliminate the systemic toxicity with a relatively

high concentration of drug only at the tumor site, when using anticancer drugs.

Controlled DDS is generally the diffusion- and dissolution-based release system

applicable to the release of drugs intended for the circulation or the localization on the

site.12 Diffusion can be defined as a mass transfer process of the individual molecules of a

substance, brought about by random molecular motion and associated with a concentration

Figure 1.1 Controlled drug delivery versus immediate release with repeated administration3


gradient. In an individual unit, the drug is mixed with the polymeric matrix and presented

either in dissolved or dispersed form. The release model follows Ficks Laws of diffusion

from the devices where the drug is involved. When the drug is dispersed in the matrix, it is

released according to square root of time kinetics until the concentration in the matrix

decreases below the saturation value, respectively.12 The desired releasing profiles of the

drugs in bulk degrading systems can be manipulated by adjusting the Mw of the polymer,

copolymer composition, crystallinity, and characteristic of the drug, interaction between

polymer and drug and loading amount of the drug, etc.

1.3.1 Polymeric spheres

Micro/nanospheres fabricated from biodegradable polymer for DDS have become

important since such systems enable the controlled-release of drug into desired sites.

Polymeric spheres protect drugs from the physiological environment where hydrophobic

drugs are not soluble, some organs within gastrointestinal tract with severely aggressive

environment like low pH or active enzyme which facilitate hydrolysis or decomposition.

Biodegradable polymers contain active groups (amides, enamine, enol-ketone, ester,

urea and urethane) along the main chains, which can be hydrolyzed and/or oxidized,

eventually to small molecules, under biological medium. The degraded fractures of the

polymers should not be toxic acutely as well as chronically for further biomedical

applications.

The biodegradation of hydrolysable polymers proceeds in a diffuse manner, with the

amorphous regions degrading prior to the complete split of the crystalline and cross-linked

regions. Polymers containing one or more hydrolysable functional groups have been

synthesized and found to be biodegradable.

Biodegradable polymers can be classified into three categories: biopolymers (e.g.

polypeptide, protein, DNA, and RNA), as well as polyesters produced by microorganism

(e.g. poly (3-hydroxybutyrate)); polysaccharides (e.g. cellulose, starch, chitosan, and

dextran etc.); synthetic polymers (e.g. polyester, polyamide, PMMA and PECA). During

the past decades, biodegradable polymers have been widely used, especially in the

fabrication of sutures and implants to support the body’s recovery systems.13 Another rapid

development is controlled-release of drugs with diverse characteristics, such as anticancer

6

drug adriamycin,14 5-fluorouracil,15 cisplatin,16 Paclitaxel,17 indomethacin,18 peptides,19 and

proteins,20-22. Due to their biocompatible property, aliphatic polyesters draw a lot of interest

in particle formation in order to act as a drug carrier.

Polyester is a kind of condensation polymer which can be produced as a result of various

types of reactions between diacid + diol, or di(acid chloride) + diol, or from

transesterification processes. Polylactide can be synthesized from the monomer lactic acid

through esterification process (Figure 1.2), as well as from monomer lactide through ROP

process (Figure 1.3). Polyesters are widely used and several studies on drug delivery have

been done and well documented.10, 23

1.3.2 Liposomes

Liposomes are a type of small vesicles, usually 50 nm to 5 µm in diameter, composed of

lipid bilayers enclosing an aqueous compartment. Liposomes have shown great potential as

DDS. An assortment of drugs including antineoplastic drugs, antimicrobial drugs, chelating

agents, steroids, vaccines, peptides and proteins has been incorporated either in the

entrapped aqueous volume of liposomes,24, 25 or in the phospholipid bilayer.26 Due to the

high degree of biocompatibility, liposomes have been considered as a DDS for intravenous

delivery. In preclinical studies, liposomes have been used for delivery of paclitaxel and its

analogs.27-29 Liposomes can also increase cellular delivery efficiency of certain drugs such

as PALA which are poorly taken up into cells.30 For example, the potency of liposomal

delivery of PALA to human ovarian tumor cell lines was 500-fold greater than that of free

PALA.31

Figure 1.2 Esterification process

Figure 1.3 Ring opening polymerization


1.3.3 Magnetic NPs

One of the major disadvantages of most therapies is that drugs are not delivered to

pathological sites specifically. The therapeutic drugs are administered intravenously leading

to general systemic distribution, resulting in deleterious side-effects as the drug attacks

normal, healthy cells in addition to the abnormal cells. Magnetic NPs have been applied in

various kinds of clinical applications32 such as targeted drug delivery systems,33-35 contrast

agents for MRI,36-38 and hyperthermia tumor therapy.39-41 In most cases, superparamagnetic

γ-Fe2O3 (maghemite) and Fe3O4 (magnetite) NPs are employed. Fine control of the particle

size and surface chemistry are of great importance because particle size distribution would

strongly affect the magnetic performance42, 43 and chemically modified particle surface

would render the particles with desired functionalities.44-46 Drug carriers comprised of

magnetic NPs have been discovered to be driven by magnetic force to desired sites since

late 1970s.47-49 Polymeric micelles with magnetite NPs inside have been reported as

magnetically guided DDS to deliver various kinds of biomolecules such as DNA and

photodynamic therapy drugs for tumors.50, 51 In addition, magnetic particles with silica

coating have been developed.52, 53 The coating acts to shield the magnetic particle from the

surrounding environment and can also be functionalized by attaching amino groups,

carboxyl groups, biotin, carbodi-imide and other functional groups.54-56 Recently, magnetic

nanotubes have been developed for targeted drug delivery.57, 58 Magnetic entities including

paramagnetic ions (Gd3+) and superparamagnetic iron oxide NPs can act as MRI markers

in DDS.59-61 Magnetic NPs are associated with temperature-sensitive polymers and/or

luminescent substances have been prepared as multi-purpose probes.62

1.3.4 Gold and silica NPs

Many inorganic materials show low toxicity and promise for controlled drug delivery.

Most importantly, inorganic NPs may possess and/or carry a reagent with unique optical,

electrical or magnetic properties which make it possible to monitor the drug carriers with

different imaging modalities and direct the migration of drug carriers. Gold NPs are

emerging recently as leading candidates in the field of tumor therapy. CYT-6091 drug is

covalently linking to the surface of colloidal gold through thiol-derivatized PEG.63 Gold

nanoshells which can be heated up by NIR have been used for thermal therapy of solid

8

tumors.64 Silica is also widely used in many biomedical applications due to its relatively

benign biocompatibility and easiness to chemically modify. More specifically, amorphous

silica particles are nontoxic and used as food additives and components of vitamin

supplements. DNA delivery has been achieved by modifying the surface of silica with

transfection reagents such as amino silanes.65 Combined with block copolymers, silica

nanoshells have fabricated and suggested for delivery drugs and biomarkers.53 Recently,

silica NPs coated with proteins,66 DNA strands,67, 68 or antigens69 have been reported for

binding of specific cells or receptors in the body.

1.3.5 Mesoporous materials

Recently mesoporous materials have been suggested for applications such as drug

delivery and tissue engineering.70 Different ceramic porous matrice such as calcium

phosphate, bioactive glasses, zeolites and so on have been explored as drug carriers.

Mesoporous silica with well-ordered pore distribution facilitates the homogeneity of

absorption and release of drugs. It has not come into practice until ibuprofen was loaded in

mesoporous silica matrix for the first time and drug release was investigated.71 SBA-15,

MCM-41 and MCM-48 are the three main kinds of porous silica materials. So far, several

reviews have featured the recent progress in this field.72-75

1.4 Multifunctional DDS

1.4.1 Functionalization with targeted ligands

By incorporating different components and surface modification, DDS are turned into be

passive and/or active targeting to the required pathological sites. A variety of methods have

been developed to attach corresponding vectors such as antibodies,76, 77 peptides,59, 78 sugar

moieties,79-81 folate,82-84 and other ligands85, 86 to the carrier surface. Modification with

specific antibodies has been applied to NPs and liposomes. The routine method to attach

antibodies includes protein covalent binding to the reactive groups on the nanocarriers.

Drug carriers with saccharides moieties on the surface can target cells with selection and

lectin receptors, for example, cerebroendothelial cells. Folate-modified nanocarriers are

used to target tumors because folate receptor expression is frequently overexpressed in

many tumor cells.87, 88 liposomal drug carriers,83 microgels,89 iron oxide NPs,90 quantum


dots91 and polymer-DNA hybrid NPs92 with folate tagging on the surface are reported to

selectively target cancer cells.

1.4.2 Visualization

It is of great interest to achieve real-time tracking of accumulation of drug carriers inside

the target by combining various imaging modalities (γ-scintigraphy, MRI, CT, ultra-

sonography) with DDS. MRI is superior due to its high resolution for soft tissue and non-

invasive so that it is suitable for in vivo applications. Therefore, there have been a lot of

studies on the development of MRI-detectable DDS. Polymeric micelles encapsulating iron

oxide NPs are fabricated as MRI-ultrasensitive markers.59 Mesoporous silica coated iron

oxide are synthesized for multifunctional DDS incorporating luminescence and MRI.

Fluorescent molecules and drugs can be encapsulated in the mesoporous silica.52 Putting

together MRI contrast agents, photodynamic sensitizers and targeting ligands, a

multifunctional nanoparticle platform for in vivo MRI and photodynamic therapy of brain

cancer has been constructed.60

1.4.3 Stimuli-responsive

For controlled delivery, it is necessary to make drug carriers sensitive to local

physiological stimuli such as pathology-associated changes in local pH, temperature, and/or

chemical environments, and external triggers such as electrical, magnetic and optical

stimuli.

Polymeric components with pH-sensitive bonds are used to produce stimuli-responsive

DDS that are usually stable at the physiological pH, however, undergo degradation upon

exposure to lowered pH in pathological sites such as tumors, infarcts, inflammation zones

or cell cytoplasm or endosomes.93-95 Various kinds of PEGylated liposomes have been

prepared for pH-sensitive drug delivery.96-99 Polymeric micelles having acid-labile bonds in

the structure have also been reported as pH-sensitive DDS.100-102 Additional, receptors such

as folate and biotin conjugated to the surface of pH-sensitive drug carriers facilitates the

specific binding to the tumor cells.82, 103

In the past two decades, much attention has been attracted by PNIPAAm due to its

structural changes at a certain temperature corresponding to the LCST.104-106 PNIPAAm has

10

this property mainly because of the dual characters in the structure that contains both a

hydrophobic isopropyl and a hydrophilic amino group. This intrinsic phase change along

with temperature variation is used in copolymers which form spheres for temperature-

sensitive DDS. PNIPAAm undergoes the sharp and reversible phase transition at LCST.104,

107-118 Up to now, a wide variety of medical implements made from PNIPAAm has been

proposed in different forms, e.g micelles,108, 109 NPs,110 hydrogel,111 and tablets.112 Not only

PNIPAAm homopolymer itself, but the application has also been expanded to other

PNIPAAm-derivative materials in combination with organic/inorganic counterparts such as

PNIPAAm-poly(butylmethacrylate),108 PNIPAAm-poly(D,L-lactide),109 PNIPAAm-

polystyrene,104 and Ag-PNIPAAm-polystyrene.119

Chemical environment can be also utilized as a trigger for controlled drug release. For

example, microgel responsive to the concentration of glucose has been developed for

glucose-sensitive DDS.120 Hydrogel based on poly(aspartic acid) and poly(acrylic acid) are

demonstrated to be able to swell and shrink due to the change of salt concentration.121 It

was shown that magnetic stimulation intrinsically enhances dopamine release from nucleus

accumbens shell of morphine-sensitized rats during abstinence.122 As an artificial intelligent

delivery matrix, ferrogels triggered by DC magnetic field which show an “on-off” manner

drug release has been reported.123 More recently, hydrogel constructed with thermosensitive

polymer and magnetic NPs has been prepared and such system is of great promise in drug

delivery applications due to the dual stimuli-sensitivity.124 Examples of other stimuli-

sensitive DDS such as ultrasonic125 and electric126 are not so many in the literatures so far,

though we should always pay attention to the exotic triggers which can be envisioned to be

used safely and simply in DDS.


1.5 MRI application

1.5.1 Physical background

When a system has two discrete energy levels, the protons in such a system will be

found to be in the high or the low energy levels according to a defined probability, which is

Boltzmann distribution:

exp( )l

u

N EN kT

∆= (2.1)

where ∆E is the energy difference between the two levels; k is the Boltzmann constant and

T is the absolute temperature. Once a strong magnetic field B0 is applied to this system, the

energy difference ∆E will increase proportionally to B0, therefore the difference of the

protons in the lower and upper levels of the energy will be increased. Since the intensity of

NMR signal is directly dependent on the population difference, the NMR signal also

increases. Strong magnetic field is commonly applied in order to increase the signal-to-

noise ratio.

In the presence of external magnetic field, the spinning moment of proton not only

rotates around its own axis, but also precesses around the direction of the external magnetic

field. Actually, for protons, two cones of precession exist: one for the nuclei in the state of

low energy and another one in the opposite direction for the nuclei in the high energy state.

The frequency ω of this precessing motion is given by the Larmor equation:

ω = γ B0 (2.2)

Where ω is the Larmor frequency (unit: MHz), γ is the gyromagnetic ratio, and B0 is the

strength of the magnetic field. A resonance phenomenon will occur when an

electromagnetic wave of appropriate frequency, equal to the Larmor frequency, excites the

nuclei to the higher energy level.

A radiofrequency pulse equal to Larmor frequency is applied to transfer an equilibrium

system to the unstable state of high energy. The process which the system needs to

discharge the excess energy, returning to the equilibrium state is called relaxation. A 90o

pulse is commonly applied to excite the magnetic moment of the protons. After the

excitation, the magnetic moment no longer aligns with the external magnetic field B0, but in

the plane perpendicular to B0. During the course of relaxation, the longitudinal fraction of

12

the magnetic moment start to restore from zero and the transverse component of the

magnetic moment start to diminish to zero. The T1 relaxation time, termed as spin-lattice or

longitudinal relaxation time as well, is the time required for the system to recover to 63% of

its equilibrium value after it has been exposed to a 90o pulse.

After a spin system has been excited by an RF pulse, it initially behaves like a coherent

system which all the components of the magnetization precess in phase around the direction

of the external field. As time passes, the observed signal starts to decrease as the spins

begin to diphase. The decay in the x-y plane is faster than the decay of the magnetization

along z-axis. This additional decay of the net magnetization in the x-y plan is due to a loss

of phase coherence of the microscopic components, which partially results from the slightly

different Larmor frequencies induced by small differences in the static magnetic fields at

different locations of the samples. This process is characterized by T2, the spin-spin or

transverse relaxation.

1.5.2 Contrast agents

The imaging contrast of MR arises from the different relaxation times of different part of

the tissue. To obtain a better image with well-defined mapping, contrast agents are utilized

during the imaging procedure. The contrast agents basically decrease the T1 and/or T2

relaxation times depending on if it is a T1 or T2 contrast agent. The relaxation times can be

manipulated by the use of T1 (e.g. gadolinium and manganese chelates) and T2 (e.g. SPIO)

contrast agents, producing brighter (T1-weighed) and darker (T2-weighed) images where

they are accumulated.127, 128 Several types of SPIO have been developed for MR contrast

enhancing agents with appropriate surface coatings.129-133 The efficiency of an MRI contrast

agent is commonly assessed in terms of its relaxivities r1 and r2, which are rates of proton

relaxations and are determined according to the following equation:

1/Ti,obs = 1/Ti,d + ri[M] (i = 1, 2) (2.3)

where 1/Ti,obs is the observed solvent relaxation rate in the presence of a contrast agent,

1/Ti,d is the relaxation rate of the pure diamagnetic solvent, and [M] is the concentration of

the contrast agent.


1.5.3 Superparamagnetic iron oxide NPs

Magnetite (Fe3O4) and maghemite (γ-Fe2O3) are two important ferrimagnetic minerals

among all kinds of iron oxides. Magnetite has an inverse spinel structure.134 The unit cell is

face-centered cubic based on 32 O2- ions, and the lattice parameter is 0.839 nm. Maghemite

has a similar structure with magnetite except all or most of the Fe ions are in the trivalent

state, resulting in cation vacancies that compensate for the oxidation of Fe2+. The lattice

parameter of maghemite is 0.834 nm.135

1.5.3.1 Superparamagnetism

Magnetism describes the way of aligning magnetic moment in matter, particularly in

magnetic materials. Basic magnetic phenomena have been well explained with quantum

mechanics. Thus we are not going to discuss the detailed theory about paramagnetism,

ferromagnetism, antiferromagnetism and ferrimagnetism which have been beautifully

described elsewhere.

The dynamic single domain size is defined as the size of a single domain particle at

which the thermal energy becomes comparable or larger than the anisotropy energy.

EA = KV sin2θ (2.4)

where K is the effective uniaxial magnetocrystalline anisotropy constant per unit volume, θ

is the angle between the magnetization direction and the easy axis of the particle, and V is

the volume of the particle.

When particles become smaller than the dynamic single domain size, anisotropy energy

becomes comparable with the thermal energy kBT where kB is the Boltzmann constant.

According to Boltzmann distribution:

∫=

20

)sin())(-(exp

)sin())(-(exp )( π

θθθ

θθθ

θθd

TkE

dTk

E

df

B

B (2.5)

we get the probability f between angle θ and θθ d+ . The relation between kBT, KV and the

magnetic moment of the particle are:

kBT << KV: )(f θ is large around 0=θ . The magnetic moment is fixed along the easy

direction of magnetization.

14

kBT ≥ KV: Thermal energy, in this case, is high enough to move the magnetic moments

away from the easy axis. This phenomenon is known as superparamagnetic relaxation,

which is characterized by the relaxation time τ. τ for a particle with uniaxial anisotropy is

approximately given by the expression:

)(exp0

TkKV

Bττ = (2.6)

From expression (2.4) we see that τ strongly depends on temperature and volume of the

particle. Consequently, when the size of the ferromagnetic or ferromagnetic particles is

reduced below a critical size (Dc), the collective behavior of such particles is the same as

that of paramagnetic atoms but with a giant magnetic moment. Dc is varied through

different kinds of materials, typically in the nanosized range.

As superparamagnetic state is achieved, the magnetic NP goes through a

superparamagnetic relaxation process, in which the magnetization direction of the NPs

fluctuates instead of align the magnetic moment along a certain direction. When lowering

the temperature until a certain point, such fluctuation is restricted so that it is not large

enough to reverse the magnetization direction. This temperature is called blocking

temperature and can be determined by FC and ZFC curves obtained via SQUID

magnetometer. The temperature-dependent magnetization M(T) exhibits a turning point in

ZFC curve at the blocking temperature.

1.5.3.2 Preparation methods of magnetic NPs

Synthetic methods of magnetic NPs have been well documented so far. Three most

common approaches used to prepare magnetic NPs are chemical/physical vapor

deposition,136 mechanical attrition,137-139 and chemical routes from solution.140 In this thesis,

chemical routes are primarily emphasized and four typical approaches are introduced in a

nutshell.

Precipitation. Precipitation is the most widely used and oldest method to prepare the

NPs. Soluble metal salts are used as precursors and precipitation occurs upon anionic

counter ions (usually oxalate, carbonate and hydroxide) are added. Nanosized precipitation

is obtained as the form of insoluble metal salts that need further calcination to obtain the

corresponding oxides. High degree of agglomeration of the product is inevitable due to the


heat treatment. Magnetite and maghemite, as exceptions, can be directly prepared by

alkalizing the Fe2+/Fe3+ and Fe3+ solutions as shown by the equations below. This method is

also used in preparation of ferrites containing cobalt, zinc, manganese and so on.

Fe2+ + 2 Fe3+ + 8 OH- → Fe3O4 + 4 H2O (2.7)

The advantage of the precipitation method is that large quantity of the NPs can be

produced per batch, though difficulties in controlling the morphology and size distribution

are encountered. Parameters such as pH, metal ion concentration, temperature and

surfactants have been played to achieve better size and morphology control.141-145 However,

rather large size distribution (σ > 30%) and roughly spherical shape can be obtained.

Massart firstly carried out the synthesis of magnetite NPs by using alkaline precipitation

technique.146 The research on magnetic NPs both on fundamental chemistry and various

applications, especially in engineering and biomedical fields, started to prosper since then.

So far this technique is still dominant in producing clinically approved iron oxide NPs.147-

150

Microemulsion. Surfactant molecules spontaneously aggregate into spherical

assemblies in solution. nm in diameter which would serve as suitable template for synthesis

of NPs.140, 151, 152 Direct micelles have the hydrophilic section of the surfactant on the

outside of the micelles while the hydrophobic parts of the surfactant appear to be on the

surface in the case of the reverse micelles. Reverse micelles are widely utilized in synthesis

of inorganic NPs. Magnetite and maghemite NPs were firstly prepared in this manner by

oxidizing Fe2+ ions within reverse micelles.153 The interior of the micelles containing

aqueous solutions of precursors can be considered as a restricted “nano-reators”. Several

kinds of surfactants have been applied for such purpose, e.g. AOT,154, 155 CTAB,156-158

SDS,159 and polyethoxylates (Tween).160, 161 The reaction of precipitation can be carried out

within reverse micelles to produce various kinds of ferrite materials such as

(Mn,Zn)Fe2O4,162 (Ni,Zn)Fe2O4,163 ZnFe2O4,164 and BaFe12O19.165 The particle size can be

easily controlled by changing the size of micelles through tuning the ratio between the

surfactant and hydrocarbon. Another advantage of microemulsion method is that the “nano-

reactors” are isolated with each other which limit the ripening process during the particle

growth. As a consequence, narrow size distribution (σ < 10%) can be achieved.

16

Sol-gel. Sol-gel method has been used to synthesize pure, stoichiometric and

monodisperse oxides NPs and nanocomposites,166-168 but it was not expanded to iron oxide

until Yamanobe et al. applied the sol-gel process to preparation of maghemite and sodium-

modified maghemite particles.169 This process was further studied by da Costa and the

coworkers.170 Sugimoto et al. invented and systematically studied the sol-gel routes to

prepare hematite particles.171 Magnetite and maghemite NPs have been derived from the

obtained hematite NPs by Sugimoto and his coworkers 10 years after his first work.172

Large size range from tens of nanometers to hundreds of nanometers and relatively narrow

size distribution can be achieved.

Pyrolysis of organometallic compounds. Organometallic compounds are usually

unstable at an elevated temperature, resulting in production of many kinds of NPs including

semiconductor quantum dots,173, 174 metallic/alloy NPs,175-178 and iron oxide/ferrite NPs.179-

181 It has been shown that polymers or organic capping agents determine the cluster size

and effectively protect the particles during the nucleation and crystal growth.182, 183

Hexadecylamine, oleylamine and oleic acid have been used in the synthesis of Co NPs by

reduction of Co(η4-C8H13)(η4-C8H12) in hydrogen. By varying the composition of ligands,

the size and shape of particles could be controlled.184 CoxPt1-x alloy has been prepared from

the organometallic compounds in the presence of PVP.185

Alivisatos et al. have shown that Co NPs can be prepared through decomposing the

cobalt carbonyl in the presence of oleic acid and TOPO.186 In these processes, oleic acid

and TOPO act as the capping agents with long alkyl chains that can stabilize the particles

during the course of nucleation and growth. Spherical maghemite NPs are synthesized

when the iron cupferron complex is rapidly injected into trioctylamine at 300 °C.187

Maghemite NPs can be synthesized by oxidizing the Fe NPs formed via decomposition of

iron pentacarbonyl or through one-step decomposition of iron pentacarbonyl in the

presence of mild oxidant.179 It was hypothesized that the iron oleate complex was the

critical intermediate to form the seeds and subsequently grow into the NPs. Peng et al.

firstly carried out the practical synthesis of iron oxide NPs by choosing iron oleate, stereate,

and laureate as precursors.188 Encouraged by this postulation, Hyeon et al. improved the

procedure and inventively suggested a large-scale synthetic route for iron oxide NPs by

pyrolysis of iron oleate complex.181 Iron oxide particles with very narrow size distribution


(σ < 5%) and high crystallinity can be obtained via pyrolysis routes. By altering the ratio

between capping molecules and precursors, size of particles can be readily controlled.

1.5.3.3 Surface modification of SPION

1.5.3.3.1 Phase transfer

Inorganic NPs are promising materials because of their unique size-dependent properties.

A variety of methods have been developed to synthesize NPs either in aqueous solution or

in organic solution.140 Water-based synthesis of NPs is fraught with problems of broad size

distribution and aggregation as a result of ionic interactions, which can be avoided by

lowering the concentration of precursors and employing certain surfactants.146, 189-191

However, NPs synthesized in organic phase, as discussed in the previous chapter, can be

normally controlled in a narrow size distribution, which is favorable for investigating

physical properties and certain applications such as high-density magnetic storage.

Although producing high concentration of NPs, the high temperature decomposition

method in organic solvents has a major drawback that particles are immiscible with water,

which greatly restrains the further development for biomedical applications. The problems

are that 1) the surface capping molecules have long alkyl chains which are highly

hydrophobic and definitely can not be dispersed in physiological environment; 2) such

capping molecules lack of functional groups, e.g. oleic acid, oleylamine, TOP and TOPO,

etc. which will not allow further chemical modification and/or conjugation of desired

functional molecules.

To take advantage of the high quality NPs prepared through high temperature

decomposition methods in organic phase, enormous amount of work has been done to

increase water-dispersibility of the particles, especially for bursting biomedical applications

of NPs. For example, CdSe, CdSe/CdS core/shell quantum dots that were synthesized via a

typical organic-based procedure, covered by TOP and oleylamine as stabilizing agents,

were transferred to aqueous solution by incorporation of surfactants and phospholipids with

capping molecules. The optical properties of quantum dots were retained and used for cell

labeling. Ag and iron oxide NPs were “pulled” to water by forming a host-guest structure

with α-cyclodextrin.192 Alternatively, surface capping molecules such as oleic acid and

18

oleylamine can be incorporated with amphiphilic surfactant/lipid and poly(maleic

anhydride alt-1-tetradecene) with their hydrophobic domain and the hydrophilic domain on

the other side of the molecules solubilize NPs in aqueous solution.193 Ligand exchange with

TMAOH,194 DMSA,195 TSC,196 PEG-terminiated dendron,197 phosphine oxide polymer and

TMA-POSS molecules198 could also achieve successful phase transfer of NPs.

1.5.3.3.2 Pluronic copolymers

Pluronic copolymers (termed “Poloxamer” or “Synperonic”) are a kind of ABA-type

triblock-copolymer consisting of EO and PO blocks in a structure: EOx-POy-EOx. Such a

structure results in an amphiphilic copolymer, in which the number of hydrophilic EO and

hydrophobic PO units can be altered to vary the size, hydrophilicity and lipophilicity of the

polymer. Pluronic copolymers with different x and y values are characterized by distinct

HLB.

The Pluronic copolymers undergo self-assembly into micelles in aqueous solutions.

These polymer molecules form dispersion in water at the concentrations below the CMC.

At concentrations of the block copolymer above the CMC, the Pluronic molecules

aggregate, forming micelles through a process called “micellization”. The PO blocks self-

assemble to form the inner core due to the hydrophobic interaction and the EO blocks form

hydrophilic corona covering the micelles. Pluronic micelles are depicted as spheres

composed of a PO core and an EO corona. It is correct for most block copolymers, which

have an EO content above 30%, especially in relative dilute solutions at body temperature.

However, additional micelle morphologies, including lamella and rods (cylinders), can also

form in Pluronic systems. The hydrodynamic diameters are in the range from about 20 to

about 80 nm for the spherical micelles depending on the type of the Pluronic polymers. The

number of block copolymers forming one micelle is referred to as the “aggregation

number”. Usually this number ranges from several to over a hundred. Table 2.1 presents a

list of selected Pluronic copolymers available from BASF Corporation, and their respective

physicochemical properties.199 Based on the core-shell structure, Pluronic micelles could

carry water-insoluble substance and this process can be referred as “solublization”.200 Such

properties have been actively studied because it is promising to use Pluronic micelles for

carrying hydrophobic drugs and polypeptides within the PO core of the micelles.201 PF127


has been selected among the list to coat SPION in this work because it has large amount of

EO units, high HLB which will maintain the particles dispersed in aqueous phase, as well

as low CMC (2.8×10-6 mol/L) which will form micellar structure with NPs encapsulated.

1.5.3.3.3 Biocompatible surface

It is crucial to control the physical and chemical properties of the drug carrier in case of

in vivo applications. Manipulated polymer NPs may be considered as biocompatible

materials, if they do not provoke an adverse reaction or hypersensitivity in the body. A

thrombus may be formed very fast when polymer contact plasma. Materials with biogenic

blood-compatible surface must be used in contact with the blood in the vessel. Therefore,

the biocompatible materials for drug delivery should be able to stay with plasma and living

cells without active interactions.

Therefore, creation of different surface functionalities on the micro/NPs is of emerging

research interest especially to enhance the biocompatibility. Polymeric coating is promising

due to the advantage of the flexibility in the controls of chemical compositions in order to

manipulate the functions of the polymers. Various approaches were reported to create a

polymeric coating on particle surfaces. LBL deposition through charge interaction between

positive and negative polyelectrolyte has been recognized as a successful method to coat

large particles (more than tens of nanometers in diameter) with polymers.202, 203 However,

direct grafting from the surfaces of NPs through the ATRP was developed as another

feasible method. Different types of NPs e.g. γ-Fe2O3,204 MnFe2O4,205 SiO2206 and Au207

were coated with various kinds of polymers such as PS,208 PNIPAAm,209 PtBA210 etc via

the ATRP. Exchange of surface capping agents with ATRP initiator is the crucial step of

the whole procedure, so that the following free radical polymerization could be carried out

on the surface of NPs. In this work, we transplant such an idea to ROP. For the first time,

poly(L,L-lactide)@SPION (PLLA@SPION) NPs have been designed and fabricated

through the SIROP. Because PLLA is recognized as a type of highly biocompatible

material with outstanding biodegradability, the PLLA@SPION NPs would be less toxic

and easy to chemically modify, for instance, with targeting molecules or therapeutic drugs.

In particular, copolymers containing hydrophilic segments can serve as surface

modification of particulates or micelles which have been considered as potential candidates

20

for DDS.211, 212 The RES plays an important role in impeding the circulation of drug

carriers around the body by recognizing and removing alien particles. Therefore, effective

drug delivery could not be achieved after directly injection. The function of RES in liver is

regulated by the presence and balance of two contrary blood components: opsonins, that

promote phagocytosis; and dysopsonins, that suppress this process. Dysopsonins prefer to

perform in a high hydrophilic milieu. Thus, modification with hydrophilic polymers can

effectively reduce the chance of uptake by macrophages. For the sake of increasing

hydrophilicity of DDS, PEG has been widely applied to modify the surface of nanocarriers

in order to avoid being captured by immune system.

Gold NPs are used to cover the surface of DDS due to the inert nature of elemental gold

which is proved to be nontoxic.213 Though recent analysis has indicated that the colloidal

gold does not cause acute cytotoxicity, it is believed that the NPs could be rapidly modified

by the surrounding cellular environment.214 This process could potentially alter the

properties of the surface of NPs and it implies that proteins would attach to the surface of

gold layer and enhance the cellular uptake which will facilitate cellular delivery of drugs.

Furthermore, the immunostimulatory effect of gold NPs on dendritic cells have been

investigated, indicating there is no significant impact on maturation of dendritic cells,

implying that they can be used as a safe component in drug carriers.


2 Experimental

2.1 Thermosensitive drug carriers

2.1.1 Synthesis of PNIPAAm-PDLA and PLLA-PEG copolymers

Synthetic pathway of diblock copolymers is depicted in Figure 2.1. Briefly, PNIPAAm

was synthesized via free radical polymerization which was initialized by BPO under

anhydrous condition. Thereafter, synthesized PNIPAAm homopolymer, D,D-lactide and

stannous 2-ethylhexanoate were mixed in anhydrous toluene and the second synthesis was

performed for 7 hours. Parallelly, PLLA-PEG copolymer was synthesized from PEG, L,L-

lactide and stannous 2-ethylhexanoate in anhydrous toluene. Upon completion of the

reaction, polymer was collected by precipitating in an excess volume of cold diethyl ether,

followed by dried in vacuo at an ambient temperature overnight.

Figure 2.1 Synthetic pathway of PNIPAAm-PDLA and PLLA-PEG block copolymers

22

2.1.2 Fabrication of PLLA-PEG@PNIPAAm-PDLA and Au@PLLA-PEG@PNIPAAm-PDLA nanospheres

An MDEM was used to prepare the w/o/w emulsion. Aqueous solution of BSA was

dropped to the chloroform containing PNIPAAm-PDLA diblock copolymer followed by

sonication. Afterwards, the obtained emulsion was mixed with PLLA-PEG chloroform

solution and added dropwise to PVA aqueous solution followed by sonication in an ice bath.

The silanization was carried out by mixing the emulsion of PLLA-PEG@PNIPAAm-

PDLA with APTMS ethanolic solution in order to functionalize the surface of the

nanospheres with amino groups. Colloidal gold NPs were synthesized by reducing [AuCl4]-

ions in the presence of reductant, NaBH4. The detailed procedure is described elsewhere.215

After the silanization, gold colloid suspension was added dropwise into the amino-modified

PLLA-PEG@PNIPAAm-PDLA emulsion.

2.2 Magnetic NPs

2.2.1 Preparation of SPION

In this work, high temperature decomposition of iron based organometallic precursors

are employed. A typical synthetic procedure to produce 12 nm iron oxide nanocrystals via

decomposition of iron oleate complex is given as an example.

Solution of ferric chloride and sodium oleate in a mixed solvent (ethanol, deionized

water and hexane) was refluxed for 4 hours. The waxy Fe oleate complex was separated

and dissolved in dioctyl ether in the presence of oleic acid, heated to reflux for 1.5 hours,

and SPION were precipitated by ethanol. Finally, the SPION were dispersed in hexane in

the presence of 100 µL oleic acid and stored at 4°C for further use and characterizations.

2.2.2 Evolution study of SPION in a hot organic solution

In a typical study, 3 g (3.34 mmol) of iron oleate complex was dissolved in 20 mL

dioctyl ether in the presence of 0.314 g (1.11 mmol) oleic acid. The reacting mixture was

heated up to 295°C at a constant heating rate of 5.5°C min-1. The first 0.5 mL sample was

withdrawn at the temperature of 220°C and the following aliquots were periodically

withdrawn from the solution. The as-prepared SPION dispersed in dioctyl ether were filled

in capillaries for XRD without purification.


2.2.3 Surface modification of SPION

2.2.3.1 Preparation of PLLA@SPION

Monodisperse SPION have been prepared through high temperature decomposition of

iron oleate complex as previously described. The coating procedure is schematically

illustrated in Figure 2.2. Briefly, L-lactic acid molecules are successfully grafted onto the

surface of SPION through carboxylic groups, hereafter abbreviated as LA@SPION. The

appending hydroxyl groups in L-lactic acid molecules serve as initiating sites for further

polymerization. Catalyzed by stannous octoate, ROP of L,L-lactide is carried out in the

presence of LA@SPION NPs. Upon completion, the poly(L,L-lactide)@SPION

(PLLA@SPION) NPs can be easily collected by centrifugation after washed with

chloroform.

2.2.3.2 Exchange with OA

The OA coated SPION prepared according to the method as described earlier was used

in phase transfer experiments. Four kinds of small molecules TMAOH, TSC, HNO3 and

DMSA are tested for ligand exchange with oleic acid.

TMAOH. 10 mg powder of SPION was mixed with 2.5 mL 10 wt.-% TMAOH and

fully dispersed after brief shaking. The black dispersion was centrifuged at 14,000 rpm for

10 minutes and washed with water 5 times in order to eliminate the excess of the TMAOH.

Finally, the particles were re-dispersed in a 2.5 mL 0.01 wt.-% TMAOH solution.

TSC. To the 2.5 mL TMAOH dispersion of SPION obtained from the previous step was

Figure 2.2 Scheme of SIROP

24

added 20 mg of TSC followed by adding HCl dropwise to the solution to bring the resulting

mixture to pH 6.5. Precipitation caused by alteration of pH was removed by filtration.

HNO3. 10 mg powder of SPION was mixed with 10 wt.-% HNO3 to produce an acidic

sol. The brownish dispersion was washed with water 5 times and the precipitate was

dispersed in 2.5 mL 1 wt.-% HNO3 solution.

DMSA. To the 2.5 mL HNO3 dispersion of SPION obtained from previous step was

added 5 mg of DMSA. The mixture was shaken vigorously for 2 hours and resulting in a

stable brownish sol.

2.2.3.3 Coating with PF127

The PF127 molecules can associate with long alkyl chains of oleic acid---the surface

capping ligands on the NPs---by the hydrophobic PO section, whilst two hydrophilic EO

tails render the particles which were originally insoluble in water with greatly enhanced

hydrophilicity. The particles after phase transfer are denoted as Pluronic@Oleic

acid@SPION, thus abbreviated as POA@SPION thereafter.

2.3 Characterizations TEM images were taken by using JEOL JEM-2000EX. XRD patterns of SPION were

recorded by a PANalytical X’Pert Pro system. The capillary XRD patterns of the particles

were measured in synchrotron (λ = 1.23 Å, MAX-lab, Lund University, Sweden). The

concentration of SPION was measured by AAS (SpectrAA-200, Varian). FT-IR spectra

were recorded on a Nicolet Avatar 360 E.S.P. spectrophotometer. TGA was measured by a

TGA Q500 system. DSC was measured by a modulated DSC 2920. ζ-potential was

measured by Zetasizer Nano ZS. The magnetization measurements were carried out at

room temperature using a VSM. 1H NMR and 13C NMR spectra were recorded on a Bruker

Avance-600 MHz and Bruker DMX 500 MHz spectrometer. The particle size distribution

was measured by DLS particlesizer. Varian Cary 100 Bio UV-Vis spectrophotometer was

used to determine the LCST and quantitate the concentration of BSA by means of BCA

assay. The Mw of PLLA was analyzed with a SEC. Relaxivities r1 (20 and 60 MHz) was

measured at 0.47 T with Minispec PC-20 and 1.41 T with Mq Series systems; r2 was

measured with Minispec (20 and 60 MHz) on spectrometer AMX-300.


2.4 Drug release In vitro release profiles from thermosensitive polymeric nanospheres were obtained as

follows. An appropriate amount of BSA-loaded Au@PLLA-PEG@PNIPAAm-PDLA was

introduced into a dialysis membrane bag and placed in 100 mL of PBS as a media

containing 0.02 wt-.% sodium azide. Two comparative experiments were simultaneously

undertaken; one at 22 °C as a control and the other at 37 °C. At the predetermined time

intervals, an aliquot of the aqueous solution was withdrawn from the media and the protein

quantity was analyzed by BCA assay.216, 217

2.5 Cytotoxicity tests

2.5.1 Thermosensitive DDS

PLLA-PEG@PNIPAAm-PDLA and Au@PLLA-PEG@PNIPAAm-PDLA were

dialyzed with cellulose membrane (Mw cut-off: 3 kDa) against water for 48 hours and then

lyophilized for 72 hours. Stock solutions were prepared at the concentration of 20 mg/mL.

Four different concentrations of the solutions were sequentially prepared by diluting the

stock solution with the appropriate amount of cell media. HeLa cells and Cos-7 cells were

purchased from American Type Culture Collection (ATCC).

Cells were prepared at a density of 5 × 104 cells per ml of medium. The prepared cells

were seeded 200 µL in each well of a 96-well microplate. The cells were incubated with

various concentrations of polymer for 48 hours. The medium was removed and replaced

with 100 µL of growth medium prior to the addition of the 20 µL/well of CellTiter 96

Aqueous One Solution Reagent based on MTS assay. The result was expressed relative to

results from untreated cells. Untreated cells were used as a positive control. The medium

without cells was used as a blank.

For TCCV assay, cells were seeded on the 6-well glass plates. Aspirating cell media

from the wells, they were filled with the working solution prepared by mixing 2 µL of 2

mM ethidium homodimer-1 solution diluted in 1 mL of PBS with 0.5 µL of calcein AM

solution. The glass plates were incubated under darkness for 30 minutes. The wells were

washed with PBS twice and filled with 1 mL of PBS per well after contained solutions

26

were aspirated. Images were taken by Axio Vert 200M fluorescence microscope (Carl Zeiss

Inc., Thornwood, NY).

2.5.2 POA@SPION

POA@SPION was lyophilized for 72 hours. Stock solutions were prepared at the

concentration of 2 mg/mL. Three different concentrations of the solutions were sequentially

prepared by diluting the stock solution with the appropriate amount of cell media. HeLa

and MCF-12A cells were purchased from American Type Culture Collection (Rockville,

MD).

Cells were prepared at a density of 5 × 104 cells per mL of medium in case of HeLa cells

and 5 × 103 cells per mL of medium for MCF-12A respectively. The prepared cells were

seeded 200 µL in each well of a 96-well microplate. Prior to the incubation with cells,

particles were diluted in cell culture medium and filtrated through 200 nm filter. The cells

were incubated with various concentrations of particles for 48 hours. Cells without any NPs

were seeded and cultivated in the same way to use as controls. The medium was removed

and replaced with 100 µL of growth medium prior to the addition of the 20 µL/well of

CellTiter 96 Aqueous One Solution Reagent (Promega) based on MTS assay for HeLa cells.

In the case of MCF-12A cells, a 10 µL of MTT dye solution was added to each well and

cells were incubated for 3 more hours. The results were expressed relative to results from

untreated cells. The medium without cells was used as a blank.

The cell viability (%) was calculated according to the following equation:

Cell viability (%) = (ODsample –ODblank) / (ODcontrol –ODblank) × 100


3 Results and discussions

3.1 Thermosensitive DDS

3.1.1 Microscopic studies

Figure 3.1 illustrates the evolution pathway in constructing Au@PLLA-

PEG@PNIPAAm-PDLA. To serve as a constituent for the vehicles to load BSA inside,

Au@PLLA-PEG@PNIPAAm-PDLA is in a form of dual-shell vesicle fabricated by

MDEM. Hydroxyl groups on the surface of PLLA-PEG@PNIPAAm-PDLA are

functionalized into amino groups and gold NPs are deposited to form Au@PLLA-

PEG@PNIPAAm-PDLA (Step I – IV). Above the LCST, it allows BSA molecules to be

released at an elevated temperature through a single outer shell ultimately, because

thermosensitive inner shells are designed to be burst and eliminated (Step V). The resulting

NPs are well-defined as shown in Figure 3.2 along with the information of size and

polydispersity index.

Figure 3.1 Evolution of Au@PLLA-PEG@PNIPAAm-PDLA: (I) Formation of the BSA-loaded PNIPAAm-PDLA nanospheres. (II) Construction of PLLA-PEG@PNIPAAm-PDLA dual-shell structure by an MDEM. (III) Functionalization of PLLA-PEG@PNIPAAm-PDLA dual-shell structure with APTMS. (IV) Immobilization of Au NPs on the surface of PLLA-PEG@PNIPAAm-PDLA. (V) Elimination of the inner shell when the temperature is above the LCST

28

Figure 3.2 (a) TEM image of PLLA-PEG@PNIPAAm-PDLA (negatively stained with 2% of ammonium molybdate aqueous solution for 2 min), (b) particle size distribution of PLLA-PEG@PNIPAAm-PDLA nanospheres, (c) TEM image of Au@PLLA-PEG@PNIPAAm-PDLA (without staining) and (d) particle size distribution of Au@PLLA-PEG@PNIPAAm-PDLA nanospheres

The size distribution of PLLA-PEG@PNIPAAm-PDLA lies in a range of 80 – 170 nm,

confirmed by DLS particlesizer. Mean diameter of PLLA-PEG@PNIPAAm-PDLA is 127

nm with 1.41 of polydispersity index. Gold layers constructed with gold NPs are clearly

seen on Au@PLLA-PEG@PNIPAAm-PDLA as dark spots in Figure 3.2c. It is noted that

particle size slightly increases up to 140 nm compared to PLLA-PEG@PNIPAAm-PDLA.

This size change results from the decreased mobility due to gold NPs’ deposition which is

reflected in calculating mean diameter by DLS particlesizer.

3.1.2 Determination of LCST

LCST of PNIPAAm-PDLA copolymer is observed at 36.7 °C from a sharp endothermic

peak in the DSC curve. (Figure 3.3). This reflects that PNIPAAm-PDLA copolymer

undergoes the phase transition at the LCST, 36.7 °C. The result is also confirmed by the

analysis by UV/Vis spectrometry. Aqueous solution of PNIPAAm-PDLA is transparent at a

lower temperature than LCST. However, as temperature increases, the solution becomes

turbid and the transmittance rate, as shown in Figure 3.4a, decreases in a sigmoid shape

owing to the predominant hydrophobicity over the hydrophilicity driven by the phase

transition. From this graph, the maximum corresponding to the LCST of PNIPAAm-PDLA

is found from the numerically differentiated curve of the transmittance rate, revealing the

LCST at 36.7 °C which is human body temperature (Figure 3.4c). It is observed that

transmittance rate of the emulsion containing Au@PLLA-PEG@PNIPAAm-PDLA also

(a)

(c)

(b)

(d)


decreases in the same fashion as temperature increases (Figure 3.4b). Whilst the

transparency of PNIPAAm-PDLA solution is reversibly regained in case the solution is

cooled down to the ambient temperature, the transmittance rate of Au@PLLA-

PEG@PNIPAAm-PDLA is not restored, suggesting that the thermosensitive inner shell of

the Au@PLLA-PEG@PNIPAAm-PDLA is irreversibly disrupted once PNIPAAm-PDLA

undergoes the phase transition by thermal stimulus above the LCST.

Figure 3.3 DSC data obtained from (a) PNIPAAm-PDLA and (b) PLLA-PEG

Figure 3.4 UV-Vis spectra at 500 nm: (a) Transparency change of the PNIPAAm-PDLA solution, (b) Transparency change of Au@PLLA-PEG@PNIPAAm-PDLA in w/o/w emulsion, and (c) A differentiated curve of (a)

30

Figure 3.5 Scheme represented burst of thermosensitive PLLA-PEG@PNIPAAm-PDLA w/o/w micelles

As schematically shown in Figure 3.5, PLLA-PEG@PNIPAAm-PDLA w/o/w micelles

demonstrate the morphology of spheres, since the shell-forming PNIPAAm shows the

character of hydrophilicity below LCST so that the inner milieu could be stabilized from

the aqueous solution outside. As soon as the temperature reaches LCST, the inner shell

comprised of PNIPAAm turns to be hydrophobic suddenly, and result in release of the

encapsulated substances.

3.1.3 Drug release

BSA release rates are monitored at two different temperatures including ambient

temperature and human body temperature. The results are demonstrated in Figure 3.6 by

plotting the released amount of BSA versus time. In Figure 3.6, two curves represent

different release rates respectively. The profile at 37 °C (Figure 3.6a) shows a quasi-linear

increase in the release rate of BSA. However the release rate at 22 °C (Figure 3.6b)

increases smoothly before 60 hours, and expedites thereafter due to induced instability of

the dual-shell structure, while the profile at 37 °C maintains a high release rate which can

be attributed to the disrupted inner shell. Therefore, the inner shell burst above the LCST

causes the main difference of the release rate, because, as temperature is elevated above the

LCST, the inner shell becomes unstable and the dual-shell structure changes into a simple

single-shell structure. In this stage, the release rate will be dominated only by various


parameters involving PLLA-PEG, the component of the outer shell. Then, the ongoing

release behavior follows the release mechanism from the single-shell structure.

3.1.4 Cytotoxicity evaluation of polymeric nanospheres

A typical graph of MTS assay results is shown in Figure 3.7, indicating that neither of

the nanospheres exerts any critical influence on HeLa cells. In addition, dead/living cells

are visualized as fluorescent microscope images taken after the TCCV assay. No

remarkable effect on the cell morphology and viability is observed. As seen in Figures 3.8,

the majority of the cells are alive, with no significant destructive effect in either case:

Au@PLLA-PEG@PNIPAAm-PDLA and PLLA-PEG@PNIPAAm-PDLA.

It is noticeable that, although cell viability of PLLA-PEG@PNIPAAm-PDLA is to some

extent lower than that of Au@PLLA-PEG@PNIPAAm-PDLA, excellent biocompatibility

is acquired. However, interestingly, Au@PLLA-PEG@PNIPAAm-PDLA exhibits

significantly enhanced cell viability even at high doses such as 1000 µg/mL or 100 µg/mL

in 48-hour incubation (p<0.05). This effect contributed by gold layer is commonly

observed in both cells. It suggests that beneficial effect of gold layer is predominantly

maximized especially at high doses.

Figure 3.6 In vitro release profiles of BSA from Au@PLLA-PEG@PNIPAAm-PDLA in PBS solution: (a) 37 °C and (b) 22 °C

32

Figure 3.7 MTS assay using HeLa cell line after incubation with (a) Au@PLLA-PEG@PNIPAAm-PDLA and (b) PLLA-PEG@PNIPAAm-PDLA for 2 days. (* denotes p<0.05 by Student's t test)

Figure 3.8 Fluorescence microscopy images of HeLa cells exposed to (a) 100 µg/mL Au@PLLA-PEG@PNIPAAm-PDLA nanospheres, (b) 1000 µg/mL PLLA-PEG@PNIPAAm-PDLA nanospheres, and (c) control, where green spots represent living cells and red ones represent dead cells.

In our study, no receptor-mediated endocytosis is proposed, and a large portion of the

nanospheres is unlikely to be internalized because no favorable size effect exists for

endocytosis. Consequently, there is no driving force to ameliorate or deteriorate cell

proliferation taking place in the cytoplasm. Nonetheless, PLLA-PEG diblock copolymer as

the outer shell component of PLLA-PEG@PNIPAAm-PDLA is presumed to reduce the

interfacial affinity between “foreign” spheres and protein molecules due to its

hydrophilicity. PEG constituent does not merely play a role to prevent the particles from

being anchored by proteins, but more importantly, it diminishes the opportunity of the

nanospheres attaching to the cell membrane which may be induced by various interactions.

In contrast, gold possesses high affinity to biomacromolecules including various

immunoglobulins, albumins, and fibrinogen residing in the in vivo condition in comparison

(a) (b)

(c)


to hydroxylated surface.218 Likewise, the exterior gold layer of Au@ PLLA-

PEG@PNIPAAm-PDLA is, at the first stage, supposedly coated with a variety of proteins

in the cell media, whereas PLLA-PEG@PNIPAAm-PDLA does not undergo the same

binding process. In the next stage, therefore, Au@PLLA-PEG@PNIPAAm-PDLA

surrounded by the proteins initiates the interaction with the surface of the cell membrane. It

is another key to explain how cell proliferation can be promoted by accelerated transport of

nutrients from media into cytoplasm. Moreover, Au@PLLA-PEG@PNIPAAm-PDLA

anchored on the cell membrane may lower the potential in transmembrane uptake and

facilitate the diffusion rate of nutrient penetrating through the cell membrane.

3.2 Magnetic NPs

3.2.1 Morphology and structure studies

The obtained particles can be readily dispersed in nonpolar solvents, e.g. hexane, toluene

and so on, forming a stable black suspension. XRD pattern with characteristic peaks of

inverse spinel structure indicates that iron oxide nanocrystals are pure ferrite phase with

high crystallinity (See appended papers). It is not possible to distinguish whether these

particles are magnetite or maghemite solely from XRD results because both of them have

the inverse spinel structure showing identical XRD patterns. Nevertheless from the

synthetic procedure, we could assume the complete transformation to maghemite because

no inert atmosphere was applied during the course of reaction. Therefore, iron oxide

species would be completely oxidized to maghemite. It was further confirmed by iron

concentration measured by AAS, which is 70.44 ± 0.67%, almost equal to the iron

concentration in maghemite, 69.94%. Since we still lack evidence to confirm the exact

phase of the obtained NPs, we would like to refer them as iron oxide other than magnetite

or maghemite.

34

Figure 3.9 (a) TEM image of SPION arranged in a 2D hexagonal array. Insets are SAED pattern and HRTEM of a single nanocrystal of SPION, (b) TEM of SPION with a larger magnification, and (c) particle size distribution. Insets in (a) are SAED and HRTEM

NPs covered with oleic acid repulse from each other due to the Van der Waals force of

the long alkyl chain of oleic acid, thus forming ordered array of particles over a short range

(~ 200 nm). The nearly spherical NPs can be clearly seen in TEM images, assembling into

hexagonal array (Figure 3.9a and b). Very narrow size distribution has been achieved which

is shown in the histogram (Figure 3.9c) with σ = 3.8%. Insets in Figure 3.9a show a SAED

pattern of SPION and rings are in a good agreement with indexed peaks in XRD pattern.

The HRTEM image (inset of Figure 3.9a) of a single particle shows distinct lattice fringe,

revealing the highly crystalline nature of the SPION.


Table 3.1 Parameters for controlling the size of SPION

Fe oleate complex (mmol)

Oleic acid (mmol)

Ratio (Fe : OA)

Average size (nm)

Standard deviation

2.23 0.78 3 : 1 11.5 3.8% 2.22 2.23 1 : 1 13.0 4.8% 2.22 4.44 1 : 2 16.0 4.8% 2.22 5.60 1 : 2.5 4.0 26% 2.22 6.70 1 : 3 - - 2.22 8.88 1 : 4 - -

The size of SPION can be manipulated by varying the molar ratio between Fe oleate

complex and oleic acid in a predetermined volume of solvent (13.75 mL). The experimental

data of size control are summarized in Table 3.1. As increasing the mole of oleic acid,

particle size is increased from 11.5 to 16.0 nm. Nevertheless, particles stop growing when

the Fe to oleic acid ratio reaches 1 : 2.5, the particle size drops down to 4.0 nm with broad

size distribution, indicating high concentration of oleic acid would suppress particle growth

process to some extent. Even no particle can be observed under TEM when the ratio of Fe

to oleic acid is increased to 1 : 3 and 1 : 4. A remarkable drop of particle size occurs at the

turning point of Fe:OA = 1 : 2.

TEM images of the samples with Fe:OA ~ 3:1, 1:1 and 1:2 are shown in Figure 3.10

where size increasing can be clearly seen. The facets of the nanocrystals have been evolved

when the particle size increases. Particles from Fe:OA ~ 3:1 can be regarded as quasi-

spherical particles while Fe:OA ~ 1:2 becomes polyhedral morphology as a result of size

increase. It was also reported elsewhere that by increasing the concentration of oleic acid,

one could obtain larger particles out of this procedure.219 Peng et al. reported the particle

size could reach and be kept at a certain value before Ostwald ripening.188 More recently,

Lin et al. discovered a reversible transformation process between the Co NPs and their

clusters by means of changing the concentration of oleic acid.220 The finding implied that

an excess of ligand molecules would work in the opposite way rather than enlarge the

particles as one expected. Such process has been applied to other particulate systems, for

instance Au221 and PbS,222 as reported as “digestive ripening” which facilitates the

dissolution of the large particles and grow the small ones in order to narrow the size

distribution. In our case, by increasing the concentration of oleic acid, maghemite NPs

36

could undergo a similar “digestive ripening” during the course of particle formation,

forming TEM-undetectable clusters.

3.2.2 Evolution of SPION

Particle growth of SPION in the dioctyl ether has been studied by TEM in the similar

manner as previously reported.188 To further confirm the TEM observation, particles in

dioctyl ether solvent without separation were filled in capillaries to check in synchrotron.

Formation of NPs are not seen when the temperature is below 290 °C after 20 minutes

heating. Although no visible particle is observed at 45 minutes, peaks with very low

intensities are seen in the corresponding synchrotron XRD patterns (Figure 3.12). Particle

size calculated through Debye-Scherrer formula (D = Kλ /Bcosθ) is rather small at this

stage (Table 3.2). After this initial stage, quasi-cube-shape was observed at 60 minutes with

rather large particle size (Figure 3.11a). The particles gradually changed to nearly

monodisperse spherical shape at 90 minutes at the same temperature (Figure 3.11b). The

particle size slightly increases when the solutions are held for another 45 minutes with

broadened size distribution, which is likely due to the Ostwald ripening process (Figure

3.11c). We could therefore conclude that there is no advantage to boil the reacting solution

longer than 2 hours. Grain size of particles calculated according to XRD patterns are close

to the size measured by TEM (Table 3.2), indicating that the particles are single crystalline.

Figure 3.10 TEM images of SPION prepared from Fe:OA is (a) 3:1, (b) 1:1 and (c) 1:2


Figure 3.11 TEM images of evolution studies. (a) to (c) are sample 6 to 8 for Fe:OA = 3:1

Figure 3.12 XRD obtained from synchrotron for (A) Fe:OA = 3:1, (B) Fe:OA = 1:1. For both graphs, (a) to (e) are corresponding to samples taken from 30 min to 135.min

Table 3.2 Size comparison between XRD and TEM results for Fe:OA = 3:1

Time (min) 30 45 60 90 135 Temp. (°C) 293 294 294 295 294 D (nm, XRD) - 4.3 13.7 15.8 17.7 D (nm, TEM) - - 13.9 16.5 18.4

3.2.3 Surface modification of SPION

3.2.3.1 PLLA@SPION

3.2.3.1.1 Microscopic observation

TEM images in Figure 3.13 show the morphology of as-synthesized SPION,

LA@SPION and PLLA@SPION. Before coated with LA and PLLA, SPION remain very

narrow size distribution. However, diameters as well as the standard deviation increase

38

gradually after LA and PLLA coating procedures as shown in the histogram, indicating a

thin layer of coating molecules are present on the particle surface.

Figure 3.13 (a) TEM image of as-synthesized SPION and the size distribution thereof (b) TEM image of LA@SPION and the size distribution thereof; (c) TEM image of PLLA@SPION and the size distribution thereof. Digital camera images show the distribution for each group of particles between hexane (upper) and chloroform (bottom)

(a)

(b)

(c)


The changing of surface chemistry has been also verified by distribution test of the

particles between hexane and chloroform. The as-synthesized SPION exhibit high

solubility in hexane while low solubility in chloroform. Conversely, the solubility of

LA@SPION and PLLA@SPION in chloroform increases dramatically because LA and

PLLA are readily dissolved in chloroform. Such a variation of solubility provides us a piece

of evidence that the surface of particles were in situ covered with PLLA.

3.2.3.1.2 Characterization of surface coatings

FTIR spectra confirm the presence of the L-lactic acid and PLLA on the surface of

particles (Figure 3.14). The amount of coating shells has been measured by

thermogravimetric analysis (TGA) (Figure 3.16). By dissolving SPION under mild

condition, pure PLLA was separated from the composite. SEC results show that PLLA

coating layer has Mw = 13.2 kDa with polydispersity 1.321. In Figure 3.18, 1H NMR (500

MHz, CDCl3): 1.59-1.62 (d, J = 8.0 Hz, 3H), 5.15-5.21 (dd, J = 8.0 Hz, 1H). 13C NMR (500

MHz, CDCl3): d 16.6, 69.0, 169.6. FTIR spectrum of separated PLLA is compared with the

standard PLLA in Figure 3.15. DSC is used to determine the Tg, Tc and Tm (Figure 3.17).

Figure 3.14 FTIR spectra of (a) oleic acid covered SPION; (b) LA@SPION; and (c) PLLA@SPION

40

Figure 3.15 FTIR spectra of (a) standard PLLA and (b) PLLA separated from PLLA@SPION

Figure 3.16 TGA curves of (a) oleic acid covered SPION with weigh loss 15.5%, (b) LA@SPION with weight loss 22.3%, and (c) PLLA@SPION with weight loss 24.0%

Figure 3.17 The DSC curve of PLLA separated from PLLA@SPION

Figure 3.18 (a) 1H NMR and (b) 13C NMR of PLLA separated from PLLA@SPION

(a) (b)


3.2.3.1.3 Magnetic properties

VSM has been used to study the behavior of magnetization at room temperature. A

typical hysteresis study for PLLA@SPION is shown in Figure 3.19. No obvious remanent

magnetization is observed when the applied field is zero, indicating the particles remain

superparamagnetic. Saturation magnetization of magnetic cores of three kinds of particles is

similar indicating the magnetic properties are retained during the procedure of SIROP.

3.2.3.2 Phase transfer of SPION

3.2.3.2.1 Microscopic observation

All the aqueous dispersions underwent 2 days dialysis against 1 L water in order to

remove the excess of ligand molecules. Upon addition of TMAOH and HNO3, charges on

the NP surface were generated, producing electrical double-layer which generated inter-

particle repulsion, as illustrated in Figure 3.20 In the case of exchange with TSC, TMAOH

were replaced with negatively charged citrate groups which can be evidenced by the change

of ζ-potential values from +52 mV to -40 mV. DMSA, with two mercapto groups and two

carboxylate groups, can form a strong surface layer of NPs and confer to the NPs negative

charges due to the carboxylate groups.

Figure 3.19 Magnetization curve of PLLA@SPION

42

Figure 3.20 Schematic illustration of TMAOH and HNO3 coated SPION

Spherical SPION with 12 nm in diameter have been synthesized via the high

temperature decomposition method and fell into ordered hexagonal array. After ligand

exchange with TMAOH, TSC, HNO3 and DMSA, particles no longer stayed in an array but

randomly distributed with a slight aggregation (Figure 3.21). Inter-particle distance was

decreased compared to the one before phase transfer where hydrophobic interaction

between oleic acid molecules impulse from each other. Smaller hydrophilic molecules such

as TMAOH, TSC, HNO3 and DMSA would not sufficiently protect the particles from

attaching to each other. The stability of such dispersions mainly depends on the electrical

double-layer on the surface of the particles which can be greatly affected by pH and ionic

strength of the dispersions. To meet the requirement of biomedical applications, either in

vitro or in vivo, neutral pH of particle suspension is the primary demand. It is always risky

to vary the pH of aqueous colloidal dispersions. Once the electrical double-layers get

perturbed, the colloidal systems will lose their stability and the precipitation occurs

eventually. Even with a great caution when tuning the pH, aggregation is inevitable; hence

decrease the concentration of the obtained colloidal dispersions at neutral pH.


Figure 3.21 TEM images of (a) as-synthesized SPION, SPION coated with (b) TMAOH, (c) TSC, (d) HNO3 and (e) DMSA

Figure 3.22 Schematic illustration of POA@SPION

SPION have been successfully transferred from hexane to water phase by PF127.

SPION after phase transfer are schematically illustrated in Figure 3.22. TEM images show

that the SPION are monodisperse in water (Figure 3.23). Comparative photographs (inset

of Figure 3.23) show the complete phase transfer from organic to aqueous phase. There is

no significant change in the size and shape of particles as a result of the phase transfer. The

particles possess homogeneous morphology in a regular round shape with an average

44

diameter of 10.1 nm (σ ~ 4.5%). The obtained particles in aqueous solution can stay stable

in a broad pH range (data not shown), indicating a great possibility for in vitro

administration.

3.2.3.2.2 Magnetic properties

The magnetic properties of the SPION before and after phase transfer have been studied

at room temperature by using a VSM. The magnetization of as-synthesized SPION,

TMAOH@SPION, DMSA@SPION and POA@SPION were measured as a function of an

applied magnetic field. The representitive curve of POA@SPION is shown in Figure 3.24.

The magnetic cores of TMAOH and DMSA coated particles are from the same batch, while

the magnetic cores of as-synthesized SPION and POA@SPION are from another batch.

None of the samples show a hysteresis loop, indicating NPs retain superparamagnetic

properties. The obtained curves can be described by the Langevin equation:

M(x) = Ms (cothx- (1/x)) (3.1)

where x = µH/kBT, Ms is the saturation magnetization, µ is the magnetic moment of a

single particle, H is the applied field, kB is Boltzmann’s constant, and T is the absolute

temperature.223 The Ms of as-synthesized SPION and POA@SPION are 41.0 and 31.7

Am2kg-1 respectively. The Ms of TMAOH@SPION and DMSA@SPION are 53.5 and 51.2

Am2kg-1. Fitting the curves in Figure 3.24 to the Langevin equation, we obtained diameters

of the magnetic cores. The size can be obtained through the following expressions:

0118

→= Hs

)dHdM(

HkTDπρ

(3.2)

Figure 3.23 SPION dispersed in water after phase transfer


where ρs is the specific magnetization of the iron oxides. The diameters of as-synthesized

SPION, POA@SPION, TMAOH@SPION and DMSA@SPION are 9.98, 11.1, 12.88 and

12.78 nm, which is rather close to the value obtained from both TEM and XRD data.

3.2.3.2.3 Cytotoxicity test of POA@SPION

It is of great significance to minimize the toxicity of the particles for in vivo

administration. The PF127 block copolymer is selected as a suitable candidate of surface

modification because it is recognized as being highly biocompatible as approved by the

Food and Drug Administration224, 225 and have long been used in experimental medicine

and pharmaceutical sciences.201, 226-228 Samples of POA@SPION with three different iron

concentrations, ranging from 1 to 100 µg/mL, were incubated with the HeLa and MCF-12A

cell lines for 48 hours. The viability of the cells was evaluated by MTS and MTT assays,

and the results showed no significant cytotoxicity to the mitochondria function for both cell

types (Figure 3.25).

-1000 -500 0 500 1000-60

-40

-20

0

20

40

60

-100 -50 0 50 100-30-15

01530

Mag

netiz

atio

n (A

m2 kg

-1)

Applied field (mT)

Figure 3.24 Room temperature magnetization curve of as-synthesized SPION (-●-) and POA@SPION (-○-)

46

Figure 3.25 Viability of HeLa and MCF-12A cells exposed to POA@SPION at various iron concentrations for 48 hours

3.3 MR studies of POA@SPION The relaxation times T1 and T2 were measured at 0.47 T (20 MHz proton Larmor

frequency, 37°C) and 1.41 T (60 MHz proton Larmor frequency, 37°C). The imaging

contrast arises from the difference between r2 and r1 values. For a T2 contrast agent, the

higher the r2/r1 ratio the better the contrast efficacy. The relaxivities of POA@SPION are

summarized in Table 4.3 and are compared to those obtained under the same conditions

using commercial T2 contrast agent Resovist® (Schering AG, Germany). Noticeably, the

r2/r1 ratios of POA@SPION are 6.6 and 15.8 folds higher than those of Resovist at 0.47 T

(20 MHz) and 1.41 T (60 MHz) respectively, indicating that POA@SPION could be

considered as a T2 contrast agent with high efficiency. Because the magnetic cores of

POA@SPION and Resovist are produced via different methods, it is also necessary to

exclude the impact of the magnetic cores. Therefore, we also investigated the relaxivities of

SPION transferred to aqueous phase by coating with small molecules e.g. TMAOH and

DMSA. From Table 3.3, it is seen that the relaxivities and r2/r1 ratios of TMAOH@SPION

and DMSA@SPION are similar with those of Resovist. Hence the enhancement of the r2/r1

ratios could be most likely attributed to the unique surface coating of POA@SPION. The

surface coatings, hydrodynamic diameters and relaxivities of POA@SPION, Resovist and

Feridex® (Advanced Magnetics, Cambridge, Massachusetts) are summarized in Table 3.3.

The r1 of POA@SPION is lower than those of Resovist and Feridex, while the r2 of


POA@SPION is higher than both commercial contrast agents. As a result, r2/r1 ratio of

POA@SPION is much higher (0.47 T, 20 MHz) than the commercial contrast agents.

Table 3.3 Surface coatings, mean hydrodynamic diameters and relaxivities of SPION with different coatings, Resovist and Feridex measured at 20 and 60 MHz (0.47 T and 1.41 T) in water (37°C).

20 MHz 60 MHz Sample name Surface coating

Mean hydrodynamic diameter (nm) r1 r2 r2/r1 r1 r2 r2/r1

POA@SPION PF127/OA 71 1.33 63.4 47 0.311 71.3 229

TMAOH@SPION TMAOH - 22.3 155.8 6.98 9.7 175.0 17.96

DMSA@SPION DMSA - 8.01 67.8 8.46 2.6 76.7 29.40

Resovist Carboxydextran 65 24.92 176.85 7.10 10.90 190.19 14.45

Feridex* Dextran 72 40 160 4 - - -

Note: The unit of ri (i = 1, 2) is s-1mM-1. * From ref. 229

Figure 3.26 Scheme of surface coating of POA@SPION. Out of the dashed line is the hydrophilic domain and inside of the dashed line is the hydrophobic domain

48

Considering that Resovist and Feridex have a thick layer of carboxydextran and dextran

respectively, it is therefore possible that the PF127/OA amphiphilic double layer may play

a dual-role so as to enhance the T2 relaxation rate and decrease the T1 relaxation rate at the

same time. As shown in Figure 3.26, the hydrophilic PEO domain would facilitates the

diffusion of large quantity of water molecules close to the magnetic core of POA@SPION,

which is in turn enhances the T2 relaxation rate.230 Furthermore, it was reported that T2

relaxation rate of ice is very high.231 Similarly, in the hierarchical structure, the mobility of

water molecules in the diffusing layer is restricted due to the hydrogen bonding with

hydrophilic chains of PEO blocks, turning into large amount of “ice-like” water. Therefore,

large r2 relaxivity of POA@SPION is owing to the “ice-like” water in the hydrophilic

blocks. The dimension of “diffusing layer” of POA@SPION can be characterized as the

mean hydrodynamic diameter which is similar with the other contrast agents (Table 3.3). It

was reported that the r2 values can be greatly increased by clustering T2 contrast agents in

reservoirs such as liposomes or cells.232 This effect results from the intrinsic mechanism of

spin-spin relaxation, which relies on the local concentration of the contrast agents. However,

POA@SPION shows no enhancement to the local Fe concentration because it forms a well-

dispersed and stable ferrofluid as been evidenced by TEM studies (Figure 3.23). Moreover,

the smaller r2 value compared with those exceedingly high ones for clustered particles in

micelles reveals additional evidence that POA@SPION are not agglomerated.232

Conversely, T1 contrast agents require close contact with the water molecules to effectively

generate T1 contrast due to the inherent mechanism of spin-lattice relaxation.233 As seen in

Figure 3.26, the magnetic core of POA@SPION is segregated from the exterior water

molecules by a compact hydrophobic layer comprised of PPO blocks and oleic acid

molecules. Water molecules diffused into PEO domain can hardly come into contact with

the magnetic core because of the hydrophobic nature of the inner layer. Such a hierarchical

hydrophobic-hydrophilic coating of POA@SPION is of substantial difference from the

conventional T2 contrast agents whose coating layers are entirely hydrophilic so that the

diffusion of water molecules to magnetic core is not restricted. The covalent bond between

carboxylic group of oleic acid and iron oxide ensures complete coverage of particle surface.

The subsequent incorporation of PF127 molecules further increases the thickness of this

hydrophobic “isolating layer”. Such a structure comprised of PPO blocks and oleic acid


molecules would effectively hinder the external water molecules from contact with the

magnetic core, which in turn kinetically limit the spin-lattice relaxation rate.

50

4 Conclusions Multifunctional drug delivery systems (DDS) including thermosensitive polymeric

nanospheres, magnetic nanoparticles and high performance magnetic resonance T2 contrast

agents have been prepared through different methodologies.

Temperature-responsive polymer is used as a key component to fabricate the

thermosensitive DDS. Biodegradable polymers are selected as the backbone to form the

nanospheres. The phase transition temperature, lower critical solution temperature (LCST),

can be tuned to 36.7°C, the human body temperature, by controlling the ratio between two

blocks in the copolymer. Drug release performance of such thermosensitive DDS have been

examined by employing the bovine serum albumin as a model drug. The results show an

enhanced releasing rate when the temperature is elevated above LCST which is in a good

agreement with phase transition results confirmed by UV-Vis and differential scanning

calorimetry. Poly ethylene glycol is used to cover the surface of drug carriers in order to

protect unspecific binding with proteins in the plasma, thus avoiding uptake by immune

system. However, nanospheres coated with small gold NPs have shown higher cell viability

compared with those without gold coating. The result implies that gold is a safe component

in fabrication of multifunctional DDS.

Monodisperse superparamagnetic iron oxide nanoparticles (SPION) are prepared

through high temperature decomposition method in an organic solution. The obtained

particles are highly crystalline. By changing different ratio between iron precursor and

surfactant, particle size can be easily manipulated. Particle growth is investigated by means

of transmission electron microscope and synchrotron X-ray diffraction on the real-time

base. Optimum refluxing time and temperature are determined on the basis of the results.

The SPION are then modified with biodegradable polymer poly(L,L-lactide) (PLLA) to use

as a drug carrier, since PLLA is easy to link with biomolecules and drugs in the future. The

surface coating layer is fully characterized to confirm the presence of PLLA. Nevertheless,

for biomedical applications, hydrophobic particles prepared in organic solution should be

transferred to aqueous solution. We have successfully transfer SPION with assistance of

small molecules. The major disadvantage of these small capping agents is that the particles


after phase transfer are not stable over broad pH range, especially at physiological pH.

Amphiphilic Pluronic F127 copolymer is used as a new phase transfer agent that can form

stable SPION suspension over large pH range. The surface of obtained particles is covered

with poly ethylene oxide domain to endow the particles with high hydrophilicity which will,

in turn, prolong the blood circulation time and increase the biodistribution. Most

interestingly, besides other promising feature for DDS, Pluronic F127 coated SPION show

a remarkably high r2/r1 ratio, indicating that these particles are potentially better T2 contrast

agent than the commercially available ones. The relaxometric data have been evaluated and

mechanism of such enhancement has been discussed.

On the basis of present results, we believe that we could eventually achieve the ultimate

goal that one single integrated system can display a controlled release manner with stimuli-

sensitive features, and can be monitored with a certain type of imaging modality.

52

Acknowledgements First of all, I would like to thank my principle supervisor, Prof. Mamoun Muhammed,

for giving me the precious opportunity to join the Materials Chemistry Division and leading

me to the world of nanobiotechnology. His enlightening ideas and kindness is always

strong support during the course of study. I have to say sincere thanks to my tutor, Dr.

Maria Mikhaylova, for her great patience, scientific discussion and professional guidance

on my first step of research.

I want to thank Yun Suk Jo for introducing me into polymer and biomedical field, and

his continuous helping with ongoing experiments. I wish him a good luck for his PhD in

ETHZ. Thanks to Prof. Do Kyung Kim for his encouragement and invaluable advices. I

wish him an outstanding academic future at Keele University in UK. I would like to thank

Dr. Muhammet Toprak for his great help from research to everyday life. I wish him a great

success in the scientific career. Many thanks to Andrea, Fei, Carmen, Sayed, Sverker and

all other members in Materials Chemistry Division for their priceless friendship.

I would like to acknowledge the fruitful collaboration and discussions with Helen

Vallhov, Dr. Susanne Gabrielsson and Prof. Annika Scheynius from Department of

Medicine; Erika Witasp and Prof. Bengt Fadeel from Institute of Environmental Medicine

at Karolinska Institutet. I would also like to thank Dr. Sophie Laurent, Dr. Alain Roch, and

Prof. Robert N. Muller from Department of General, Organic and Biomedical Chemistry at

University of Mons-Hainaut in Belgium for the productive collaboration and great help on

MR experiments. Many thanks to Johanna Öberg, Ivan Bednar, Rouslan Sitnikov and Prof.

Christian Spenger at MR Centrum of Karolinska University Hospital for their kind help

with MRI measurements. Thanks to Prof. Josep Nogues from Department of Physics at

Universitat Autònoma de Barcelona for helping on the magnetic measurements. Thanks to

Dr. Tiezhen Ren and Prof. Xiaodong Zou at Department of Chemistry in Stockholms

Universitetet for synchrotron XRD measurements.

Special thanks to Peter, my best friend, for kind encouragement and support during the

past few years. I wish him a nice future and a happy family.

Last but not least, I would like to thank my parents for their endless support and my wife

Chuanmin Hu for her deep love and a strong support for my career.


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