SYNTHESIS OF SUPERPARMAGNETIC NANOSTRUCTURES AND THEIR MAGNETIC RESONANCE IMAGING APPLICATIONS CHOO SHI GUANG, EUGENE NATIONAL UNIVERSITY OF SINGAPORE 2012 SYNTHESIS OF SUPERPARMAGNETIC NANOSTRUCTURES AND THEIR MAGNETIC RESONANCE IMAGING APPLICATIONS CHOO SHI GUANG, EUGENE (B APPL SCI., HONS), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (PH.D) DEPARTMENT OF MATERIALS SCIENCE AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 ACKNOWLEDGMENTS To Dr Xue Jun Min I wish to give to you my deepest thanks It is an honor to be your first graduating Ph.D student You had taught me during my undergraduate studies, and given advice on my FYP even though you were not my assigned advisor You have since then been the key motivator of my graduate studies I must thank you again for responding so positively to all my bad experimental results I deeply appreciate all your contributions of time, ideas, and financial support to make my Ph.D experience both productive and stimulating You are more than a teacher to me To Dr Borys Shuter I am particularly indebted to you in my later work regarding MRI measurements I thank you for unselfishly putting aside your work to patiently carry out MRI measurements with me and then discuss the results I particularly dedicate this thesis to you, for without your expertise and guidance to kickstart my MRI studies, this thesis would not have come to fruition I wish you a long and happy retirement! I wish to thank Dr Chuang Kai-Hsiang and his research team of the Magnetic Resonance Imaging group in the Singapore Bioimaging Consortium (SBIC) I am grateful for all their help in conducting the MR spectroscopic imaging in the final part of my Ph.D studies I would like to thank the NMR laboratory (Department of Chemistry, NUS) for their help in my NMR measurements Particularly, I would like to thank Mdm Han Yanhui for attending to my frequent service requests and then performing the measurements and processing the results ever so promptly i I would like to thank the entire team of lab technologists in the Advanced Materials Characterization Laboratory in my department I thank Serene Chooi for coordinating the safety of the labs I thank Mr Chan for keeping the computers and facilities running smoothly I thank Mdm He Jian for providing a clean and tidy biological lab for my cell assays I thank Agnes for all her help for SEM and Zetasizer I thank Chen Qun for his assistance on my XRD and VSM experiments I thank Yeow Koon for all his help on FT-IR and UV-Vis experiments I thank Henche for all his help on the TGA experiments I thank Roger for maintaining the smooth running of our wet-lab Additionally, I would also like to thank Yin Hong and Brenda for all their technical assistance before they left the department I would also like to thank Dr Zhang Jixuan of the TEM lab for all her assistance, guidance, and freedom granted in the use of the TEM equipment I am always looking forward to the TEM sessions To Xiaosheng, Jiaquan, Sheng Yang, Li Meng, Chen Yu, and Erwin of the Nanostructured Biomedical Materials Lab I thank all my good labmates for being cooperative during my time as the safety representative of the group I am grateful for all the meal gatherings that were organized within our group, and it just seems amazing how we could come up with so many excuses to go out together for a good meal I had a great time working and chilling out with all of you! Lastly, I would like to thank all my other close friends (both research and nonresearch) for all their support and encouragement during my life as a research student I thank you all for giving me a semblance of normal life in the stereotypical lifeless life of a Ph.D student Mazel tov! ii TABLE OF CONTENTS Acknowledgments i  Table of Contents iii  Summary vi  List of Related Publications viii  List of Tables x  List of Figures .xii  List of Abbreviations xix  CHAPTER 1:  Introduction 1  1.1  Nanoprobes for Clinical Diagnostic Imaging 1  1.2  Magnetic Resonance Imaging 5  1.2.1  T1 Contrast Effect 7  1.2.2  T2 Contrast Effect 8  1.3  Advances in Contrast Agents for Magnetic Resonance Imaging 11  1.3.1  Off-Resonance Saturation Contrast Enhancement 11  1.3.2  Chemical Exchange Saturation Transfer (CEST) 13  1.3.3  Heteronuclei Magnetic Resonance Spectroscopy Imaging 14  1.3.4  Magnetic Field-Induced Magnetic Resonance Spectroscopic Imaging 15  1.4  Project Motivations and Designs 16  1.5  Research Objectives 20  CHAPTER 2:  Experimental 22  2.1  Materials 22  2.2  Materials Synthesis 23  2.2.1  Synthesis of nm Fe3O4 Nanoparticles 23  2.2.2  Synthesis of nm Fe3O4 Nanoparticles 24  2.2.3  Synthesis of MnFe2O4 Nano-Octahedrons 24  2.2.4  Synthesis of Silver Nanoparticles 25  2.2.5  Synthesis of Functional Amphiphilic Brush Copolymer 26  2.2.6  Synthesis of Fluorescent Amphiphilic Brush Copolymer 27  2.2.7  Synthesis of PEG-Conjugated Amphiphilic Brush Copolymer 29  iii 2.3  Phase Transfer of Single Hydrophobic Nanoparticles 30  2.4  Preparation of Nanoparticle/Polymer Spherical Nanocomposites 31  2.5  Characterization 32  2.5.1  Chemical Analysis 32  2.5.2  Morphological Study 33  2.5.3  Magnetic Properties 35  2.5.4  Optical Properties 36  2.5.5  Thermogravimetric Analysis 36  2.5.6  Colloidal Stability 37  2.5.7  Water Absorption Studies 37  2.5.8  Cell Viability Assays 38  2.5.9  Cell Labelling 39  2.5.10  Magnetic Relaxivity Studies 39  2.5.11  MRI Phantom Studies 40  CHAPTER 3:  Synthesis of Magnetic Nanoparticles and Functional Amphiphilic Brush Copolymer 41  3.1  Motivations and Design of Experiment 41  3.2  Synthesis of Iron Oxide Nanoparticles 47  3.2.1  Characterization of Iron Oxide Nanoparticles 49  3.3  Synthesis of Manganese Ferrite Nano-Octahedrons 52  3.3.1  Characterization of Manganese Ferrite Nano-Octahedrons 55  3.4  Synthesis of Functional Amphiphilic Brush Copolymer 58  3.4.1  3.4.2  FT-IR Analysis of Functional Brush Copolymer 61  H NMR Analysis of Functional Brush Copolymer 59  3.5  Remarks 63  CHAPTER 4:  Formation of Composite Superparamagnetic Nanoclusters 65  4.1  Motivations and Design of Experiment 65  4.2  Effect of Oil-to-Water Ratio 68  4.3  Effect of Polymer Concentration 71  4.4  Effect of Nanoparticle Concentration 75  4.5  Effect of SPION Size 78  4.6  Thermogravimetric Determination of Loading Density 81  4.7  Colloidal Stability Studies 83  4.7.1  Colloidal Stability in Water and PBS 84  iv 4.7.2  Colloidal Stability in Tesla Magnet 85  4.7.3  Stability Against Protein Adsorption 86  4.8  pH Stability Studies 88  4.9  Remarks 90  CHAPTER 5:  Superparamagnetic Nanocomposite Structures for Enhanced T2 Contrast Effect and Fluorescent Imaging 92  5.1  Motivations and Design of Experiment 92  5.2  Preparation and Characterization of Iron Oxide Nanocomposites 95  5.3  Magnetic Properties of Iron Oxide Nanoclusters 98  5.4  Calculation of Intra-Particle Separation 103  5.5  Relationship Between Magnetic Properties and Intra-Particle Separation in IONCs 105  5.6  MRI Relaxivity Studies 109  5.7  Fluorescent Tagging of Cells for Dual Modal Imaging 121  5.8  Remarks 125  CHAPTER 6:  Study of Magnetic Nanostructures for Off-Resonance MR Spectroscopic Imaging 127  6.1  Motivations and Design of Experiment 127  6.2  Preparation and Characterization of Manganese Ferrite Nanocomposites 134  6.3  1H NMR Spectroscopy Study of Magnetic Nanocomposites 142  6.4  MR Spectroscopic Imaging Study 149  6.5  Water Absorption Studies of the MFNC Nanocomposites 150  6.5.1  Water Swelling Study 151  6.5.2  Water Permeability Tests 152  6.5.3  Tuning of Hydrophilicity by Conjugation with PEG 155  6.6  pH Stability 157  6.7  Colloidal Stability 159  6.8  Cell Cytotoxicty 160  6.9  Remarks 161  CHAPTER 7:  Conclusions and Future Work 163  7.1  Project Conclusions 163  7.2  Possible Improvements for Future Work 166  Biobliography 170  v SUMMARY In this thesis, the study was aimed at improving current superparamagnetic contrast agents utilized in magnetic resonance imaging (MRI) For this purpose, the synthesis of well-defined spherical magnetic nanocomposite structures was proposed The objective was to study the magnetic behaviour of such a nanostructure based on superparamagnetic nanocrystal (SPMN) assemblies in a polymeric matrix By varying the packing density of SPMNs within a sphere, the separation distance between the nanocrystals could be readily controlled Through examining the properties of such nanostructures, it was possible to gain deeper insights on the magnetic interaction behavior between SPMNs Hence, the research work was mainly focused on investigating and exploiting unique magnetic behaviours of SPMNs for enhancing MRI contrast effects The key advantage of SPMNs over other MRI contrast agents is its high molar relaxivity Due to the strong magnetic fields induced by SPMNs, they distort local field patterns, which is useful in MRI because it affects the spin behaviour of protons The most common source of protons in the body is hydrogen in water, with a net spin of ½ When placed in a magnetic field, protons precess at a frequency that is dependent on the magnitude of the external field Hence, by disturbing the local field through the introduction of SPMNs, the protons experience changes in precession frequency and consequently lose phase coherence with respect to the bulk pool This phenomenon is known as dephasing and results in the spreading of MR spectral signals Based on such magnetic behaviour, two mechanisms were proposed to enhance MRI contrast using SPMNs vi The first was simply to maximise the rate at which protons dephase The region that undergoes rapid spin dephasing would appear dark in a bright MR image so that the SPMN-targeted location could be distinguished Assembled secondary structures of SPMNs were found to display unusually good proton dephasing effects However, past studies were based on uncontrolled aggregation of SPMNs that were generally irregular in shape and size, which made it impossible to correlate the MR effects with the structure of magnetic contrast agents Herein, the proposal of wellstructured and uniform magnetic composite nanospheres addressed this issue The second way could be termed as “rephasing” SPMNs typically cause random shifts in the precession frequencies of protons, which results in a wide range of frequencies However, if one can recover the loss in phase coherence and restore it at a frequency that is different from the natural precession frequency of water, an alternative signal could be employed for detection of the contrast agent The proposed magnetic nanocomposites structure could hypothetically produce such a unique effect and be potentially useful for dual modal MR spectroscopic imaging As such, this thesis was premised on fabricating uniform and well-dispersed superparamagnetic nanocomposite structures as the key materials component The particles were broadly characterized in terms of size, morphology, composite structure, magnetic properties, pH stability, colloidal stability and cytotoxicity Finally, they were analyzed for its potential as multifunctional and multimodal imaging probes for detection based on the MRI platform vii LIST OF RELATED PUBLICATIONS  E S G Choo, E Peng, R Rajendran, P Chandrasekharan, C T Yang, J Ding, K H Chuang, J M Xue, “Superparamagnetic Nanostructures for Off-Resonance Magnetic Resonance Spectroscopic Imaging”, DOI: Advance Functional Materials 10.1002/adfm.201200275, accepted on 13th Aug 2012  E S G Choo, X S Tang, Y Sheng, B Shuter, J M Xue, “Controlled Loading of Superparamagnetic Nanopaticles in Fluorescent Nanogels as Effective T2Weighted MRI Contrast Agents”, Journal of Materials Chemistry, 21, 2310-2319 (2011)  E S G Choo, B Yu, J M Xue, “Synthesis of Poly(acrylic acid) (PAA) Modified Pluronic P123 Copolymers for pH-Stimulated Release of Doxorubicin”, Journal of Colloid and Interface Science, 358, 462-470 (2011)  E Peng, E S G Choo, P Chandrasekharan, C T Yang, K H Chuang, J M Xue, “Synthesis of Manganese Ferrite/Graphene Oxide Nanocomposites for Biomedical Applications”, DOI: Small 10.1002/smll.201201427, accepted on 21st Aug 2012  X L Liu, H M Fan, J B Yi, Y Yang, E S G Choo, J M Xue, D D Fan, J Ding, “Optimization of Surface Coating on Fe3O4 Nanoparticles for High Performance Magnetic Hyperthermia Agents”, Journal of Materials Chemistry, 22, 8235-8244 (2012)  X S Tang, K Yu, Q H Xu, E S G Choo, G K L Goh, J M Xue, “Synthesis and Characterization of AgInS2-ZnS Heterodimers with Tunable Photoluminescence”, Journal of Materials Chemistry, 21, 11239-11243 (2011)  X S Tang, E S G Choo, L Li, J Ding, J M Xue, “Synthesis of ZnO Nanoparticles with Tunable Emission Colors and Their Cell Labeling Applications”, Chemistry of Materials, 22, 3383-3388 (2010)  J Q Yuan, E S G Choo, X S Tang, Y Sheng, J Ding, J M Xue, “Synthesis of ZnO–Pt Nanoflowers and their Photocatalytic Applications”, Nanotechnology, 21, 185606-185615 (2010)  L Li, E S G Choo, X S Tang, J Ding, J M Xue, “Ag/Au-Decorated Fe3O4/SiO2 Composite Nanospheres for Catalytic Applications”, Acta Materialia, 58, 3825-3831 (2010)  X S Tang, E S G Choo, L Li, J Ding, J M Xue, “One-Pot Synthesis of Water-Stable ZnO Nanoparticles via a Polyol Hydrolysis Route and their Cell Labeling Applications”, Langmuir, 25, 5271-5275 (2009)  L Li, E S G Choo, X S Tang, J Ding, J M Xue, “A Facile One-Step Route to Synthesize Cage-Like Silica Hollow Spheres Loaded with Superparamagnetic viii general preparation route of Folate–NH2 can be proposed as follows: PEG–bisamine, folic acid, and dicyclohexylcarbodiimide are dissolved in dimethyl sulfoxide in the presence of triethylamine as catalyst [125] The mixture reacts for 24 h in the dark at room temperature under nitrogen gas The mixture is centrifuged to separate the byproduct dicyclohexylurea and then purified by precipitation in anhydrous diethyl ether Folate–NH2 can then be reacted with PBMA in the same way as the grafting of dodecylamine Hence, the preparation of folate-decorated magnetic nanocomposites is possible and this method could be used for specific targeting and detection of cancer cells In Chapter 6, it was shown that the PEG-modified copolymer, PBMA-g(C12/PEG) could similarly be used in the formation of nanoclusters The incorporation of PEG to the polymer matrix is advantageous because PEG is a well-known biocompatible polymer that has anti-fouling properties and has become an established means to improve water solubility and biocompatibility of material and particle surfaces [57,126,127] This could also increase blood circulation times by reducing uptake by the body’s reticuloendothelial system (RES) Furthermore, the 1H NMR results had shown that PEG modified magnetic nanocomposites generated a stronger secondary peak as compared to the regular MFNCs This was because the increased hydrophilicity of the polymer allowed for greater diffusion and exchange of water molecules in and out of the nanocomposites spheres However, a few issues remained to be studied regarding the incorporation of PEG for the formation of the nanocomposites spheres Firstly, the increased hydrophilicity due to the addition of PEG could become detrimental if the polymer is susceptible to dissolution The stability of the nanocomposites structure in water would be compromised and it would lead to the disintegration of the sphere within a short period of time There are two 167 w ways to ntrol the hy ydrophilicit of the co ty opolymer The first is by control T s lling the p percentage grafting of PEG whi the seco is by using PEG of differen chain f ile ond u nt l lengths The aim would be to imp e prove the water absorp w ption of the nanospher while e res m maintaining structure st g tability of th particle/p he polymer composite Another issue th was not addressed in this thes was the m hat t sis manipulatio of the on f frequency s shift of water Experim ments so far have show that the overall siz of the r wn ze n nanocomposites neither had an eff on the degree of fr r fect d requency sh nor the intensity hift i o the secon of ndary peak as shown in Figure 7-1 a n F Figure 7-1: Effect of MFNC size on the 1H NMR spectra of wat Peak in M e ter ntensities w norma were alized agains the main water peak st The magnetic particle load p ding compo osition and core partic size also had no cle o s substantial e effect on th frequenc shift as discussed in section For mos of the he cy d n st 168 experiments performed, the position of the secondary peak remained relatively unchanged at approximately -3.2 ppm It is believed that the type and shape of the magnetic nanocrystal could also have different effects on the frequency shift Thus, nanostructures with more anisotropic structures may be the key to producing variable shifts in the water peak For this purpose, magnetic nanocrystals with other morphologies could be investigated Possible morphologies include nanocubes [128,129] and nanoplates [130,131,132] Such anisotropic structures have large faceted surfaces that could produce unique effects for using a similar nanocomposite design Nanorings or nanotubes [133,134,135] could also be suggested for such a study as they are similar to the micromachined cylinder structures reported by Zabow et al [42] If different frequency shifts can be obtained for each contrast agent, they can be colorcoded according to degree of the frequency shift With the excellent colloidal 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GUANG, EUGENE (B APPL SCI., HONS), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. .. Study of Magnetic Nanostructures for Off -Resonance MR Spectroscopic Imaging 127  6.1  Motivations and Design of Experiment 127  6.2  Preparation and Characterization of Manganese... image of IONC-b5 (Inset: SAED ring patterns of IONC-b5); (h) XRD profile of IONC-b5; and (i) VSM profiles of core SPION-7nm (∆) and IONC-b5 (□) (Page 96) Figure 5-3: (a) M(H) curves of SPION-4nm and