SYNTHESIS AND FUNCTIONALIZATION OF MICRO/NANO-PARTICLES FOR MALIGNANT CELLS DETECTION AND ELIMINATION HU FEIXIONG NATIONAL UNIVERSITY OF SINGAPORE 2009 SYNTHESIS AND FUNCTIONALIZATION OF MICRO/NANO-PARTICLES FOR MALIGNANT CELLS DETECTION AND ELIMINATION HU FEIXIONG (B. E., M. S., Tianjin University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS First of all, I would like to express my deepest gratutide to my supervisors, Professor Neoh Koon Gee and Professor Kang En-Tang, who have been instrumental in guiding this research and providing useful advice throughout the long period of this research work. I would like to thank them for their patience and unfaltering commitment to their students; for their inspired ideas, which always make me go ahead on my way. Their enthusiasm, sincerity and dedication to scientific research have greatly impressed me and will benefit me in my future career. I would like to thank all my friends and all group members for their valuable helps in both academic issues and in other issues. Particular thanks go to Dr. Cen Lian, Dr. Li Yali and Dr. Shi Zhilong for sharing with me their invaluable experience in the research field. In addition, I have many thanks to give to all technologists, specifically Miss Chew Su Mei and Mr Yuan Zeliang, thanks for kindly help during my research. The financial support for Ph.D study from National University of Singapore is also greatly appreciated. Finally, I would like to express my deepest gratitude and indubtedness to my parents, my grandfather and other relatives for their constant concern love, patience and surpport. Special thanks to my wife, Ma Lanfang, for her love and encouragement. i TABLE OF CONTENTS ACKNOWLEDGEMENTS .i TABLE OF CONTENTS ii SUMMARY .vi NOMENCLATURE viii LIST OF FIGURES ix LIST OF TABLES xiii CHAPTER INTRODUCTION CHAPTER LITERATURE SURVEY 2.1 Effects of Biocides on Bacteria 2.1.1 Potential Targets and Effects of Biocides 2.1.2 Interactions between Cell Membrane and Disinfectants .8 2.1.3 Mechanisms of Antibacterial Action of Quaternary Ammonium Salts.9 2.1.4 Factors Affecting Biocidal Activity .10 2.1.4.1 Electrostatic Interaction between the Cells and the Disinfectants .10 2.2 2.1.4.2 Hydrophobic Chain Length .11 2.1.4.3 Morphological Effect of Disinfectants 12 Magnetic Nanoparticles for Cancer Detection and Treatment .14 2.2.1 Basic Concepts of Magnetism .15 2.2.2 Synthesis Methods of Magnetic Nanoparticles 17 2.2.2.1 Precipitation 18 2.2.2.2 Microemulsions .20 2.2.2.3 Polyols .21 2.2.2.4 High-temperature Decomposition of Organic Precursors .24 2.2.3 Functionalization Magnetic Nanoparticles for Biomedical Applications 25 2.2.3.1 Coprecipitation 25 2.2.3.2 Encapsulation Method 26 2.2.3.3 Deposition Methods 27 2.2.3.4 Surface Initiated Polymerization Processes 29 2.2.4 Magnetic Nanoparticles for Biomedical Applications .30 ii 2.2.4.1 Magnetic Targeted Drug Delivery 30 2.2.4.2 Contrast Agents for Magnetic Resonance Imaging 33 2.2.4.3 Magnetically Induced Hyperthermia Treatment for Malignant Cells 36 CHAPTER ANTIBACTERIAL AND ANTIFUNGAL EFFICACY OF SURFACE FUNCTIONALIZED POLYMERIC BEADS IN REPEATED APPLICATIONS 38 3.1 Introduction 39 3.2 Materials and Methods 42 3.2.1 Materials 42 3.2.2 Preparation of Microbeads .42 3.2.3 Quaternization of the P4VP .42 3.3 3.4 3.2.4 Bulk and Surface Analysis .43 3.2.5 Antibacterial Assay on E. coli .44 3.2.6 Antifungal Assay on A. niger .46 Results and Discussion .49 3.3.1 Properties of Microbeads .49 3.3.2 Antibacterial Characteristics of Beads .54 3.3.3 Antifungal Characteristics of Beads 61 Conclusions .68 CHAPTER SYNTHESIS EVALUATION OF AND IN VITRO TAMOXIFEN-LOADED ANTI-TUMORAL MAGNETITE/PLLA COMPOSITE NANOPARTICLES 69 4.1 Introduction 70 4.2 Materials and Methods 73 4.2.1 Materials 73 4.2.2 Preparation of Magnetic Nanoparticles .73 4.2.3 Preparation of Tamoxifen-loaded Magnetite/PLLA Composite Nanoparticles (TMCN) 74 4.2.4 Characterization of the Magnetic Carrier 75 4.2.4.1 Particle Size and Surface Properties .75 4.2.4.2 Fe3O4 Loading and Tamoxifen Encapsulation Efficiencies 75 4.2.5 In vitro Tamoxifen Release Studies .76 4.2.6 Cell Culture Assay .77 iii 4.3 4.2.6.1 Nanoparticle Uptake by MCF-7 Cells 77 4.2.6.2 Inhibition of MCF-7 Cells Proliferation .77 Results and Discussion .79 4.3.1 Characterization of the Magnetic Carrier 79 4.3.1.1 Fe3O4 Loading and Distribution 79 4.3.1.2 Drug Loading and in vitro Release .83 4.3.1.3 Particle Size, Morphology and Surface Properties .85 4.3.2 4.4 Cell Culture Assay .89 4.3.2.1 Cell Uptake of TMCN 89 4.3.2.2 Cytotoxicity of TMCN against MCF-7 Cells .92 Conclusions .95 CHAPTER SYNTHESIS OF FOLIC ACID FUNCTIONALIZED PLLA- b-PPEGMA POLYMERIC NANOPARTICLES FOR CANCER CELL TARGETING 96 5.1 Introduction 97 5.2 Materials and Methods 100 5.2.1 Materials 100 5.2.2 Preparation of Double-headed Initiator .100 5.2.3 Preparation of PLLA-b-PPEGMA .101 5.2.3.1 2-bromo-2-methylpropionyl End Functionalized Poly(L-lactic acid) .101 5.2.3.2 ATRP of PEGMA .101 5.2.3.3 Conversion of the Terminal Hydroxyl Groups to Chloride 102 5.2.4 Preparation Polymeric of Folic Nanoparticles Acid Functionalized (PNP) with PLLA-b-PPEGMA Encapsulated Magnetic Nanoparticles .103 5.3 5.2.4.1 PNP with Encapsulated Magnetic Nanoparticles .103 5.2.4.2 PNP Surface Functionalized with Folic Acid .104 5.2.5 Cell Culture Assay .105 5.2.6 Characterization .107 Results and Discussion .108 5.3.1 Characterization of PLLA-b-PPEGMA .108 5.3.2 Characterization of PNP 113 5.3.3 Surface Functionalization of the PNP with Folic Acid 115 iv 5.3.4 5.4 Cell Culture Assay .117 Conclusions .125 CHAPTER CELLULAR NANOPARTICLES ATRP RESPONSE “PEGYLATED” VIA TO MAGNETIC SURFACE-INITIATED 126 6.1 Introduction 127 6.2 Materials and Methods 130 6.3 6.4 6.2.1 Materials 130 6.2.2 Surface Initiated Atom Transfer Radical Polymerization 130 6.2.3 Cell Culture 131 6.2.4 Characterization .132 Results and Discussion .133 6.3.1 Physical Properties of the Magnetic Nanoparticles .133 6.3.2 Surface-initiated ATRP of PEGMA 135 6.3.3 Cell Uptake 144 Conclusions .150 CHAPTER CONCLUSIONS 151 CHAPTER RECOMMENDATIONS FOR FURTHER STUDY 155 REFERENCES 159 LIST OF PUBLICATIONS 178 v SUMMARY Micro/nano size particles are very useful vehicles for surface and bulk functionalization due to their high surface/volume ratio and their ability to encapsulate other agents. In this thesis, different approaches of surface and bulk functionalization of micro/nanoparticles for diagnosing or eliminating malignant cells were developed. At the same time, other important properties of particles such as the cytotoxicity and cell uptake were investigated after the functionalization process. A simple technique was first developed for preparing polymeric microparticles for antimicrobial applications. Poly (4-vinyl pyridine)/poly (vinylidene fluoride) (P4VP)/(PVDF) microparticles prepared by the phase inversion technique were used as the substrate. P4VP contributed the antimicrobial groups while PVDF provided the mechanical strength of the beads. The N-alkylation of the P4VP was carried out with alkyl chains of different lengths since the length of the carbon side chains has been shown to affect the antibacterial efficacy of pyridinium-type polymers. Two microorganisms, a Gram-negative bacteria Escherichia coli (E. coli) and a fungi spore Aspergillus niger (A. niger), were chosen to test the antimicrobial efficacy of the microparticles. To obtain a better understanding of the difference in efficacy against these two microbial species, the effect of surface pyridinium groups on cellular components was studied. This technique for preparing antibacterial microparticles has the advantages of ease of mass production and scale up, and the microparticles possess stability for repeated usage. In the second part of the work, magnetic nanoparticles were developed for the detection and elimination of malignant mammalian cells. For in vivo applications, the vi particles to be introduced must be small enough, such that they not clog the blood vessels through which they are guided to the target organ. A new magnetic targeted drug delivery carrier was developed by encapsulating magnetic Fe3O4 seeds and tamoxifen, a drug for human breast cancer, in a biodegradable polymer, poly(L-lactic acid) (PLLA), in the form of nanoparticles. These magnetic nanoparticles can also be used as contrast agents for magnetic resonance imaging (MRI), with which the distribution of the carrier can be visualized in vivo. The encapsulation of tamoxifen in the polymer matrix can extend the release profile over that of other reported methods. The anti-cancer activity of the nanoparticles was evaluated with MCF-7 breast cancer cells. Subsequently, a block polymer, poly(L-lactic acid)-block-poly(poly(ethylene glycol) monomethacrylate) (PLLA-b-PPEGMA), was synthesized for encapsulating the magnetic seeds, and the composite polymer-Fe3O4 nanoparticles were then surface functionalized with folic acid. The uptake of the folic acid functionalized nanoparticles by cancer cells was shown to be enhanced compared to that of nanoparticles without folic acid functionalization. Finally, a new PEGylation strategy was developed to increase the circulation time of magnetic nanoparticles in the blood stream via surface initiated atom transfer radical polymerization (ATRP). A silane initiator was first immobilized on the magnetic nanoparticles surface. Then, copper-mediated ATRP technique was used to graft polymerize poly(ethylene glycol) monomethacrylate (PEGMA) on the magnetic nanoparticles surface. 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G. Neoh and E. T. Kang. Synthesis of folic acid functionalized PLLA-bPPEGMA nanoparticles for cancer cell targeting. Macromol. Rapid Commun., 30, pp.609-614. 2009. 178 [...]... magnetic nanoparticles; (c-f) XPS wide scan and C 1s, Cl 2p and Si 2p core level spectra of CTS-immobilized nanoparticles 138 Figure 6.6 (a) XPS wide scan and (b) C 1s core level spectra of PPEGMAimmobilized nanoparticles after polymerization time of 4 h 140 Figure 6.7 TGA curves of (a) pristine magnetic nanoparticles, (b) CTSimmobilized nanoparticles, (c), (d), (e) PPEGMA-immobilized nanoparticles... nanoparticles The response of macrophage cells to pristine and PPEGMA-immobilized nanoparticles was compared The results showed that the macrophage cells are very effective in cleaning up the pristine magnetic nanoparticles With the PPEGMA-immobilized nanoparticles, the amount of nanoparticles internalized into the cells is greatly reduced to . SYNTHESIS AND FUNCTIONALIZATION OF MICRO/ NANO- PARTICLES FOR MALIGNANT CELLS DETECTION AND ELIMINATION HU FEIXIONG NATIONAL UNIVERSITY OF SINGAPORE. NATIONAL UNIVERSITY OF SINGAPORE 2009 SYNTHESIS AND FUNCTIONALIZATION OF MICRO/ NANO- PARTICLES FOR MALIGNANT CELLS DETECTION AND ELIMINATION HU FEIXIONG (B. E., M. S., Tianjin. technique for preparing antibacterial microparticles has the advantages of ease of mass production and scale up, and the microparticles possess stability for repeated usage. In the second part of