MULTIFUNCTIONAL FOLATE CONJUGATED POLYMERIC MICELLES FOR ACTIVE INTRACELLULAR DRUG DELIVERY ZHAO HAIZHENG NATIONAL UNIVERSITY OF SINGAPORE 2007 MULTIFUNCTIONAL FOLATE CONJUGATED POLYMERIC MICELLES FOR ACTIVE INTRACELLULAR DRUG DELIVERY ZHAO HAIZHENG (B. Eng. & M. Eng., TIANJIN UNIVERSITY, PRC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements Firstly, I would like to thank my supervisor, Dr. Lanry Yung, for his support, guidance and inspiration during my Ph.D. work in these four years. His rigorous methodology, objectivity and enthusiasm in research showed me the qualities required to succeed in the research field. He also created a scientific and amiable lab environment for the students. His advice will serve me in all aspects of life. Special thanks are also given to National University of Singapore for its financial support. I would also like to thank the faculty and stuff in the Chemical and Biomolecular Engineering for their support, especially Ms. Li Xiang, Ms. Li Fengmei, Mr. Han Guangjun, Mr. Boey Kok Hong and Ms. Tay Choon Yen. I also appreciate the support of lab officers from other departments of NUS, Ms. Kho Jia Yann from the Insitu Hybridization Lab, Mr. Ong Ling Yeow and Mr. Toh Kok Tee from the Flow Cytometry Lab, Mr. James Low from the Animal Holding Unit. I am grateful to my colleagues and fellow graduate students, Mr. Zhong Shaoping, Mr. Qin Weijie, Mr. Zhu Xinhao, Mr. Khew Shih Tak, Mr. Tan Chau Jin, Weiling, Deny and phuoc. They not only help me on my research, but also offer great friendship which is so important to me. I would also like to thank my friends from Tianjin University who are also doing Ph.D. in our department. With their friendship and support, I have a wonderful time while I am far away from China. Finally, I am most grateful to my parents and my brother whose love, support and understanding made this work possible. I also deeply appreciate my husband, without his support and encouragement, I would not have completed this Ph.D. research. I Table of Contents Acknowledgements I Table of Contents II Summary VI List of Figures IX List of Tables XIV Chapter Introduction .1 1.1 Background . 1.2 Aims and scope of this project Chapter Literature Review 2.1 Tumor specific chemotherapy . 2.1.1 Side effects of traditional chemotherapy 2.1.2 Anatomical, physiological and pathological considerations .6 2.1.3 Tumor Targeting .10 2.2 Multidrug resistance of Chemotherapy . 24 2.3 Drug Delivery Systems . 26 2.3.1 Liposomes .27 2.3.2 Polymeric microparticles/nanoparticles 29 2.3.3 Polymer-drug conjugates 30 2.3.4 Polymeric Micelles .32 Chapter The synthesis and characterization of PLGA-PEG-FOL micelles 34 3.1 Introduction . 34 3.2 Materials and methods 35 3.2.1 Materials .35 3.2.2 Synthesis of Conjugates 35 3.2.3 Characterization of polymers 41 3.2.4 Preparation of DOX-loaded PLGA-PEG-FOL micelles .42 3.2.5 Characterization of polymer micelles .42 3.2.6 In vitro drug release 45 3.3 Results and discussion 46 3.3.1 Synthesis and characterization of PLGA-PEG-FOL conjugate 46 3.3.2 Properties of copolymer micelles 52 3.3.3 In vitro drug release 60 3.4 Conclusions . 61 Chapter Selectivity of folate conjugated polymer micelles for anticancer drug delivery 62 4.1 Introduction . 62 4.2 Materials and methods 63 4.2.1 Materials .63 4.2.2 Preparation and characterization of DOX-loaded micelles 63 4.2.3 In vitro cell culture studies 64 II 4.2.4 Statistical analysis .67 4.3 Results and discussion 67 4.3.1 Characterization of DOX-loaded micelles 67 4.3.2 Expression of folate receptors in different cell lines 68 4.3.3 Cytotoxicity Test .68 4.3.4 Cellular uptake of DOX 71 4.3.5 Cell cycle analysis .73 4.3.6 The effect of folate content on micelles to targeting efficiency .78 4.4 Conclusions . 80 Chapter pH-triggered drug release for active intracellular drug delivery .81 5.1 Introduction . 81 5.2 Materials and methods 83 5.2.1 Materials .83 5.2.2 Synthesis of the poly(β-amino ester)-PEG-FOL conjugate 83 5.2.3 Characterization of polymers 84 5.2.4 Preparation and characterization of DOX-loaded micelles 86 5.2.5 Acid-base titration .86 5.2.6 Physicochemical properties of polymer micelles .86 5.2.7 In vitro cell experiments .87 5.2.8 Statistical analysis .87 5.3 Results and discussion 87 5.3.1 Characterization of poly(β-amino ester)-PEG-FOL copolymer .87 5.3.2 Buffering capacity of the polymers .89 5.3.3 The effects of pH values on the physicochemical properties of micelles .90 5.3.4 Critical association concentration (CAC) .92 5.3.5 In vitro drug release 95 5.3.6 Cytotoxicity Test .97 5.3.7 Cellular uptake of DOX 100 5.4 Conclusions . 102 Chapter Folate conjugated polymer micelles formulated with TPGS 103 6.1 Introduction . 103 6.2 Materials and methods 104 6.2.1 Materials .104 6.2.2 Preparation of DOX-loaded micelles 105 6.2.3 Physicochemical properties of polymer micelles .106 6.2.4 Drug release study .106 6.2.5 In vitro cellular assays 107 6.2.6 Statistical analysis .109 6.3 Results and discussion 110 6.3.1 Physicochemical properties of DOX-loaded PLGA-PEG-FOL micelles.110 6.3.2 In vitro drug release 112 6.3.3 Cellular DOX uptake 113 6.3.4 Cytotoxicity Test .118 6.3.5 Apoptosis assay .122 III 6.3.6 Accumulation of rhodamine in Caco-2 cells 123 6.4 Conclusions . 126 Chapter The pharmacokinetics and tissue distribution of folate conjugated polymer micelles 127 7.1 Introduction . 127 7.2 Materials and methods 128 7.2.1 Materials .128 7.2.2 In vitro cell culture studies 128 7.2.3 Subcutaneous tumor growth .129 7.2.4 Pharmacokinetics of the four drug formulations 129 7.2.5 Biodistribution study .130 7.3 Results and discussion 131 7.3.1 Tumor growth .131 7.3.2 Pharmacokinetics of the DOX-loaded micelles 133 7.3.3 Biodistribution 135 7.3.4 Tumor and heart morphology analysis .142 7.4 Conclusions . 146 Chapter Potential use of cholecalciferol polyethylene glycol succinate as a novel pharmaceutical additive .147 8.1 Introduction . 147 8.2 Materials and methods 148 8.2.1 Materials .148 8.2.2 Synthesis of cholecalciferol polyethylene glycol succinate (CPGS) 149 8.2.3 Characterization of CPGS .151 8.2.4 Preparation of DOX-loaded PLGA nanoparticles 151 8.2.5 Characterization of polymer nanoparticles .151 8.2.6 Accumulation of rhodamine in Caco-2 cells 152 8.2.7 Cytotoxicity assay .153 8.2.8 Statistical analysis .153 8.3 Results and discussion 154 8.3.1 Characterization of CPGS .154 8.3.2 Critical micelles concentration (CMC) of CPGS 158 8.3.3 The effect of CPGS on physicochemical properties of nanoparticles 160 8.3.4 Drug release 162 8.3.5 Accumulation of rhodamine in Caco-2 cells 164 8.3.6 Cytotoxicity test 166 8.4 Conclusions . 169 Chapter Conclusions & Recommendations 170 9.1 Conclusions . 170 9.2 Recommendations for future work . 173 9.2.1 Effects of PEG length of folate conjugates on targeting ability and antitumor effects .173 9.2.2 The antitumor efficacy of different drug formulations .175 IV 9.2.3 The mechanism of TPGS or CPGS on the MDR inhibition .176 9.2.4 Using the current delivery system as magnetic resonance imaging (MRI) contrast agents 178 Reference .180 Appendix Ι .195 Appendix II 198 V Summary Many chemotherapy treatments have significant side effects because non-specific delivery of anticancer drugs damages healthy organs. Folate or folic acid has been employed as a targeting moiety of various anticancer agents to increase their cellular uptake within target cells since folate receptors (FRs) are vastly overexpressed in many human tumors. In this thesis, a biodegradable polymer poly(D,L-lactide-co-glycolide)poly(ethylene glycol)-folate (PLGA-PEG-FOL) was used to form micelles for encapsulating anticancer drug doxorubicin (DOX). The difference of cytotoxicity, cellular uptake and apoptosis percentage between different cancer cells and healthy cells implies that the folate conjugated micelles has the ability to selectively target cancer cells that overexpress FRs on their surface. Furthermore, the amount of folate on the micelles was optimized at 40%-65% in order to kill cancer cells but, at the same time, have minimal effect on normal healthy cells. To accelerate the drug release in endosome, a pH-sensitive block copolymer poly(βamino ester)-PEG-FOL was synthesized. This copolymer is hydrophilic at endosomal pH of 5-6. However, under physiological environment (pH 7.4), the poly(β-amino ester) block is hydrophobic but the PEG-FOL block is hydrophilic, resulting in the formation of polymer micelles with poly(β-amino ester) in the core and PEG-FOL at the shell. To control the drug release from the micelles, mixed micelles of PLGA-PEG-FOL and poly(β-amino ester)-PEG-FOL were fabricated. The incorporation of pH-sensitive polymer in the micelles increased the buffering ability and changed physicochemical properties at the endosomal pH. The release of DOX in the micelles was accelerated at VI pH 5.0, which resulted in increased cytoplasmic concentration of DOX and improved cytotoxicity. This formulation would be useful as an effective intracellular delivery carrier of hydrophobic therapeutic agents. Another serious problem associated with cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells during the course of treatment. To overcome MDR, a new drug formulation - PLGA-PEG-FOL formulated with d-α-tocopheryl polyethylene glycol succinate (TPGS), known as mixed micelles, was fabricated. Compared with the PLGA-PEG-FOL formulation, the addition of TPGS showed higher cellular uptake of DOX, and subsequently a higher degree of DNA damage and apoptosis, and eventually a higher cytotoxicity to drug resistant cells. The enhanced cellular uptake of mixed micelles was related to the P-glycoprotein (P-gp) inhibition function of TPGS. In addition, the formulations with TPGS also selectively enhance the cytotoxicity of drug resistant cancer cells with overexpressed folate receptors and affect normal cells at minimum. The pharmacokinetics and biodistribution of this new formulation was also evaluated with rat tumor xenograft models. The mixed micelles formulation showed enhanced drug accumulation in drug resistant tumors. 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The fabrication of iron oxide (Fe3O4) nanoparticles Materials: FeCl3.6H2O, oleic acid, octadecene, methanol and NaOH The synthesis of Fe3O4 requires two steps. Step is to synthesize the intermediate product, Fe(III) Oleate; and Step is to form Fe3O4 from the intermediate product. In Step 1, 0.54g of FeCl3.6H2O was dissolved in 10ml of methanol (solution 1). Separately, 0.24g of NaOH was dissolved in 20ml of methanol (solution 2). Under vigorous stirring, 1.7 ml oleic acid was added into solution 1. Oleic acid is a long chain surfactant which acts as a stabilizer in ferrofluid suspension to prevent agglomeration. After homogeneous mixing, solution was added into solution under stirring. The intermediate product Fe(III) oleate was formed after the brown precipitate was dried under vacuum. Subsequently, Step was carried out to produce Fe3O4. The intermediate product was placed on a hot plate with magnetic stirrer and 15ml of octadecene was added in. The mixture was stirred while being heated at 80◦C until the precipitate was dissolved completely. At this point, 1.7ml of oleic acid was added into the mixture and the solution was heated at 300◦C for 30 mins and cool down to room temperature. The final product is Fe3O4 in oleic acid. 2. The preparation of Fe3O4 loaded PLGA-PEG-FOL micelles Known amount of Fe3O4 and PLGA-PEG-FOL was dissolved in ml of DCM and stirred until the mixture was well-mixed. Subsequently, the oil mixture was dripped into DI water by drop wise. The mixture was stirred overnight to evaporate the organic solvents. 195 Appendix 3. FT-IR spectrum of the Fe3O4 loaded PLGA-PEG-FOL micelles 130 %T Characteristic of Fe3O4 Characteristic of PLGA-PEG 80 30 3900 3400 2900 2400 1900 1400 900 400 -1 (cm ) Wave Number (cm-1) Figure A1 FT-IR spectrum of the Fe3O4 loaded PLGA-PEG-FOL micelles 4. The morphology of Fe3O4 loaded PLGA-PEG-FOL micelles Figure A2 TEM images of iron oxide nanoparticles encapsulated in micelles Since TEM can not focus on both nanoparticles and the micelles, only iron oxide nanoparticles were observed as small black dots. However, from the TEM images, it is 196 Appendix believed that the iron oxide clusters are encapsulated in the micelles. Due to the limitation of equipment, micelles were not visible under TEM. From the images, the average size of iron oxide encapsulated PLGA-PEG is 100-200 nm. (a) (b) Figure A3 FESEM images of (a) Fe3O4 encapsulated in polymeric micelles, (b) a zoomed-in micelle. The FESEM images also show that micelles size is around 100 to 200 nm. The zoomedin image demonstrates that iron oxide nanoparticles were encapsulated within the polymer micelles. 197 Appendix Appendix II List of Publications Journal publication: 1. Haizheng Zhao and Lanry Yung, Selectivity of folate conjugated polymer micelles against different tumor cells, International Journal of Pharmaceutics, 2008, 349: 258-268. 2. Haizheng Zhao, Engcheong Tan and Lanry Yung, Potential use of cholecalciferol polyethylene glycol succinate as a novel pharmaceutical additive, Journal of biomedical materials research Part A, 2008, 84(4): 954-964. 3. Haizheng Zhao and Lanry Yung, The effect of TPGS on folate conjugated polymer micelles for selective tumor targeting, Journal of biomedical materials research Part A, 2008, Under revision. 4. Haizheng Zhao and Lanry Yung, Folate conjugated polymeric micelles with pHtriggered drug release property for active intracellular drug delivery, Journal of biomedical materials research Part A, 2008, Under review. 5. Haizheng Zhao and Lanry Yung, The pharmacokinetics and tissue distribution of folate conjugated polymer micelles, Pharmacertical Research, 2008, Submitted. Conference publication: 1. Haizheng Zhao and Lanry Yung, Folate receptor mediated targeting delivery of antineoplastic drugs, 32nd annual meeting of the controlled release society 2005, Miami, USA. 2. Haizheng Zhao and Lanry Yung, Folate conjugated polymer micelles for targeting drug delivery, 33rd annual meeting of the controlled release society 2006, Vienna, Austria. 198 [...]... copolymer micelles were fully evaluated, such as micelles size, morphology, stability, surface properties, drug loading content and drug release 2 Study the selectivity of the folate conjugated micelles between cancer cells and normal cells Until now, the selectivity of the folate conjugated micelles has not been addressed Therefore, in this thesis, the in vitro selectivity of the targeting delivery. .. cellular uptake of drugs in the cancer cells by inhibiting P-gp mediated MDR 12-14 Folate conjugated micelles formulated with TPGS was fabricated to evaluate the effects of TPGS on the physicochemical properties, cellular uptake and selective cytotoxicity of the folate conjugated micelles In particular, whether the addition of TPGS to the micelles can increase the cellular uptake of DOX in the drug resistant... target for the delivery of anticancer drugs It has also been shown that folate achieves deeper penetration than normal antibodies as receptor ligands 37 The exploitation of FR-mediated drug delivery has been referred to as a molecular Trojan horse approach where drugs attached to folate are shuttled inside a targeted FR-positive cell in a stealth-like fashion Folate displays extremely high affinity for. .. vivo effects of the multifunctional polymeric micelles The pharmacokinetics and biodistribution was evaluated with rat tumor xenograft models Two different tumor models, drug sensitive model and drug resistant model, were compared Different drug formulations were evaluated to compare their targeting ability and MDR inhibition Tumor and heart histology was also performed to study the drug accumulation... cells that are actively dividing If the treatments could be targeted specifically to cancer cells, side effects would drastically decrease, improving 5 Chapter 2 the quality of life for patients Therefore, scientists focus on the research of drug delivery systems (DDS) to decrease the side effects The design of an effective drug delivery system must meet two primary criteria First, the drug delivery vehicle... DOX, DOX-loaded PLGA-PEG-FOL micelles and mixed micelles with 10% TPGS 116 Figure 6.5 Confocal images of KB/DOX cells treated with drug formulations (a) free DOX, (b) DOX-loaded PLGA-PEG micelles, (c) DOX-loaded PLGA-PEG-FOL micelles, (d) DOX-loaded mixed micelles with 5% TPGS, (e) DOX-loaded mixed micelles with 10% TPGS and (f) Fibroblasts treated with mixed micelles with 10% TPGS 117... of the micelles, the synergistic effects between the tumor targeting and P-gp inhibition, and the in vivo therapeutic effects of the micelles The specific objectives of this thesis include: 1 Design a tumor targeting delivery system A biodegradable block copolymerpoly(D,L-lactide-co-glycolide)-poly(ethylene glycol) -folate (PLGA-PEG-FOL) was prepared to form micelles for encapsulating anticancer drug. .. process that mediates folate- targeted drug delivery is identical to that for the free vitamin 34, 35 As illustrated in Figure 2.4, exogenous folate- drug conjugates bind to externally oriented FRs located on the plasma cell membrane This is a highly specific event, i.e., analogous to a key (folate) inserting into a lock (FR) Immediately after binding, the plasma membrane surrounding the folate conjugate/FR... challenge is to develop a new delivery system which has targeting ability to cancer cells and at the same time overcome MDR in cancer cells This PhD work aims to fabricate multifunctional polymeric micelles which could specifically target to cancer cells and overcome MDR The scope of this thesis include the fabrication of multifunctional polymeric micelles, the selectivity of the micelles between cancer... pH-sensitive polymer to increase the drug cytotoxicity After folatemediated endocytosis, in order to accelerate the drug release in early endosome, a pHsensitive polymer- poly(β-amino ester)-PEG-FOL conjugate was prepared The effects of the pH- sensitive polymer to drug release, cytotoxicity and cellular uptake were investigated 4 Fabricate multifunctional polymeric micelles which could specifically target . MULTIFUNCTIONAL FOLATE CONJUGATED POLYMERIC MICELLES FOR ACTIVE INTRACELLULAR DRUG DELIVERY ZHAO HAIZHENG NATIONAL UNIVERSITY OF SINGAPORE 2007 MULTIFUNCTIONAL. MULTIFUNCTIONAL FOLATE CONJUGATED POLYMERIC MICELLES FOR ACTIVE INTRACELLULAR DRUG DELIVERY ZHAO HAIZHENG (B. Eng. & M. Eng., TIANJIN UNIVERSITY, PRC) A THESIS SUBMITTED FOR THE. Properties of copolymer micelles 52 3.3.3 In vitro drug release 60 3.4 Conclusions 61 Chapter 4 Selectivity of folate conjugated polymer micelles for anticancer drug delivery 62 4.1 Introduction