MULTIFUNCTIONAL NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR DIAGNOSIS AND TREATMENT OF CANCER PAN JIE NATIONAL UNIVERSITY OF SINGAPORE 2009 MULTIFUNCTIONAL NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR DIAGNOSIS AND TREATMENT OF CANCER PAN JIE (M. ENG., TIANJIN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENT First of all, I would like to express my deepest gratitude to my supervisor, A/P Feng Si-Shen, for his heartfelt guidance, valuable suggestions, profound discussion and encouragement throughout the entire period of this research work. His enthusiasm, sincerity and dedication to scientific research have greatly impressed me and will benefit me in my future career. I am also thankful to my all colleagues and laboratory officers for their support and assistance. In particular, thanks are due to Dr. Dong Yuancai, Dr. Zhang Zhiping, Ms. Sun Bingfeng, Mr. Prashant Chandrasekharan, Ms. Anitha Panneerselvan, Mr. Liu Yutao, Ms. Anbharasi Vanangamudi, Mr. Gan Chee Wee, Ms. Tan Mei Yee, Dinah, and many other colleagues for their kind help and assistance. It is my great pleasure to work with all of them. I am also indebted to the technical staff of Department of Chemical & Biomolecular Engineering, especially Dr. Yuan Zeliang, Mr. Boey Kok Hong , Mdm Lee Chai Keng, Mdm Li Xiang, Mdm Li Fengmei for their help and support. The research scholarship provided by National University of Singapore is also gratefully acknowledged. Finally, I would like to express my deepest gratitude and indebtedness to my wife, my parents, my daughter, my brother for their love and support. I TABLE OF CONTENTS ACKNOWLEDGEMENT I TABLE OF CONTENTS II SUMMARY X NOMENCLATURE XIII LIST OF FIGURES XV LIST OF TABLES XX Chapter Introduction . 1.1 General background . 1.2 Objective and thesis organization . Chapter Literature Review 2.1 Nanocarriers for Cancer Therapy 2.1.1 Passive and active targeting . 2.1.2 NP carriers for targeted therapy . 10 2.1.2.1 Biodegradable polymer NPs 11 2.1.2.2 Liposomes 12 2.1.2.3 Dendrimers . 13 2.1.2.4 Nucleic-acid-based NPs 13 2.1.2.5 Nanoshells 14 2.1.3 Targeting molecules for the development of targeted NPs 15 2.1.3.1 Folate-based targeting molecules 15 2.1.3.2 Monoclonal antibodies 16 2.1.3.3 Aptamer targeting molecules . 17 2.1.3.4 Oligopeptide-based targeting molecules 19 II 2.2 Molecular Imaging . 20 2.2.1 Introduction . 20 2.2.2 Imaging modalities 22 2.2.2.1 Nuclear imaging 23 2.2.2.2 MR Imaging 25 2.2.2.3 Ultrasound 26 2.2.2.4 Optical Imaging 27 2.2.3 Molecular imaging of cancer . 29 2.2.3.1 Cancer diagnosis and staging 29 2.2.3.2 Tumor characterization . 31 2.2.3.3 Therapy assessment 31 2.2.4 Conclusions . 32 2.3 QDs as Cellular Probes 33 2.3.1 Properties of QDs 33 2.3.2 Synthesis of quantum dots and QDs solubilization . 35 2.3.3 Conjugating QDs with biomolecules 37 2.3.4 Cellular imaging and tracking 40 2.3.5 Tumor targeting and imaging . 41 2.3.6 Cytotoxicity . 42 2.3.7 Prospective 43 Chapter Formulation, Characterization and In Vitro Evaluation of Quantum Dots Loaded in Poly(lactide)-vitamin E TPGS Nanoparticles for Cellular and Molecular Imaging 45 3.1 Introduction . 45 3.2 Materials and Methods . 50 III 3.2.1 Materials . 50 3.2.2 Synthesis of PLA-TPGS copolymer . 51 3.2.3 Preparation of QDs-loaded PLA-TPGS NPs and MAA-coated QDs 51 3.2.4 Characterization of QDs-loaded PLA-TPGS NPs . 52 3.2.4.1 Particle size analysis . 52 3.2.4.2 Surface morphology 52 3.2.4.3 Surface chemistry of QDs-loaded PLA-TPGS NPs . 53 3.2.5 Photophysical characterization . 54 3.2.5.1 Fluorescent images 54 3.2.5.2 Emission spectra . 54 3.2.5.3 The photostability . 54 3.2.6 QDs encapsulation efficiency 55 3.2.7 Cell line experiment 55 3.2.7.1 Cell culture . 55 3.2.7.2 In vitro cellular uptake of QDs-loaded PLA-TPGS NPs 56 3.2.7.3 In vitro cytotoxicity of QDs-loaded PLA-TPGS NPs . 56 3.3 Result and Discussion 57 3.3.1 Characterization of the PLA-TPGS copolymer 57 3.3.2 Characterization of QDs-loaded PLA-TPGS NPs . 58 3.3.2.1 Size and size distribution . 58 3.3.2.2 Surface morphology 59 3.3.2.3 Surface chemistry of QDs-loaded PLA-TPGS NPs . 61 3.3.3 Photophysical characterization . 64 3.3.3.1 Fluorescent colors and emission spectra 64 3.3.3.2 Photostability 67 IV 3.3.4 QDs encapsulation efficiency 69 3.3.5 In vitro evaluation . 69 3.3.5.1 Cellular uptake of nanoparticles 69 3.3.5.2 In vitro cytotoxicity of QDs-loaded PLA-TPGS NPs . 70 3.4 Conclusion . 73 Chapter Targeted Delivery of Paclitaxel Using Folate-decorated Poly(lactide)-vitamin E TPGS Nanoparticles . 75 4.1 Introduction . 75 4.2 Materials and Methods . 78 4.2.1 Materials . 78 4.2.2 Synthesis and characterization of PLA-TPGS copolymer . 79 4.2.3 Synthesis of TPGS-COOH and FOL-NH . 80 4.2.4 Formulation of paclitaxel-loaded NPs with folate-decoration . 81 4.2.5 Characterization of paclitaxel-loaded NPs with folate decoration . 82 4.2.5.1 Particle size and size distribution 82 4.2.5.2 Surface charge 83 4.2.5.3 Surface morphology 83 4.2.5.4 Drug encapsulation efficiency . 83 4.2.6 Surface chemistry 84 4.2.7 In vitro drug release kinetics 84 4.2.8 Cell cultures 84 4.2.9 In vitro cellular uptake of NPs . 85 4.2.10 In vitro cytotoxicity . 86 4.3 Results and Discussion . 87 4.3.1 Characterization of PLA–TPGS copolymers 87 V 4.3.2 Characterization of folate-decorated NPs . 89 4.3.2.1 Size and size distribution . 89 4.3.2.2 Surface charge 89 4.3.2.3 Surface morphology 90 4.3.2.4 Drug encapsulation efficiency . 91 4.3.3 Surface chemistry 91 4.3.4 In vitro drug release . 92 4.3.5 In vitro cellular uptake of NPs . 93 4.3.6 In vitro cytotoxicity . 97 4.4 Conclusion . 99 Chapter Targeting and Imaging Cancer Cells by Folate Decorated, Quantum Dots Loaded Nanoparticles of Biodegradable Polymers 101 5.1 Introduction . 101 5.2 Materials and Methods . 106 5.2.1 Materials . 106 5.2.2 Synthesis of TPGS-COOH and folate-NH 107 5.2.3 Formulation of QDs-loaded NPs with folate-decoration and free QDs . 107 5.2.4 Characterization of QDs-loaded NPs with folate decoration . 109 5.2.4.1 Particle size and size distribution 110 5.2.4.2 Surface charge 110 5.2.4.3 Surface morphology 110 5.2.4.4 Emission spectrum 110 5.2.4.5 QDs encapsulation efficiency 111 5.2.5 Surface chemistry 111 VI 5.2.6 Cell line experiment 112 5.2.6.1 Cell cultures 112 5.2.6.2 In vitro cellular uptake of NPs . 112 5.2.6.3 In vitro cytotoxicity . 113 5.3 Results and Discussion . 114 5.3.1 Characterization of QDs-loaded NPs with folate decoration . 114 5.3.1.1 Size and size distribution . 114 5.3.1.2 Surface charge 115 5.3.1.3 Surface morphology 115 5.3.1.4 Emission Spectrum . 116 5.3.1.5 QDs encapsulation efficiency 116 5.3.2 Surface chemistry 116 5.3.3 In vitro cellular uptake of NPs . 117 5.3.4 In vitro cytotoxicity . 119 5.4 Conclusion . 123 Chapter Multifunctional QDs/ Docetaxel -loaded Poly(lactic-co-glycolic acid) Targeted Nanoparticles for Cancer Cell imaging and Therapy 125 6.1 Introduction . 125 6.2 Materials and Methods . 129 6.2.1. Materials 129 6.2.2 Synthesis of TPGS-COOH 130 6.2.3 Synthesis of FOL-NH 131 6.2.4 Preparation of QDs/docetaxel-loaded PLGA/TPGS-COOH NPs 131 6.2.5 Formulation of QDs/docetaxel-loaded PLGA/TPGS-COOH NPs with FOL conjugation 132 VII 6.2.6 Characterization of TC and FD NPs . 133 6.2.6.1 Particle size and size distribution 133 6.2.6.2 Drug encapsulation and loading efficiency 134 6.2.6.3 Surface charge 134 6.2.6.4 Surface morphology 135 6.2.6.5 Surface chemistry . 135 6.2.7 In vitro drug release . 136 6.2.8 Photophysical characterization . 136 6.2.8.1 Fluorescent images 136 6.2.8.2 Emission spectra . 136 6.2.9 QDs encapsulation and loading efficiency . 137 6.2.10 Cell line experiment . 137 6.2.10.1 Cell cultures 137 6.2.10.2 In vitro cellular uptake of NPs . 138 6.2.10.3 In vitro therapeutic effect and targeting effects 139 6.3 Results and Discussion . 140 6.3.1 Characterization of QDs/docetaxel-loaded NPs 140 6.3.1.1 Particle size and size distribution 140 6.3.1.2 Drug encapsulation and loading efficiency 141 6.3.1.3. 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Generic strategy of preparing fluorescent conjugated polymer loaded poly(DL-lactide-co-glycolide) nanoparticles for targeted cell imaging，Li Kai, Pan Jie, Liu Yutao, Feng SS, Liu Bin，Submitted to Advanced Functional Materials. (NUS Journal Tier 1, JIF=6.808). 4. Multifunctional docetaxel/QDs-loaded poly (lactic-co-glycolic acid) targeted nanoparticles for diagnosis and cancer therapy, Pan Jie, Liu Yutao, Feng SS, to be submitted to Small. 5. Targeting and imaging cancer cells by folate decorated, quantum dots loaded nanoparticles of biodegradable polymers, Pan Jie, Feng SS, Biomaterials, 30 (2009) :1176-1183. (NUS Journal Tier 1, JIF=6.646). (Ranked by SienceDirect.com as Top 25 Hottest Paper at 17th position in the journal of Biomaterials, Q1 2009) 6. Formulation, characterization and in vitro evaluation of quantum dots-loaded poly(lactide)-vitamin E TPGS nanoparticles for cellular and molecular imaging, Pan Jie, Wan Yan, Feng SS, Biotechnology and Bioengineering, 2008, 101(3) :622-633. (NUS Journal Tier 1, JIF=2.936). 7. Targeted delivery of paclitaxel using folate-decorated poly (lactide)-vitamin E TPGS nanoparticles, Pan Jie, Feng SS, Biomaterials，29 (2008) :2663-2672. (NUS Journal Tier 1, JIF=6.646). (Ranked by SienceDirect.com as Top 25 Hottest Paper at 6th position in the journal of Biomaterials, Q2 2008) 188 [...]... degradation rate and hydrophobic/hydrophilic balance and develop the copolymer nanoparticles for cancer chemotherapy and imaging, as well as to prepare targeted nanoparticles for cancer therapy and imaging using a new strategy that is the contents of targeting ligands on the surface of nanoparticles can be controlled through adjusting the component ratio of two polymers First of all, a novel system of poly(lactide... targeted delivery of the drug to the corresponding cancer cells and thus enhance its therapeutic effects and reduced its side effects In Chapter 5, a new strategy was developed to prepare folate-decorated nanoparticles of biodegradable polymers for QDs formulation for targeted and sustained imaging for cancer diagnosis at its early stage Two polymers of poly(lactide)-vitamin E TPGS (PLA-TPGS) and vitamin... effects, and promote a sustainable imaging 2) to synthesize nanoparticles (NPs) of the blend of two component polymers for targeted chemotherapy with paclitaxel used as model drug 3) to develop a new strategy to prepare folate-decorated nanoparticles of biodegradable polymers loading QDs for targeted and sustained imaging for cancer diagnosis at its early stage 4) to prepare biodegradable polymeric multifunctional. .. imaging and medical diagnostics, especially the cancer detection in its early stage Finally, multifunctional NPs of biodegradable polymers loaded with QDs and docetaxel for targeting, therapy and imaging were prepared and characterized In this study, docetaxel and quantum dots were employed as the model anticancer drug and imaging agent, respectively Nanoparticles of polymer blend containing PLGA and TPGS-COOH... pristine drug and the folate-decoration can significantly promote targeted delivery of the drug to the corresponding cancer cells and thus enhance its therapeutic effects and reduced its side effects Next, a new strategy was developed to prepare folate-decorated nanoparticles of biodegradable polymers for QDs formulation for targeted and sustained imaging In order to reduce the cytotoxicity of QDs, two... is feasible for targeted imaging to improve imaging specificity and sensitivity as well as to reduce side effects of QDs to normal cells In Chapter 6, folate-decorated nanoparticles of biodegradable polymers loaded with QDs and docetaxel for targeting, therapy and imaging were prepared and characterized In this study, docetaxel and quantum dots were employed as the model anticancer drug and imaging... drugs Novel biodegradable polymers/ copolymers with desired prolonged residence time in the circulation system and enhanced therapeutic efficacy of the loaded drug are thus needed Currently, surgical intervention, radiation and chemotherapeutic drugs are widely used methods for cancer treatments But these approaches often also kill healthy cells 2 and lead to toxicity for the patients Therefore, the development... blood) (http://en.wikipedia.org/wiki /Cancer) Different from cancer, benign tumors are self-limited, do not invade or metastasize Cancer may affect people at all ages, even fetuses Cancer is the first-leading cause of death in Singapore, and the proportion of cancer deaths in all causes of death was 27.7% in 2007 (http://www.moh.gov.sg) Nanoparticles of biodegradable polymers are prepared from natural... cytotoxicity for both of MCF-7 cells and NIH 3T3 cells Additionally, our findings indicated that under same conditions, cytotoxicity of QDs formulated in the PLA-TPGS/TPGS-COOH NPs is lower for normal cells such as NIH 3T3 cells than that for beast cancer such as MCF-7 breast cancer cells due to folate targeting effect Our study showed that QDs formulated in folate-decorated nanoparticles of PLA-TPGS/TPGS-COOH... against cancer 1.2 Objective and thesis organization The overall purpose in this thesis is to develop nanoparticles of biodegradable polymers for molecular imaging and targeted therapy In particular, the objectives of this thesis include: 3 1) to prepare a novel system of poly(lactide acid) - d-α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) nanoparticles (NPs) loading quantum dots (QDs) formulation . MULTIFUNCTIONAL NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR DIAGNOSIS AND TREATMENT OF CANCER PAN JIE NATIONAL UNIVERSITY OF SINGAPORE 2009 MULTIFUNCTIONAL. MULTIFUNCTIONAL NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR DIAGNOSIS AND TREATMENT OF CANCER PAN JIE (M. ENG., TIANJIN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. degradation rate and hydrophobic/hydrophilic balance and develop the copolymer nanoparticles for cancer chemotherapy and imaging, as well as to prepare targeted nanoparticles for cancer therapy and imaging