Trastuzumab decorated nanoparticles of biodegradable polymers for targeted small molecule chemotherapy

TRASTUZUMAB-DECORATED NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR TARGETED SMALL MOLECULE CHEMOTHERAPY SUN BINGFENG (M Eng., Tianjin University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISION OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my deepest appreciation to my supervisor, Professor Feng SiShen, for his wise guidance, patient encouragement and unconditional support, throughout the period of this research His great passion to science, serious style of work and rational logical thinking leave me a deep impression that will benefit me endlessly From all these, I have learnt how to overcome the difficulties in research and how to carry out research work independently I appreciate their continuous support and useful advice of my colleagues in Chemotherapeutic Engineering Lab, Ms Tan Mei Yee Dinah, Dr Balu Ranganathan, Dr Zhang Zhipingi, Dr Dong Yuancai, Dr Zhao Lingyun, Mr Pan Jie, Mr Chandrasekharan Prashant, Mr Liu Yutao, Mr Gan Chee Wee, Miss Panneerselvan Anitha, Miss Vanangamudi Anbharasi, Mr Phyo Wai Min and many other colleagues I may neglect to mention here Thanks also go to my parents, my husband, my sister and my friends Without their help and encouragement, this work would have been more difficult Finally, I wish to express my gratitude to National University of Singapore for providing me such a good chance and research scholarship to pursue my research in Singapore Being exposed to the frontier of bioengineering, I have thus enriched my knowledge and enhanced my ability for future work i Table of Contents Acknowledgements i Table of Contents ii Summary viii Nomenclature x List of Tables xii List of Figures xiii List of Publications xv Chapter Introduction .1 1.1 Background .1 1.2 Objective 1.3 Thesis organization Chapter Literature review .5 2.1 Cancer .5 2.1.1 Introduction to cancer 2.1.2 Causes of cancer 2.1.3 Cancer treatments .6 2.2 Cancer chemotherapy 2.2.1 Chemotherapy 2.2.2 Problems in chemotherapy .10 2.2.2.1 Toxicity 11 2.2.2.2 Dosage form 11 2.2.2.3 Pharmacokinetics 12 2.2.2.4 Drug resistance 12 ii 2.2.3 Anticancer drugs 13 2.2.3.1 Paclitaxel .13 2.2.3.2 Docetaxel 16 2.3 Nanoparticles 18 2.3.1 Nanotechnology 20 2.3.2 New-concept chemotherapy .21 2.3.3 Nanoparticle fabrication 22 2.3.3.1 Solvent extraction/evaporation technique .22 2.3.3.2 Nanoprecipitation method .23 2.3.3.3 Dialysis method 24 2.3.3.4 Salting-out method 25 2.3.4 Nanoparticle properties 26 2.4 Vitamin E TPGS 28 2.4.1 Introduction to Vitamin E TPGS .28 2.4.2 TPGS as a bioavailability enhancer 30 2.4.3 TPGS as drug absorption enhancer 31 2.4.4 Other applications 31 2.5 Targeted therapy 32 2.5.1 Introduction to targeted therapy .32 2.5.2 Passive and active targeting .35 2.5.2.1 Passive targeting .35 2.5.2.2 Active targeting .37 2.5.3 Small molecule tyrosine kinases inhibitors 38 2.5.4 Monoclonal antibody 41 2.6 HER2 targeted therapy 42 iii 2.6.1 Assessment of HER2 status .43 2.6.1.1 IHC 44 2.6.1.2 FISH 44 2.6.2 Trastuzumab (Herceptin®) 45 2.6.2.1 Mechanisms of action of trastuzumab 45 2.6.2.2 Clinical efficacy of Herceptin .47 2.6.3 Trastuzumab-functionalized nanoparticles 49 Chapter Trastuzumab-decorated biodegradable nanoparticles for targeted delivery of paclitaxel 53 3.1 Introduction 53 3.2 Materials and methods 54 3.2.1 Materials 54 3.2.2 Preparation of nanoparticles 55 3.2.3 Characterization of nanoparticles 56 3.2.3.1 Size and size distribution 56 3.2.3.2 Surface morphology 56 3.2.3.3 Surface charge .57 3.2.3.4 Drug encapsulation efficiency 57 3.2.3.5 Thermal gravimetric analysis 57 3.2.3.6 Surface chemistry analysis 58 3.2.3.7 SDS-PAGE analysis 58 3.2.4 In vitro drug release kinetics 58 3.2.5 Cell cultures .59 3.2.6 In vitro cellular uptake study .59 3.2.6.1 Qualitative study through confocal laser scanning microscopy .59 iv 3.2.6.2 Quantitative study through microplate ready analysis 60 3.2.7 In vitro cytotoxicity 61 3.3 Results and discussions .62 3.3.1 Size, size distribution and drug encapsulation efficiency 62 3.3.2 Surface charge 62 3.3.3 Surface morphology 63 3.3.4 Surface chemistry .64 3.3.5 Stability of HER2 antibody 65 3.3.6 Thermal gravimetric analysis 66 3.3.7 In vitro drug release 67 3.3.8 Cellular uptake of nanoparticles 70 3.3.9 Confocal microscopy 74 3.3.10 In vitro cytotoxicity 75 3.4 Conclusions 79 Chapter Targeted delivery of docetaxel using trastuzumab-functionalized nanoparticles of biodegradable copolymers 81 4.1 Introduction 81 4.2 Materials and methods 82 4.2.1 Materials 82 4.2.2 Synthesis of PLA-TPGS and TPGS-COOH copolymers 83 4.2.3 Preparation of nanoparticles 84 4.2.4 Characterization of nanoparticles 88 4.2.4.1 Size and size distribution 88 4.2.4.2 Surface charge .88 4.2.4.3 Quantification of trastuzumab .88 v 4.2.4.4 Surface morphology 89 4.2.4.5 Surface chemistry 89 4.2.4.6 Drug encapsulation efficiency 89 4.2.4.7 SDS-PAGE analysis 90 4.2.5 In vitro drug release kinetics 90 4.2.6 Cell cultures .91 4.2.7 In vitro cellular uptake study .91 4.2.7.1 Qualitative study: confocal laser scanning microscopy (CLSM) 91 4.2.7.2 Quantitative study: microplate reader analysis .92 4.2.8 In vitro cell cytotoxicity 92 4.3 Results and discussions .93 4.3.1 Characterization of PLA-TPGS copolymer .93 4.3.2 Size & size distribution 94 4.3.3 Quantification of trastuzumab 95 4.3.4 Surface charge 96 4.3.5 Drug encapsulation efficiency 97 4.3.6 Surface morphology of nanoparticles 97 4.3.7 Surface chemistry .98 4.3.8 Stability of HER2 antibody 100 4.3.9 In vitro drug release kinetics 101 4.3.10 Cellular uptake of nanoparticles 103 4.3.10.1 Qualitative study 103 4.3.10.2 Quantitative study .105 4.3.11 In vitro cytotoxicity 107 4.4 Conclusions .114 vi Chapter Conclusions and recommendations .116 5.1 Conclusions .116 5.2 Recommendations .117 References 119 vii Summary Targeted chemotherapy has been a challenge in Nanomedicine Nanoparticles (NPs) of biodegradable polymers can serve as effective drug delivery systems to carry the chemotherapeutic agents to cancer cells These drug delivery systems can be further functionalized with targeting ligands which can bind to specific receptors overexpressing on the surface of cancer cells, thus achieving highly-specific targeting function This work developed two novel drug delivery systems of trastuzumabdecorated nanoparticles (NPs) of biodegradable polymers for targeted chemotherapy with Taxoids (Paclitaxel and Docetaxel) as model small molecule therapeutics Trastuzumab (Herceptin®) is a FDA-approved humanized monoclonal antibody drug which is effective for the cancer of human epidermal growth factor receptor-2 (HER2) overexpression It is also found synergistic with Taxoids The first system is paclitaxel-loaded poly(D,L-lactide-co-glycolide)/montmorillonite (PLGA/MMT) NPs with trastuzumab physically adhered on the NP surface The NPs were prepared by a modified solvent extraction/evaporation method and characterized by state-of-the art equipment for their physicochemical and pharmaceutical properties such as size and size distribution, surface morphology, surface chemistry, surface charge, drug encapsulation efficiency and in vitro drug release kinetics Both of the quantitative and qualitative investigation showed that the paclitaxel-loaded PLGA/MMT NPs with trastuzumab-decoration achieved significantly higher cellular uptake efficiency than the NPs without trastuzumab-decoration The results of in vitro cytotoxicity experiment on SK-BR-3 cells further proved the targeting effects of trastuzumab decoration on the PLGA/MMT nanoparticles Judged by IC50 of SK-BR- viii cells after 24 h culture, the therapeutic effects of the Pac-PLGA/MMT-HER NP formulation could be 12.74 times higher than that of the Pac-PLGA/MMT NP formulation and 13.11 times higher than Taxol® The second system is the docetaxel-loaded NPs of a blend of two novel copolymers One is poly(lactide)-D-α-tocopheryl polyethylene glycol succinate (PLA-TPGS), which is of ideal hydrophobic-lipophilic balance for high drug encapsulation efficiency and high cellular adhesion, and another is carboxyl group-terminated TPGS (TPGSCOOH), which facilitates the antibody conjugation on the nanoparticle surface The targeting effect can be quantitatively controlled by adjusting the copolymer blend ratio, which was testified by in vitro viability experiments on SK-BR-3 cells and MCF-7 cells Judged by cellular mortality, the trastuzumab-functionalized NP formulation can be 1.215-, 1.215- and 1.073-fold more effective for MCF-7 cells, and 1.697-, 1.886and 1.126-fold more effective for SK-BR-3 cells than the NP formulation without trastuzumab-functionalization after 24, 48 and 72 h treatment, respectively The trastuzumab-functionalized NPs have great potential to be applied as targeted therapeutics against the HER2-overexpressing cancer ix Iannello A, Ahmad A Role of antibody-dependent cell-mediated cytotoxicity in the efficacy of therapeutic anti-cancer monoclonal 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(PLGA/MMT) nanoparticles decorated by trastuzumab for targeted chemotherapy of breast cancer Biomaterials 2008; 29(4): 475-486 Sun B, Feng SS Trastuzumab decorated nanoparticles for targeted chemotherapy. .. conjugation of trastuzumab on NP surface for targeted drug delivery Experiments were carried out to investigate the feasibility of the obtained NPs for targeted delivery of small molecule drugs The trastuzumab. .. objective of this research is to study the effectiveness of two systems of trastuzumabfunctionalized nanoparticles for targeted drug delivery In the first part, a novel system of trastuzumab- decorated