Development and Application of Modified Hylauronic Acid
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DEVELOPMENT AND APPLICATION OF MODIFIED HYLAURONIC ACID DERIVED NANOPARTICLES FOR IMAGE GUIDED SURGERY AND DRUG DELIVERY BY TANNER KINKADE HILL A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Biomedical Engineering May, 2015 Winston-Salem, North Carolina Approved By: Aaron M. Mohs, PhD, Advisor and Chair Lissett R. Bickford, PhD Nicole H. Levi, PhD Frank C. Marini, PhD, Thaddeus J. Wadas, PhD DEDICATION AND ACKNOWLEDGEMENTS I would first like to thank my advisor Dr. Aaron M. Mohs for the extensive support, guidance, and encouragement he has provided, without which this work would not have been possible. Throughout the time spent in his lab, Dr. Mohs has had an incredibly supportive, innovative, and driving role as my advisor and this has been critical in terms of learning, productivity, and well-being. I would also like to thank the other members of my committee, Dr. Frank C. Marini, Dr. Nicole H. Levi, Dr. Lissett R. Bickford, and Dr. Thaddeus J. Wadas for their thoughtful critiques and advice how to improve this research. Amanda Davis, Dr. Steve Kridel, and Dr. Todd Lowther also deserve special thanks for their work on the Nano-ORL collaboration, as without their contribution the work would not have come to completion. Dr. Colleen Webb and Dr. Joseph von Fischer both deserve thanks for their incredible influence on my choice of a scientific career and the research training they provided me with during my time at Colorado State University. I would also like to give a very special thanks to Dr. Kenneth Klopfenstein of the Colorado State University Department of Mathematics, whose influence on me personally and scientifically cannot be overstated. Dr. K. was a wonderful mentor to me, his classes and teaching not only taught me calculus but about the world and myself as well. I would also like to thank the Mike and Lucy Robbins family for their generous financial support of my graduate career through their fellowship. Finally, I would like to thank my parents, whose support has been a significant boon to me during my graduate career. i TABLE OF CONTENTS DEDICATION AND ACKNOWLEDGEMENTS............................................... i TABLE OF CONTENTS................................................................................. ii LIST OF FIGURES AND TABLES................................................................. viii LIST OF ABBREVIATIONS............................................................................ x ABSTRACT.................................................................................................... xiii CHAPTER 1: INTRODUCTION…………………………………………………. 1 1.0 Nanoparticles in Medicine and Biomedical Research…….……………… 1 Liposomal Nanoparticles ……………………………………………………….... 3 Metallic Nanoparticles …………………………………………………………… 5 Quantum Dots ……………………………………………………………………. 7 Polymeric Nanoparticles ………………………………………………………… 8 Hyaluronic Acid Derived Nanoparticles ……………………………………….. 9 2.0 Biodistribution and Toxicity of Nanoparticles…………………………….. 16 The Reticuloendothelial System ……………………………………………….. 17 Tumor Structure and the Enhanced Permeability and Retention Effect …… 17 The Mechanical Environment ………………………………………………….. 19 Nanoparticle Properties Affecting Delivery ……………………………………. 19 Size ………………………………………………………………………………… 20 Shape……………………………………………………………………………... 22 Charge…………………………………………………………………………….. 23 Surface Modification...................................................................................... 24 Toxicity……………………………………………………………………………. 26 ii 3.0 Clinical Imaging in Oncology………………………………………………. 27 X-ray and Computed Tomography…………………………………………….. 28 PET and SPECT………………………………………………………………… 30 Magnetic Resonance Imaging…………………………………………………. 31 Non-fluorescence Optical Imaging…………………………………………….. 32 Non-fluorescence Intraoperative Tumor Detection………………………… ... 33 4.0 Fluorescence Imaging and Image Guided Surgery……………………… 34 The Clinical Need for Image Guided Surgery………………………………… 35 Physics and Chemistry of Fluorescence……………………………………… 37 Fluorescence Imaging in Cancer……………………………………………… 39 Fluorescence Image Guided Surgery Instrumentation……………………… 45 5.0 Fatty Acid Synthase in Cancer……………………………………………. 47 Biology of FASN………………………………………………………………… 47 FASN Inhibitors and ORL………………………………………………………. 49 6.0 Conclusions…………………………………………………………………. 51 CHAPTER 2: SYNTHESIS OF NOVEL HYALURONIC ACID DERIVED NANOPARTICLES……………………………………………………………… 53 1.0 Introduction…………………………………………………………………. 53 2.0 Materials and Methods…………………………………………………….. 54 Materials…………………………………………………………………………. 54 Aminopropyl-1-pyrenebutanamide Synthesis……………………………….. 54 Aminopropyl-5β-cholanamide Synthesis…………………………………….. 56 Conjugation of 5βCA or PBA to HLA…………………………………………. 56 iii Conjugation of ODA to HLA…………………………………………………… 57 Hydrophobic Conjugate Characterization……………………………………. 58 Confirmation of NP Formation………………………………………………… 58 3.0 Results and Discussion…………………………………………………… 59 Hydrophobic Moiety Synthesis and Conjugation to HLA…………………... 59 4.0 Conclusions………………………………………………………………… 65 CHAPTER 3: IN VITRO CHARACTERIZATION OF INDOCYANINE GREEN-LOADED NANOPARTICLES……………………………………….. 67 1.0 Introduction…………………………………………………………………. 67 2.0 Materials and Methods…………………………………………………….. 68 ICG Loading into Nanoparticles………………………………………………. 68 Nanoparticle Characterization………………………………………………… 69 NanoICG Serum Stability……………………………………………………… 69 Photostability……………………………………………………………………. 70 NanoICG Interaction with Serum Proteins…………………………………… 70 In Vitro Cellular Uptake of NanoICG………………………………………….. 71 Cytotoxicity………………………………………………………………………. 72 Statistical Analysis……………………………………………………………… 72 3.0 Results and Discussion……………………………………………………. 73 Physical, Chemical, and Optical Characterization…………………………... 76 NanoICG Interaction with Serum Proteins…………………………………… 82 In Vitro Cellular Uptake of NanoICG………………………………………….. 85 Cytotoxicity………………………………………………………………………. 87 iv 3.0 Conclusions………………………………………………………………… 89 CHAPTER 4: TUMOR UPTAKE, BIODISTRIBUTION, AND POTENTIAL FOR IMAGE GUIDED SURGERY OF NANO-ICG IN XENOGRAFT TUMOR........................................................................................................ 91 1.0 Introduction…………………………………………………………………. 91 2.0 Materials and Methods…………………………………………………….. 92 iRFP Transfection of MDA-MB-231 Cells……………………………………. 92 Initial NIR Fluorophore Enhanced IGS……………………………………….. 93 Extension of In Vivo Biodistribution and IGS……………………………….... 94 Tissue Phantoms……………………………………………………………….. 95 Statistical Analysis……………………………………………………………... 96 3.0 Results and Discussion…………………………………………………... 96 Initial In Vivo Investigation Using Subcutaneous Flank Tumors………….. 96 Image Guided Surgery………………………………………………………… 99 Further In Vivo Investigation with Orthotopic Breast Tumors and an Additional Contrast Agent……………………………………………………… 102 Analysis with Tissue Mimicking Phantoms …………………………………... 109 4.0 Conclusions…………………………………………………………………. 113 CHAPTER 5: APPLICATION OF HYALURONIC ACID DERIVED NANOPARTICLES TO DRUG DELIVERY…………………………………... 114 1.0 Introduction………………………………………………………………….. 114 2.0 Materials and Methods……………………………………………………... 115 Materials………………………………………………………………………….. 115 v Drug Loading and Quantification……………………………………………… 116 Physical Characterization………………………………………………………. 118 FASN Inhibition………………………………………………………………….. 118 Inhibition of Lipid Synthesis……………………………………………………. 118 Cytotoxicity………………………………………………………………………. 119 Chemical Stability………………………………………………………………. 120 Mitochondrial Respiration Assays…………………………………………….. 121 Statistical Analysis ……………………………………………………………… 122 3.0 Results and Discussion…………………………………………………… 123 Drug Loading and Physical Characterization………………………………... 123 Inhibition of FASN and Lipid Synthesis………………………………………. 125 Cytotoxicity of Nano-ORL……………………………………………………… 127 Chemical Stability………………………………………………………………. 132 Mitochondrial Respiration Assays…………………………………………….. 134 4.0 Conclusions ………………………………………………………………… 140 CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS……………... 141 Summary of NanoICG Studies……………………………………………….... 141 Biodistribution and ICG Release to Serum Proteins……………………….... 142 An IGS-Specific Animal Model is Needed……………………………………. 145 Extension of NanoICG to SLN Mapping……………………………………… 146 Summary of Nano-ORL Studies……………………………………………….. 147 Diameter of Nano-ORL…………………………………………………………. 148 In Vivo Testing of Nano-ORL………………………………………………….. 149 vi HLA Remains a Suitable Biopolymer for NP Research……………………… 150 Conclusions………………………………………………………………………. 150 WORKS CITED…………………………………………………………………... 152 APPENDIX………………………………………………………………………... 180 CURRICULUM VITAE…………………………………………………………… 186 vii LIST OF ILLUSTRATIONS AND TABLES Figure 1-1…………………………………………………………………………….2 Figure 1-2……………………………………………………………………………10 Table 1-1……………………………………………………………………………..14 Figure 1-3…………………………………………………………………………….38 Figure 1-4…………………………………………………………………………….41 Figure 1-5…………………………………………………………………………….45 Figure 1-6…………………………………………………………………………….49 Figure 1-7…………………………………………………………………………….51 Scheme 2-1…………………………………………………………………………..55 Figure 2-1…………………………………………………………………………….61 Figure 2-2…………………………………………………………………………….62 Table 2-1……………………………………………………………………………...63 Figure 2-3…………………………………………………………………………….65 Figure 3-1…………………………………………………………………………….74 Table 3-1……………………………………………………………………………...75 Figure 3-2…………………………………………………………………………….77 Figure 3-3…………………………………………………………………………….78 Figure 3-4…………………………………………………………………………….79