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UNIVERSITY of CALIFORNIA SAN DIEGO Comparing Different Formulations for Delivering Arginine Deiminase Drug a Dissertation Submit UNIVERSITY OF CALIFORNIA SAN DIEGO Comparing Different Formulations for Delivering Arginine Deiminase Drug A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in NanoEngineering by Li-Chang Chen Committee in charge: Professor Sadik Esener, Chair Professor Yi Chen Professor Michael Heller Professor Yu-Hwa Lo Professor Shankar Subramaniam 2019 Copyright Li-Chang Chen, 2019 All rights reserved. The Dissertation of Li-Chang Chen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California San Diego 2019 iii DEDICATION I dedicate this dissertation to my mother, my father, and my sister for their endless support, encouragements, and love. To my grandpa, Aunt Angel, and Uncle Kenny for always believe in me and support my decisions. Lastly, to my future wife and family. iv EPIGRAPH There’s Plenty of Room at the Bottom -Richard Feynman v TABLE OF CONTENTS Signature Page ............................................................................................... iii Dedication ..................................................................................................... iv Epigraph ........................................................................................................ v Table of Contents .......................................................................................... vi List of Figures ............................................................................................... ix Acknowledgements ....................................................................................... xi Vita .............................................................................................................. xiv Abstract of the Dissertation............................................................................. xv Chapter 1 Introduction ................................................................................... 1 Chapter 2 Native Arginine Deiminase ............................................................ 7 2.1 Arginine Deiminase ........................................................................... 7 2.2 Materials ............................................................................................ 12 2.3 Methods ............................................................................................. 12 2.3.1 Molecular Cloning of Arginine Deiminase ................................ 12 2.3.2 Protein Expression and Renaturing of Arginine Deiminase ........ 14 2.3.3 Protein Purification of Arginine Deiminase ............................... 16 2.3.4 Characterization of Arginine Deiminase .................................... 16 2.4 Results ............................................................................................... 17 2.5 Discussion.......................................................................................... 23 2.6 Conclusion ......................................................................................... 27 2.7 Acknowledgments.............................................................................. 27 Chapter 3 PEGylated Arginine Deiminase ..................................................... 28 3.1 PEGylation ........................................................................................ 28 3.2 Materials ............................................................................................ 30 3.3 Methods ............................................................................................. 31 3.3.1 PEGylation Arginine Deiminase ................................................ 31 3.3.2 Characterization of PEGylated Arginine Deiminase .................. 31 3.4 Results ............................................................................................... 33 3.5 Discussion.......................................................................................... 35 3.6 Conclusion ......................................................................................... 38 3.7 Acknowledgments.............................................................................. 39 vi Chapter 4 SHELS Arginine Deiminase .......................................................... 40 4.1 Synthetic Hollow Enzyme Loaded Nanospheres (SHELS) ................. 40 4.2 Materials ............................................................................................ 42 4.3 Methods ............................................................................................. 43 4.3.1 Synthetic Hollow Mesoporous Nanospheres Synthesis .............. 43 4.3.2 Characterization of Synthetic Hollow Mesoporous Nanospheres 44 4.3.3 Loading Arginine Deiminase into Synthetic Hollow Mesoporous Nanospheres .............................................................................. 45 4.3.4 Characterization of SHELS Arginine Deiminase ....................... 46 4.4 Results ............................................................................................... 46 4.5 Discussion.......................................................................................... 50 4.6 Conclusion ......................................................................................... 54 4.7 Acknowledgments.............................................................................. 55 Chapter 5 Silica Coated Arginine Deiminase Liposome ................................. 56 5.1 Liposome ........................................................................................... 56 5.2 Materials ............................................................................................ 59 5.3 Methods ............................................................................................. 60 5.3.1 Synthesis of Arginine Deiminase Liposome .............................. 60 5.3.2 Characterization of Arginine Deiminase Liposome .................... 62 5.3.3 Synthesis of Silica Coated Arginine Deiminase Liposome ......... 63 5.3.4 Characterization of Silica Coated Arginine Deiminase Liposome 64 5.4 Results ............................................................................................... 64 5.5 Discussion.......................................................................................... 69 5.6 Conclusion ......................................................................................... 72 5.7 Acknowledgments.............................................................................. 72 Chapter 6 In vitro/In vivo Study of Different Formulations of Arginine Deiminase ...................................................................................... 73 6.1 Comparison of Different Formulated Arginine Deiminase .................. 73 6.2 Materials ............................................................................................ 74 6.3 Methods ............................................................................................. 75 6.3.1 Endotoxin Treatment of Arginine Deiminase ............................. 75 6.3.2 Endotoxin Treatment of PEGylated Arginine Deiminase ........... 76 6.3.3 Endotoxin Treatment of Silica Coated Arginine Deiminase Liposome .................................................................................. 77 6.3.4 Characterization of Different Formulations of Arginine Deiminase ................................................................................. 77 6.3.5 Cell Assay of Different Formulations of Arginine Deiminase .... 78 6.3.6 Animal Study of Different Formulations and Administration Routes of Arginine Deiminase ................................................... 79 vii 6.3.7 Determination of Arginine and Citrulline Levels by Mass Spectrometry ............................................................................. 80 6.4 Results ............................................................................................... 81 6.4.1 In vitro Comparison between the Different Formulations........... 81 6.4.2 In vivo Comparison between the Different Formulations ........... 85 6.5 Discussion.......................................................................................... 88 6.5.1 In vitro Comparison between the Different Formulations........... 88 6.5.2 In vivo Comparison between the Different Formulations ........... 91 6.6 Conclusion ......................................................................................... 94 6.7 Acknowledgments.............................................................................. 96 Chapter 7 Conclusions and Future Directions ................................................ 97 7.1 Conclusions ....................................................................................... 97 7.2 Future Directions ............................................................................... 98 References ................................................................................................... 100 viii LIST OF FIGURES Figure 2.1: A schematic diagram of the arginine metabolism ......................... 8 Figure 2.2: Mycoplasma hominis arginine deiminase structure ....................... 11 Figure 2.3: Q Sepharose Fast Flow purification chromatogram ...................... 18 Figure 2.4: Q Sepharose Fast Flow protein gel ............................................... 19 Figure 2.5: Phenyl Sepharose High Performance purification chromatogram . 20 Figure 2.6: Phenyl Sepharose High Performance protein gel .......................... 20 Figure 2.7: SuperDex 200 Prep Grade calibration
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