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Study of the Mechanisms of Microbubble-Facilitated Sonoporation in Vitro in Controlled Environments

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Study of the Mechanisms of Microbubble-Facilitated Sonoporation in Vitro in Controlled Environments Study of the mechanisms of microbubble-facilitated sonoporation in vitro in controlled environments by Zhenzhen Fan A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biomedical Engineering) in The University of Michigan 2012 Doctoral Committee: Associate Professor Cheri X. Deng, Chair Professor J. Brian Fowlkes Professor Shuichi Takayama Associate Professor Joseph L. Bull To my parents and my husband for their love and support ii Acknowledgements I would first like to thank my mom and dad for their love, encouragement and support, for their teaching me the value of good spirit and hard working. Without them, I would not be able to accomplish what I have achieved, or even one tenth of that. I would like to express my deepest gratitude to Dr. Cheri Deng, my advisor, for her patience and kindness, for her understanding and confidence in me. The dissertation would not have seen the light of day without her constant inspiring mentoring. My enormous debt of gratitude can hardly be repaid to her for her guidance and support during every stage in my graduate study. I would like to thank my dissertation committee for their inspiring guidance and support. Their invested time, support, and helpful suggestions in shaping my research and dissertation are greatly appreciated. I would like to thank Dr. Ron Kumon, Dr. Juyoung Park and Dr. Yun Zhou for their countless efforts in helping me with my projects. I am extremely privileged to have you as mentors and friends. Many thanks go to lab members in Dr. Deng’s lab, Dr. Cain’s lab and Dr. Xu’ lab for their friendship and help, as well as my collaborators outside ultrasound group. Finally, I would like to thank my husband, Xuan Wu, for his unwavering support and encouragement. Thanks for all your love. iii Table of Contents Dedication ........................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Figures ................................................................................................................... vii Abstract ............................................................................................................................ xiv Chapter 1 Introduction ........................................................................................................ 1 1.1 Specific aims ............................................................................................................. 2 1.2 Background ............................................................................................................... 4 1.2.1 Current techniques for intracellular drug and gene delivery .............................. 4 1.2.2 Therapeutic application of ultrasound combined with microbubbles ................ 7 1.2.3 Dynamic behaviors of microbubbles driven by ultrasound ............................... 8 1.2.4 Disruption of the cell membrane ...................................................................... 10 1.2.5 Transmembrane transport by sonoporation and down-stream effects ............. 11 1.3 Rationale and significance of this study ................................................................. 12 Chapter 2 Intracellular Delivery and Calcium Transients Generated in Sonoporation Facilitated by Microbubbles ............................................................................................. 14 2.1 Introduction ............................................................................................................. 14 2.2 Materials and methods ............................................................................................ 16 2.2.1 Cell culture ....................................................................................................... 16 2.2.2 Microbubbles ................................................................................................... 17 2.2.3 Measurement of intracellular calcium concentration ....................................... 18 2.2.4 Determination of membrane permeabilization with propidium iodide ............ 21 2.2.5 Optical imaging and data analysis ................................................................... 22 2.2.6 Ultrasound application and calibration ............................................................ 23 2.2.7 Experimental protocols .................................................................................... 24 2.2.8 Curve fitting ..................................................................................................... 25 2.3 Results ..................................................................................................................... 26 2.3.1 Ultrasound-driven microbubble activities, sonoporation, and calcium transients ................................................................................................................................... 26 2.3.2 Spatiotemporal evolution of intracellular marker and calcium ........................ 28 2.3.3 Delayed calcium transport in cells without pore formation ............................. 30 iv 2.3.4 Concentration-gradient driven, bidirectional transport of markers and pore closure ....................................................................................................................... 32 2.3.5 Cell viability ..................................................................................................... 36 2.4 Discussion ............................................................................................................... 37 2.5 Conclusion .............................................................................................................. 41 Chapter 3 Dynamic Activities of Microbubbles Driven by Ultrasound Exposures Correlated with Delivery Outcomes by Sonoporation ...................................................... 42 3.2 Materials and methods ............................................................................................ 43 3.2.1 Cell culture ....................................................................................................... 43 3.2.2 Assessments of delivery outcomes .................................................................. 43 3.2.3 Ultrasound exposure and microbubbles ........................................................... 44 3.2.4 Optical imaging ................................................................................................ 44 3.2.5 Data analysis .................................................................................................... 45 3.3 Results ..................................................................................................................... 45 3.3.1 Characterization of the bubble dynamics correlated with sonoporation outcomes ................................................................................................................... 45 3.3.2 Mapping sonoporation outcomes with bubble dynamics ................................. 55 3.3.3 Effects of ultrasound parameters on bubble dynamics .................................... 56 3.4 Discussion ............................................................................................................... 59 3.4.1 Sonoporation correlated with bubble dynamics ............................................... 59 3.4.2 High speed imaging and ultra-fast imaging ..................................................... 60 3.4.3 Biologic relevance besides intracellular delivery ............................................ 61 3.4.4 Different experimental setting results in different bubble dynamics ............... 61 3.5 Conclusion .............................................................................................................. 62 Chapter 4 Controlled and Enhanced Delivery Facilitated by Targeted Microbubbles ..... 63 4.1 Introduction ............................................................................................................. 63 4.2 Materials and methods ............................................................................................ 63 4.2.1 Cell culture ....................................................................................................... 63 4.2.2 Preparation of microbubbles ............................................................................ 64 4.2.3 Plasmid labeling and transfection .................................................................... 65 4.2.4 Experimental setup and ultrasound application ............................................... 66 4.2.5 Optical imaging ................................................................................................ 67 4.2.6 Data analysis .................................................................................................... 67 4.3 Results ..................................................................................................................... 69 4.3.1 Controlled delivery by targeted microbubbles ................................................. 69 v 4.3.2 Comparison of bubble dynamics between targeted and non-targeted microbubbles ............................................................................................................. 73 4.3.3 Delivery outcomes by sonoporation using targeted and non targeted microbubbles ............................................................................................................
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