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Study of Single Cell Sonoporation in Real Time

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Study of Single Cell Sonoporation in Real Time STUDY OF SINGLE CELL SONOPORATION IN REAL TIME USING ELECTROPHYSIOLOGY TECHNIQUES by Yun Zhou Submitted in partial fulfilment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Prof. Cheri X. Deng Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedicated to my parents and my wife for their love, support and patience Table of Content Table of Content .................................................................................................................1 List of Tables ......................................................................................................................5 List of Figures .....................................................................................................................6 Acknowledgements ............................................................................................................9 List of Abbreviations .......................................................................................................11 Abstract .............................................................................................................................12 CH1 INTRODUCTION...................................................................................................14 1.1 Specific aims of this research ...................................................................................14 1.2 Background and significance of this study ..............................................................16 1.2.1 Targeted drug delivery .......................................................................................16 1.2.2 Targeted gene delivery ......................................................................................17 1.2.3 Ultrasound mediated intracellular drug and gene delivery ................................18 1.2.4 Significance of this study ..................................................................................21 CH2 MEASUREMENT OF SINGLE CELL SONOPORATION USING VOLTAGE CLAMP TECHNIQUE ..............................................................................25 2.1 Introduction ..............................................................................................................25 2.1.1 Monitoring of sonoporation using voltage clamp technique in real time ..........25 2.1.2 Microbubbles to facilitate sonoporation ............................................................27 2.1.3 Effects of vitelline membrane in sonoporation of oocytes ...............................28 2.1.4 Effects of ultrasound parameters in sonoporation process ................................29 2.2 Methods ....................................................................................................................29 2.2.1 Experimental setup ............................................................................................29 2.2.2 Data acquisition .................................................................................................30 2.2.3 Xenopus oocyte preparation and animal protocol .............................................30 1 2.2.4 Voltage clamp procedure ...................................................................................31 2.2.5 Procedure to measure bubble concentration as a function of time ....................32 2.2.6 Experimental design to examine the effects of vitelline membrane in sonoporation ...............................................................................................................33 2.2.7 Experimental design to examine the effect of duty cycle on sonoporation process ........................................................................................................................35 2.3 Results ......................................................................................................................36 2.3.1 Microbubble concentration in solution during experiment ...............................36 2.3.2 The effects of vitelline membrane of Xenopus oocytes in sonoporation ..........37 2.3.3 Duty cycle effect on sonoporation .....................................................................38 2.3.4 Time constant of voltage clamp system ............................................................41 2.4 Discussion ................................................................................................................41 2.5 Conclusion ................................................................................................................43 CH3 EFFECTS OF EXTRACELLULAR CALCIUM ON CELL MEMBRANE RESEALING IN SONOPORATION .............................................................................61 3.1 Introduction ..............................................................................................................61 3.2 Methods ....................................................................................................................62 3.3 Results ......................................................................................................................62 3.3.1 Extracellular Ca2+ is required for resealing in sonoporation .............................63 3.3.2 Multiple dynamic processes and [Ca2+] dependent early-stage resealing .........65 3.3.3 Ca2+ dependent late -stage resealing ..................................................................67 3.3.4 Effects of extracellular Mg2+ on resealing process ............................................69 3.3.5 The effects of TMC amplitude ..........................................................................69 3.4 Discussion ................................................................................................................71 3.5 Conclusion and future work .....................................................................................77 CH4 DYNAMICS OF SONOPORATION CORRELATED WITH ACOUSTIC CAVITATION ACTIVITIES .........................................................................................87 2 4.1 Introduction ..............................................................................................................87 4.2 Methods ....................................................................................................................88 4.2.1 Animal protocol and cell preparation ................................................................88 4.2.2 Experimental setup ............................................................................................88 4.2.3 Data acquisition procedure and processing algorithms .....................................89 4.3 Results ......................................................................................................................91 4.4 Discussion ................................................................................................................93 4.5 Conclusion and future work .....................................................................................94 CH5 TRANSIENT PORE SIZE ESTIMATION IN SONOPORATION .................101 5.1 Introduction ............................................................................................................101 5.2 Methods ..................................................................................................................102 5.2.1 Experiments where a single pore is generated .................................................102 5.2.2 Experiment where multiple pores are generated .............................................104 5.2.3 Electro-Diffusion model to estimate pore size from transmembrane current .104 5.2.4 Experimental materials and experimental setup ..............................................107 5.3 Results ....................................................................................................................107 5.4 Discussion ..............................................................................................................108 5.5 Conclusion and future work ...................................................................................111 CH6 FUTURE WORK ..................................................................................................118 6.1 Specific topics for future investigation ..................................................................118 6.1.1 Study of single cell sonoporation of mammalian cells using patch clamp technique ...................................................................................................................118 6.1.2 Controlled microbubble interaction with ultrasound and cell membrane ......119 6.1.3 Deterministic measurement of pore size .........................................................119 6.2 Benefits to future applications on drug and gene delivery .....................................120 3 REFERENCE .................................................................................................................122
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