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THE ROLE of ULTRASOUND CONTRAST AGENTS in PRODUCING SONOPORATION Monica Mary Forbes, Ph.D. Department of Bioengineering Universi

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THE ROLE of ULTRASOUND CONTRAST AGENTS in PRODUCING SONOPORATION Monica Mary Forbes, Ph.D. Department of Bioengineering Universi THE ROLE OF ULTRASOUND CONTRAST AGENTS IN PRODUCING SONOPORATION Monica Mary Forbes, Ph.D. Department of Bioengineering University of Illinois at Urbana-Champaign, 2009 William D. O’Brien, Jr., Adviser Sonoporation uses ultrasound (US) and ultrasound contrast agents (UCAs) to enhance cell permeabilization, thereby allowing delivery therapeutic compounds noninvasively into specific target cells. The objective of this dissertation was to elucidate the biophysical mechanism of sonoporation, specifically the role of the UCA. Monolayer cells were exposed in a solution of UCA, permeability agent, and saline. Exposure-effect studies varied the peak rarefactional pressure from 4 kPa to 4.14 MPa. Two UCAs (OptisonTM and Definity®), three US frequencies (1, 3, and 5 MHz), three cells lines (Chinese hamster ovary cells, mouse fibroblasts, and mouse bone marrow stromal precursor), and three transfection agents (FITC-Dextran, Calcein, and FluoSpheres carboxylate-modified microspheres) were examined. Exposure duration, pulse repetition frequency, number of pulses, and UCA concentration were also varied. The experimental observations demonstrated that inertial cavitation was not the physical mechanism for sonoporation. Microstreaming due to linear or nonlinear oscillations of the UCA was principally responsible. This microstreaming, when produced near a cell, resulted in shear stress on the cell membranes, causing the permeability change that allowed for the uptake of macromolecules into the cells. Experimental results also showed that the closer the exposure frequency to the resonance frequency the greater the sonoporation activity; the longer the ED the greater the sonoporation activity; and increasing UCA concentration increased sonoporation activity. Various permeability agents and cell lines displayed the same major characteristics of sonoporation response, confirming that a single physical mechanism was involved. However, structural characteristics of a particular cell line influenced the susceptibility of a cell line to sonoporation. Finally, a computational model was created that described shear stress on a cell membrane due to microstreaming. The theoretical results accurately described the maximum sonoporation activity, drop off in sonoporation activity, and relative differences between maximum activity and activity after drop off. Therefore, the model supported the conclusions made in this dissertation. This dissertation successfully elucidated the physical mechanism of sonoporation. Oscillation of UCAs near a cell produced microstreaming that resulted in shear stress on the cell membranes. This shear stress resulted in sonoporation, a permeability change that allowed for the uptake of macromolecules into the cells. © 2009 by Monica Mary Forbes. All rights reserved. THE ROLE OF ULTRASOUND CONTRAST AGENTS IN PRODUCING SONOPORATION BY MONICA MARY FORBES B.S., University of Illinois at Urbana-Champaign, 2003 M.S., University of Illinois at Urbana-Champaign, 2004 DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Bioengineering in the Graduate College of the University of Illinois at Urbana-Champaign, 2009 Urbana, Illinois Doctoral Committee: Professor William D. O’Brien, Chair Professor Michael F. Insana Professor Philip M. Best Assistant Professor Michael L. Oelze ABSTRACT Sonoporation involves the use of ultrasound (US) to enhance cell permeabilization. With this method it is possible, by using US and ultrasound contrast agents (UCAs), to deliver therapeutic compounds noninvasively into specific target cells. Sonoporation activity was proven to be mediated by UCA activity. Therefore, the objective of this dissertation was to elucidate the relationship between the UCA and sonoporation. A series of approaches were used to study the biophysical mechanism of sonoporation. Monolayer cells were exposed in a solution of UCA, permeability agent, and phosphate buffered saline. Exposure-effect studies varied the peak rarefactional pressure (Pr) over a range from 4 kPa to 4.14 MPa, and five independent replicates were performed at each pressure. Two UCAs, OptisonTM and Definity®, and three US frequencies, 1 MHz, 3 MHz, and 5 MHz, were examined. A series of factorial-based studies varied 2 or 3 variables. The first factorial study looked at the interaction between exposure duration (ED), pulse repetition frequency (PRF), and number of pulses. The second factorial study varied UCA concentration and Pr. Three cells lines, Chinese Hamster Ovary cells (CHO), Mouse Fibroblasts (3T3-L1), and Mouse Bone Marrow Stromal Precursor (D1), were used to determine impact of different cell lines on sonoporation. Three transfection agents, FITC-Dextran, Calcein, and FluoSpheres carboxylate-modified microspheres, were studied to ensure therapeutic effectiveness of sonoporation. The experimental observations provided from the 3.15-MHz CHO study using OptisonTM and the three CHO studies using Definity® (0.9, 3.15, and 5.6 MHz) support a single conclusion; inertial cavitation (IC) was not the physical mechanism for sonoporation. Microstreaming due to linear or nonlinear oscillations of the UCA was principally responsible for sonoporation. This microstreaming, when produced near a cell, resulted in shear stress on the cell membranes, which caused the permeability change that allowed for the uptake of macromolecules into the cells. The maximum sonoporation activity was impacted by several factors. The closer the exposure frequency was to the resonance frequency the greater the sonoporation ii activity. Additionally, longer EDs resulted in greater percentages of sonoporated cells. Increasing UCA concentration increased sonoporation activity, however not without limit as the ultrasonic attenuation of the UCAs came into play at higher concentrations. In addition to the conclusions regarding the physical mechanism of sonoporation, this dissertation provided some insights into the biological characteristics of sonoporation. For studies involving two permeability agents, FITC-dextran and calcein, and two cell lines, CHO and D1 cells, the same major characteristics of the sonoporation response occurred, confirming that the same physical mechanism was involved for the sonoporation results observed in this dissertation. However, structural characteristics of a particular cell line influenced the susceptibility of a cell line to sonoporation. Finally, a theoretical study was conducted to determine if a computational model that described shear stress on a cell membrane due to microstreaming successfully described the sonoporation results regarding the major responses with respect to Pr. The theoretical results were compared to the sonoporation results for each exposure condition and were found to accurately describe the maximum sonoporation activity, drop off in sonoporation activity, and relative differences between maximum activity and the activity after drop off. Therefore, the model supported the conclusions made in this dissertation. This dissertation successfully elucidated the physical mechanism of sonoporation. Oscillation of UCAs near a cell produced microstreaming that resulted in shear stress on the cell membranes. This shear stress resulted in sonoporation, a permeability change that allowed for the uptake of macromolecules into the cells. iii Dedicated to my parents, Richard and Sheva Forbes, and my siblings. iv ACKNOWLEDGEMENTS I am most grateful to my advisor, Dr. William D. O’Brien, Jr., for giving me the opportunity to study, research, and pursue my doctorate at the University of Illinois at Urbana-Champaign. His mentoring, support, and ability to guide me have been invaluable for this work. His enthusiasm in embracing a new project in the lab with its own required equipment, supplies, and cell culture requirements was fundamental in allowing me to conduct this research, in addition to teaching me how to start a project from the very beginning. Also, Dr. O’Brien’s support and encouragement was essential for me to be awarded an NIH NRSA fellowship and to be a successful MD/PhD student. Finally, he has been a personal friend seeing me through several life events. I would like to thank the members of my doctoral committee: Dr. Michael Oelze, Mr. Michael Insana, and Dr. Philip Best. Their invested time, support, and helpful suggestions in shaping my research are greatly appreciated. I would especially like to thank Dr. Oelze who helped me a great deal at the beginning when I knew nothing about ultrasound or acoustics. Many thanks go to the members of the Bioacoustics Research Lab. Dr. Rita Miller has been essential for guiding me through the research, making sure I had everything that I needed (equipment, personnel, and cheerleading), and providing me with assistance. A special thank you goes to Sue Clay for her help and unwavering patience in compiling all my fellowship applications and dealing with so many last minute FITC-Dextran orders. Thank you to Dr. Raymond Fish and Dr. Sandhya Sarwate for their questions and insights which served to strengthen my project. Thank you to Jim Blue whose endless conversations regarding basketball and football kept the days interesting. I would like to acknowledge my fellow students for their friendship and help, in particular, Ryan Steinberg and Ellora Sen-Gupta. Ryan was my partner in crime for this project and went through every up and down with me. He was always ready to try and solve a problem, come in early, and perform above and beyond what was asked. Ellora’s
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