Subcellular Response to Microbubble-Mediated Sonoporation

This dissertation, "Subcellular Response to Microbubble-mediated Sonoporation" by Weijing, Zhong, 钟文静, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Sonoporation, being an ultrasound-induced membrane perforation phenomenon, has received considerable interest in view of its therapeutic potential and is rapidly emerging as a promising approach to facilitate drug delivery. This event generally occurs when acoustic cavitation develops in the vicinity of living cells, as the mechanical interactions between ultrasound and microbubble would exert a force that is substantial enough to create pores on the cell membrane. The resulting increase in cell membrane permeability is transient in nature, and short-term survival of sonoporated cells is generally assumed. However, it remains unclear as to whether sonoporation would affect the cell fate in the long run. In particular, the contemporary mechanistic understanding of sonoporation has lacked account of the cellular response at a subcellular level. This inherently raises concerns on the general therapeutic applicability of sonoporation in mediating drug delivery. This thesis first addressed the question of whether cell fate may be affected on time-lapse basis as a result of sonopopration. As observed our analysis of DNA contents and cytoplasmic signaling proteins, some cells were found to commit apoptosis (programmed cell death) after sonoporation while the remaining viable cells may enter into cell-cycle arrest that disrupted normal cell proliferation. These findings should carry two major implications from a drug-delivery standpoint. First, cellular protection strategies should be developed when using sonoporation for drug delivery in cases where cell viability should be maintained. Second, for cancer therapy where cell death is required, the cytotoxic impact of sonoporation may represent a complementary factor that can be leveraged upon in facilitating the delivery of anti-cancer drugs. Further investigations were conducted to gain insight into the intermediate transduction mechanism in which sonoporation has entailed to bring about various cytoplasmic signaling changes that promote cell-cycle arrest and apoptosis. Our results reveal a transient enhancement of intracellular Ca2+ concentration in sonoporated cells. This bioelectrical disruption event is often recognized as a central messenger to instigate a series of cell-fate regulation pathways. In addition, observations on cell membrane repair revealed an exocytotic patching mechanism, accumulation of internal vesicles and increased activities in the Golgi apparatus. Given that the elevated Ca2+ level were observed in sonoporate cells, a follow-up study was conducted to investigate the potential role of endoplasmic reticulum (ER) and mitochondria in sonoporation-induced bioeffects. These two organelles were found to be activated in succession and in ways connected to the initiation of pro-apoptotic signaling. In particular, stress response was found to be active in the ER, and this in turn induced the dysfunction of mitochondria. Also, our time-lapse observations on the mitochondrial membrane potential have confirmed that this organelle is involved in facilitating sonoporation-induced apoptosis. In summary, investigations of time-lapse dynamics of cellular and subcellular responses mediated by sonoporation are so important in elucidating the fate of the sonoporated cells and understanding the mechanism in which sonoporation has entailed to instigate the sequential signaling pathways that bring cells into such conditions, thereby refining t