Sonoporation, referring to transient membrane permeation phenomenon generated by acoustic cavitation, has spurred significant scientific interests for its potential applications in facilitating uptake of drugs and genes into living cells. With an increasing level of technical maturity in realizing sonoporation, scientists are trying to gain a deeper understanding of the cellular responses related to this biophysical phenomenon from the standpoint for drug/gene delivery. However, challenges and difficulties remain to be overcome including providing direct evidences for the microbubble-cell wave matter interaction mechanism, obtaining controllable sonoporation at the desired locations on the cell membrane, maintaining the viability of the sonoporated cells with high efficiency delivery outcomes and so on. Such a lack of scientific foundations has been recognized as a fundamental obstacle in substantiating the application merit of sonoporation. In this study, the overall objective is to stepwise unravel the cellular stress induced by microbubble-mediated sonoporation after resealing. To achieve it, two kinds of well-calibrated ultrasound exposure platforms are designed. One of them can be used for the in situ observation of the wave matter interaction ways during sonoporation via the confocal microscope. The other ultrasound exposure setup can be used for the studies of the sonoporation induced bio-effects which need many cells for analysis. With these designed and well calibrated ultrasound exposure platforms, new insights for the cellular impacts induced by sonoporation are provided. As demonstrated in vitro, sonoporation may inadvertently induce repressive cellular features even whilst enhancing exogenous molecule uptake. Both suspension-type (HL-60) and adherence-type (ZR-75-30) cells were employed in this investigation. They were routinely exposed to 1 MHz pulsed ultrasound with calibrated acoustic field profile and in the presence of microbubbles. The post-exposure morphology and the intracellular actin cytoskeletons dynamics of sonoporated cells were examined in situ using confocal microscopy. Furthermore, the cell-cycle progression kinetics of the viable sonoporated cells were analyzed using flow cytometer. Results show that, for both investigated cell types, viable sonoporated cells would exhibit membrane and nucleus shrinkage, intracellular lipid accumulation and actin deploymerization over a two hours period. On the other hand, as compared to the sham control cells, the deoxyribonucleic acid (DNA) synthesis duration of sonoporated cells is significantly lengthened as indicative of a delay in cell-cycle progression. These features are known to be characteristics of a cellular stress response, suggesting that sonoporation indeed constitutes itself as a cellular stress to living cells even after the cells are resealed. In terms of the implication of this work, this study has shown that sonoporation can be a significant cellular stress both short term and long term after ultrasound exposure. In particular, the intracellular homeostasisis found disrupted even with membrane resealing. Therefore, if sonoporation is to be used for drug delivery, efficiency may be a problem that really needs to be solved in optimizing sonoporation for drug/gene delivery purposes. On the other hand, it raises opportunities for developing other therapeutic applications via sonoporation. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

Therapeutic ultrasound employs the acoustic energy carried by high-frequency mechanical wave to induce beneficial effects on living systems. This therapeutic approach is advantageous in that its energy could be remotely focused on the targeted tissue in a non-invasive manner. Although ultrasound therapy has been shown to be feasible and effective in both laboratory experiments and clinical trials, its safety and efficacy are still challenged by the lack of fundamental knowledge of how ultrasound wave exerts physical effects on the cell system and how the cell functionally responds to the ultrasound stimulation. Motivated by the above insight, this thesis aims to provide direct experimental evidence for illustrating the biophysical details of how ultrasound wave (alone or combined with microbubble) interacts with live cells. An acoustic experimental platform with well-calibrated ultrasound field and live-cell imaging modality was developed to observe ultrasound-cell interaction. Based on this platform, a series of single-cell studies was then conducted to monitor the structural and functional changes of the live cell as well as its fluorescently-labelled components over the course of ultrasound exposure. Results obtained in this thesis provided image-level evidence for characterizing the ultrasound-cell interactions in the following three aspects. First, it was found that low-intensity ultrasound pulsing could directly perturb the plasma membrane, the cytoskeletal network and the inner nucleus of live neuroblastoma cells. This cytomechanical perturbation would result in reversible and structural alternations of subcellular components. Second, low-intensity pulsed ultrasound, when applied on neuronal cells, could exert morphological impact through inducing neurite retraction and cell body displacement, and electrophysiological impact in the form of membrane depolarization and calcium influx. This finding verified the potential of ultrasound in modulating neuronal development and excitability. Last, the cell membrane perforation and resealing dynamics induced by the ultrasound-activated microbubble were visualized and characterized. The subsequent cellular responses to this ultrasound-induced sonoporation were also identified at both membrane and cytoskeleton levels. The significance of this study is to provide direct and solid experimental evidence for understanding the biophysical interactions between ultrasound wave and live cell. This advanced scientific interpretation is definitely crucial for establishing the cellular mechanisms of therapeutic ultrasound and for providing technical insights into ultrasound treatment. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

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