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 the therapeutic role of this biophysical phenomenon and making it more efficient in facilitating drug delivery. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

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

Dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Chiropractic, Technikon Natal, Durban, 1998. / The aim of this study was to determine the effectiveness of adjustments versus the use of ultrasound in the treatment of mechanical neck pain. It was hypothesized that treatment with adjustments over a four week period, with a further four week follow-up period, would be more effective than ultrasound in terms of improving patients' cervical ranges of motion and their perceptions of pain and disability. Thirty consecutive patients suffering from mechanical neck pain were randomly assigned to either the adjustment or ultrasound groups. An experimental design was employed, whereby both groups received treatment twice a week for four weeks. After a follow-up period of a month, the patients were re-assessed. Measurements of the cervical spine ranges of motion with the CROM goniometer, algometer readings, and the completion of the Numerical Pain Rating Scale-101, CMCC Neck Disability Index and the Short Form McGill Pain questionnaires were performed before the first, fourth and final treatments as well as at the one month follow-up consultation. The data were then transferred to spreadsheets and underwent statistical analyses, using a 95 % confidence level. Analyses within each group were performed, using the Wilcoxon Signed Rank test and various readings were compared. The reading taken before the first treatment was compared to the reading taken before the final treatment. The initial reading was then again compared with the reading taken at the one month follow-up consultation. Comparison of the results of both treatment groups was statistically evaluated, using the Mann-Whitney U-Test. The comparison was made using the readings of the first, fourth and final treatments, as well as the one month follow-up consultation. This was done for all measurement parameters. / M

published_or_final_version / Endodontics / Master / Master of Dental Surgery

The purpose of this research was to investigate the feasibility of using high temperature ultrasonic pulses to administer therapeutic hyperthermia treatments independent of changes in blood perfusion and tissue properties. The use of a computer simulation program was used to study the effects of blood perfusion, tissue properties, transducer characteristics, and treatment geometry on the temperature elevation and thermal dose delivered by short high temperature ultrasonic pulses. Experiments were conducted in vitro and in vivo to investigate the effects of blood perfusion changes. Other experiments were carried out in dog thigh muscle to determine the effects of changes in tissue properties. A final study was done where murine melanoma in mice were treated with high temperature ultrasound. Results show that shorter pulse lengths (≤ 2 s) and smaller focal diameters (≤ 3 mm) give practically perfusion independent temperature elevation and thermal dose. Normal fluctuations in tissue properties should not have a significant effect on the treatment provided that proper choice of transducer is made for each individual application. High temperature ultrasonic pulses have also been shown to induce tumor responses. Based on this research, this technique is a feasible means of administering hyperthermia for cancer therapy.

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A dissertation presented in partial compliance with the requirements for the Master's Degree in Technology: Chiropractic, Technikon Natal, 2001. / The purpose of this study was to determine the relative efficacy of chiropractic manipulation used in conjunction with detuned ultrasound over the gluteal muscles compared to manipulation used in conjunction with proprioceptive neuromuscular facilitation stretching of the gluteal muscle group in the treatment of sacroiliac syndrome. It was hypothesised that both treatment groups would be effective in the treatment of sacroiliac syndrome but that manipulation used in conjunction with proprioceptive neuromuscular facilitation of the gluteal musculature would be more effective than manipulation used in conjunction with detuned ultrasound, in terms of subjective and objective clinical findings. / M

Dissertation submitted in partial compliance with the requirements for the Master’s Degree in Technology: Chiropractic Durban University of Technology, 2009 / Aim: To investigate the effect of therapeutic and sham ultrasound on radial artery blood flow (m.s-1) and radial arterial lumen diameter (mm). Subjects: Fifty healthy asymptomatic volunteers between the ages of 18-38 years. Methodology: The subjects were randomly allocated into one of five intervention groups (A-E). Group A received continuous ultrasound at 0.2 W.cm-² for 5 minutes, Group B received pulse ultrasound at 0.2 W.cm-² for 5 minutes, Group C received continuous ultrasound at 1.5 W.cm-² for 5 minutes, Group D received pulse ultrasound at 1.5 W.cm-² for 5 minutes and Group E received sham ultrasound at 0 W.cm-² for 5 minutes. Baseline radial artery blood flow (m.s-1) and radial artery lumen diameter (mm) readings were taken prior to the commencement of the therapeutic or sham ultrasound application using a Doppler ultrasound. At four minutes of application (during the therapeutic or sham ultrasound application), another set of blood flow and arterial lumen diameter measurements were taken. The final blood flow and arterial lumen diameter measurements were taken one minute after the therapeutic or sham ultrasound application was stopped. Results: The mean (± SD) radial artery blood flow and radial artery lumen diameter at baseline was 0.197 (± 0.060) m.s-1 and 2.4 (± 0.6) mm respectively. In Group A, the mean (± SD) radial artery blood flow during ultrasound application and one-minute after ultrasound application was 0.193 (± 0.070) m.s-1 and 0.179 (± 0.073) m.s-1 respectively. The mean (± SD) radial artery lumen diameter in Group A at the two time intervals was 2.2 (± 0.5) mm and 2.2 (± 0.3) mm respectively. In Group B, the mean (± SD) radial artery blood flow during ultrasound application and one-minute after ultrasound application was 0.187 (± 0.067) m.s-1 and 0.195 (± 0.041) m.s-1 respectively. The mean (± SD) radial artery lumen diameter in Group B at the two time intervals was 2.4 (± 0.4) mm and 2.3 (± 0.5) mm respectively. In Group C, the mean (± SD) radial artery blood flow during ultrasound application and one-minute after ultrasound application was 0.225 (± 0.088) m.s-1 and 0.186 (± 0.071) m.s-1 respectively. The mean (± SD) radial artery lumen diameter in Group C at the two time intervals was 2.4 (± 0.7) mm and 2.7 (± 0.8) mm respectively. In Group D, the mean (± SD) radial artery blood flow during ultrasound application and one-minute after ultrasound application was 0.215 (± 0.080) m.s-1 and 0.200 (± 0.081) m.s-1 respectively. The mean (± SD) radial artery lumen diameter in Group iv D at the two time intervals was 2.4 (± 0.8) mm and 2.4 (± 0.7) mm respectively. In Group E, the mean (± SD) radial artery blood flow during ultrasound application and one-minute after ultrasound application was 0.200 (± 0.067) m.s-1 and 0.182 (± 0.075) m.s-1 respectively. The mean (± SD) radial artery lumen diameter in Group E at the two time intervals was 2.5 (± 0.7) mm and 2.3 (± 0.5) mm respectively. There was no significant change in radial artery blood flow and radial artery lumen diameter over time in any individual group or between groups (p > 0.05; repeated measures ANOVA). There was an overall weak positive correlation between radial artery blood flow and radial artery lumen diameter at baseline (r = 0.508), during (r = 0.541) and after (r = 0.532) the therapeutic or sham ultrasound application. Conclusion: The results of this study showed that continuous, pulse or sham ultrasound had no significant effect on radial artery blood flow and radial artery lumen diameter. Furthermore, active ultrasound (continuous and pulse) was not superior to sham ultrasound in significantly affecting blood flow in a muscular artery.

A transient three-dimensional simulation program was developed to study the effects of scanning speed, scanning pattern, blood perfusion, transducer choice and multiple tilted transducers with overlapping foci during scanned focussed ultrasound hyperthermia. The results showed that (1) the temperature fluctuations increase linearly with decreasing scanning speed, (2) the temperature fluctuations are a weak, increasingly exponential function of the blood perfusion rate, and (3) that the largest temperature fluctuation is always located at the acoustical focal depth on the scan path independently of focal plane depth. Simulations using multiple scan paths showed that relatively uniform average temperature distributions can be achieved at the focal zone as long as the spacing between the concentric scans was not greater than the diameter of the focus of the power field. Finally, the results showed that using multiple tilted transducers with overlapping foci, increased focussing can be obtained at the focal depth.