Microbubbles have been used for several decades as ultrasound contrast agents in diagnostic ultrasound imaging. However, their application in gene therapy as delivery vehicles has only recently been realised. The presence of microbubbles in close proximity to cells during ultrasound insonation can increase the efficacy of drug or gene delivery by inducing formation of transient, non-lethal perforations in the cell membrane, a process termed sonoporation. In order to develop techniques for successful delivery of therapeutic agents, it is necessary to quantify the composition and physical characteristics of microbubbles in order to be able to determine how these affect the sonoporation process as required. Although several microbubbles are available commercially, the components of the shell of these proprietary microbubbles have not been disclosed. In order to study sonoporation and the possibility of delivering drugs and genes it became necessary to develop a formulation for in-house experimental microbubbles. These experimental in-house microbubbles have not been previously investigated with regard to their interaction with cells, their potential for sonoporation and / or their bioeffects. Characterisation of the in-house microbubbles was necessary prior to any attempts to use them as delivery vehicles in vitro, or indeed, in vivo. Confocal laser scanning microscopy (CLSM) was used in order to determine the size distribution of both in-house microbubbles and Definity® a commercially available contrast agent. Confocal imaging and 3-D reconstruction of in-house microbubbles indicated the structure, morphology and size-distribution of these membrane-bound microbodies. Microbubbles were later separated according to size using a density gradient. It was concluded that the distribution of sizes of the microbubbles was in part due to the multi-lamellar nature of the microbubble shell. Cells were initially cultured in Petri dishes and insonated in the presence and absence of in-house microbubbles, in order to assess any bioeffects emerging from the application of ultrasound alone or in the presence of the microbubble constructs. Cells were cultured subsequently on an acoustically-transparent Mylar membrane, which was then “sandwiched” between two acetal homopolymer (Derlin) rings and placed in a specially designed ultrasound tank. Ultimately, cells were grown in an OptiCell™, an acoustically-transparent parallel membrane environment, where delivery of molecules of various sizes, in the presence of both in-house and Definity® microbubbles was investigated. Sonoporation was achieved with insonication of SK Hep-1 cells with a “physiotherapy machine” applying a power of 2.54 W / cm2 for 2-3 secs in the presence of Definity® microbubbles and passage of Calcein, an impermeable molecule, into the cells was detected using flow cytometric analysis. In addition, expression of enhanced green fluorescent protein (EGFP) was also detected 24 hours after insonication of SK Hep-1 cells in the presence of Definity® microbubbles and a linearised plasmid pCS2, encoding EGFP, under the same ultrasonic conditions. Sonoporation was also investigated with the use of a diagnostic ultrasound scanner, since it is more clinically relevant. Although several acoustic and non-acoustic parameters were investigated, sufficient sonoporation was not attained using this scanner. The bioeffects of ultrasound on cells both in vivo and in vitro have been extensively investigated. However, the exact cellular mechanisms that are affected by the application of ultrasound waves are not understood. In this study, the effects of ultrasound on a number of pathways were investigated. Expression of Hsp70, a cell stress protein often associated with heat-shock, during application of continuous wave ultrasound, suggests that cells may undergo heat stress. During application of continuous wave ultrasound in the presence of Definity® microbubbles, expression of Hsp70 was shown to decrease compared to when ultrasound was applied in the absence of Definity® microbubbles. In addition, expression of HO-1, a protein associated with hypoxic pathways was also present during application of ultrasound in the absence of microbubbles. These results suggest that in the absence of ultrasound contrast agents, insonation can cause the expression of proteins associated with different forms of cell stress such as heat-shock and hypoxia, thus initiating the apoptotic process. In this thesis, it has been shown that the mean size of the in-house microbubbles is comparable to that of commercially available microbubbles such as Definity®. In addition, it has been shown that sonoporation and successful delivery of small molecules in the presence of Definity® microbubbles is achievable with the equipment and the specific system which was developed. This reinforces the promising role of in-house microbubbles as delivery vehicles for therapeutic agents. Finally, an investigation on the possible bioeffects of ultrasound in the presence and absence of ultrasound contrast agents, revealed that under acoustic conditions identical to those used for achieving sonoporation, cells experience stress, instigating pathways that could potentially lead to cell death.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 172-185).

An investigation of turbulent structure modification of a boundary layer for a fully developed channel flow by microbubble injection close to the upper wall was carried out using Particle Image Velocimetry (PIV). Two-dimensional velocity components in an x-y plane at Reynolds number of 5128 based on the half height of the channel and bulk velocity were measured. Microbubbles, with an average diameter of 30 ??m were produced by electrolysis and injected in the buffer layer. Different values of the void fraction were attained and used to evaluate the effects of the presence of microbubbles and their concentration within the boundary layer. A reduction in drag was observed due to the injection of microbubbles. Drag reduction augments as the value of the void fraction increases. Furthermore, increases in both the non-dimensional values of streamwise and normal turbulent intensities, normalized by the friction velocity were observed with the void fraction growth. A gradual decrease in the Reynolds shear stresses was achieved as the void fraction increases. This effect is due to a ??decorrelation?? or ??decoupling?? between the streamwise and normal fluctuating velocities. Modifications in the length and time scales due to the presence of microbubbles were detected by calculating two-point correlation coefficients in one and two dimensions and the autocorrelation coefficient at various locations within the measurement zone. Streamline length and time scales were increased. On the contrary, the normal length and time scales were decreased. The vorticity and strain rate values decreased with the injection of microbubbles. Turbulent energy production was also decreased within the boundary layer. Quadrant analysis was used to find out the contribution of the u?? and v?? fluctuating velocity components to the Reynolds stress. The presence of microbubbles reduces the contribution to the Reynolds stresses by Q4 events (sweeps), which are responsible for the production of skin friction. Vortical structure detection in the measurement area was pursued. The structure with and without the microbubble injection is compared. In this study the presence of microbubbles within the boundary layer has produced several modifications in the flow structure as well as reduction in the drag.

Gas microbubbles have a high compressibility, which make them very efficient sound scatterers. As another consequence of their high compressibility, microbubbles can be compressed by the pressure of the fluid around them, which affects their scattering properties. Due to recent progress in shelled ultrasound contrast agents and the development of almost monodispersed microbubbles, we believe it could now be possible to measure blood pressure using microbubbles as non-invasive manometers, an idea first suggested more than 30 years ago. In this thesis, both simulations and in vitro experiments will be used to investigate the changes related to the resonance of bubbles and how the concept of bubble size population affects the accuracy of this technique. In particular, it will be shown how shell dynamics dominates the response of microbubbles to blood pressure.

Gas microbubbles have a high compressibility, which make them very efficient sound scatterers. As another consequence of their high compressibility, microbubbles can be compressed by the pressure of the fluid around them, which affects their scattering properties. Due to recent progress in shelled ultrasound contrast agents and the development of almost monodispersed microbubbles, we believe it could now be possible to measure blood pressure using microbubbles as non-invasive manometers, an idea first suggested more than 30 years ago. In this thesis, both simulations and in vitro experiments will be used to investigate the changes related to the resonance of bubbles and how the concept of bubble size population affects the accuracy of this technique. In particular, it will be shown how shell dynamics dominates the response of microbubbles to blood pressure.

Intrapulmonary arteriovenous anastomoses (IPAVA) are vascular conduits through which blood flow bypasses the pulmonary circulation, and does not participate in pulmonary gas exchange. Blood flow through IPAVA (QIPAVA) is known to increase with increasing cardiac output, such as exercise, and while breathing normobaric, hypoxic gas at rest or during exercise. Previous studies demonstrate that QIPAVA is decreased at rest and during exercise in hypobaria compared to equivalent normobaric conditions. Studies involving postural changes have shown that QIPAVA may change with body position. In human studies, QIPAVA is measured either by transthoracic saline contrast echocardiography (TTSCE) or by injection of 99mTc-labeled macroaggregates of albumin (99mTc-MAA). It is unknown if discrepancies in measuring QIPAVA in normobaria and hypobaria, and in different body positions, represent real physiological changes or if they are methodological artifacts. In Chapter IV, the effect of hypobaria on QIPAVA was investigated. QIPAVA was reduced during exercise in hypobaria in normoxia and hypoxia compared to normobaric conditions, however gas exchange efficiency was unimpaired. This suggests that pulmonary blood flow may change in hypobaria such that blood flow is directed away from IPAVA. Alternatively, it may suggest that saline contrast is less stable at high altitude and not detected by TTSCE. In Chapter V, the effect of changing body position on QIPAVA as detected by TTSCE was investigated in human subjects at rest. No significant changes were observed in QIPAVA with postural changes. In Chapter VI, a perfusion model was used to investigate behavior of saline contrast microbubbles, MAA, and microspheres (20 µm and 50 µm diameter) encountering a vertical bifurcation. The results indicated that microbubbles and 20 µm microspheres tend to enter the upper branch of the bifurcation, whereas MAA and 50 µm microspheres tend to enter the lower branch. In Chapter VII, the effect of atmospheric pressure on the initial microbubble radius (Ro) of agitated saline contrast microbubbles was investigated. The results of this study demonstrated that the Ro of microbubbles created at sea level pressure was significantly smaller than Ro of microbubbles created at higher altitudes (1,668 m and 5,260 m). / 2019-01-09

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

Objective: In this study, I have directly tracked the progression of angiogenesis for three different types of breast cancer cell lines; MDA-MB-231, MCF-7, and MDA-MB-468. Each of these cell lines is known to overexpress different receptors, which may affect a tumor’s growth rate and perhaps its ability to undergo angiogenesis. Here, I measure and compare the growth, extent, and time of onset for angiogenesis. Methods: I used SCID mice to profile each of the different breast cancer cell lines. The growth rate of each tumor, along with its blood vessel development, was monitored and imaged using lipid-coated microbubbles and contrast-enhanced ultrasound (CEUS). A Vevo 2100 pre-clinical ultrasound machine was used for the imaging experiments. To track development of angiogenesis, mice were injected with perfluorobutane gas microbubbles of 1-2 microns diameter. Bubble perfusion into the tumor is an indicator of the presence of blood vessel formation. A custom image analysis program was developed in Matlab™ to eliminate breathing artifacts and track microbubble motion based on their high temporal frequency signature ("flicker"). Results: My experiments demonstrated that, although different cell lines grow at different rates, microbubbles begin to penetrate the tumor when it reaches approximately a size of approximately 3 mm in diameter. Therefore, the onset of angiogenesis occurred at different times (MCF-7 occurring first at around 9 days, MDA-MB-468 occuring at 12 days post inoculation, and MDA-MB-231 occurring at 17 days post tumor cell inoculation). Matlab™ analysis demonstrates consistent angiogenic behavior among the three cell lines. Conclusion: For all cell lines, angiogenesis started when the volume of the tumor was approximately 21.76 mm³, consistent with previous studies. As angiogenesis progressed, there was a drop in tumor blood flow. This can be explained by the sudden influx of oxygen when angiogenesis first begins. This momentarily inhibits new blood vessel formation while the tumor continues to steadily grow. After this sudden drop, tumor vascularization resumes a steady increase.

In an effort to develop an optical-acoustical understanding of ultrasound contrast agent microbubble dynamics in a micro-environment that resembles blood vessels, this thesis presents experimental work on optical trapping and acoustical probing of ultrasound contrast agent microbubbles confined in regenerated cellulose capillaries. First, we showed by acoustical means that the pressure threshold of an individual microbubble shell rupture increases significantly when confined in regenerated cellulose capillaries. We report that the shell rupture threshold in regenerated cellulose capillaries increased by at least 0.3 MPa from 0.8 MPa for unconfined microbubbles. Second, we achieved optical trapping and manipulation of ultrasound contrast agent microbubbles confined in capillaries using Hermite-Gaussian laser beams. / Graduation date: 2012