Therapeutic efficacy of drugs depends on their ability to reach the treatment target. Drugs that exert their effect within cells are constrained by an inability to cross the cell membrane. Methods are being developed to overcome this barrier including biochemical and biophysical strategies. The application of ultrasound with microbubbles increases the permeability of cell membranes allowing molecules, which otherwise would be excluded, to enter the intracellular space of cells; a phenomenon known as sonoporation. This thesis describes studies aimed at improving our understanding of the mechanism underpinning sonoporation and of the exposure parameters affecting sonoporation efficiency. Cancer cells (KHT-C) in suspension were exposed to ultrasound and microbubbles – total of 97 exposure conditions. The effects on cells were assessed through uptake of cell-impermeable molecules (10 kDa to 2 MDa FITC-dextran), cell viability and microscopic observations of the plasma membrane using flow cytometry, colony assay and electron microscopy techniques. Sonoporation was a result of the interaction of ultrasound and microbubbles with the cell membrane. Disruptions (30-100 nm) were generated on the cell membrane allowing cell impermeable molecules to cross the membrane. Molecules up to 2 MDa in size were delivered at high efficiency (~70% permeabilisation). Sonoporation was short lived; cells re-established their barrier function within one minute, which allowed compounds to remain inside the cell. Following uptake, cells remained viable; ~50% of sonoporated cells proliferated. Sonoporation efficiency depended on ultrasound and microbubble exposure conditions. Microbubble disruption was a necessary but insufficient indicator of ultrasound-induced permeabilisation. The exposure conditions can be tailored to achieve a desired effect; cell permeability of ~70% with ~25% cell death versus permeability of ~35% with ~2% cell death. In addition, sonoporation depended on position in the cell cycle. Cells in later stages were more prone to being permeabilised and killed by ultrasound and microbubbles. This study indicated that sonoporation can be controlled through exposure parameters and that molecular size may not be a limiting factor. However, the transient nature may necessitate that the drug be in close vicinity to target cells in sonoporation-mediated therapies. Future work will extend the investigation into in vivo models.

Therapeutic efficacy of drugs depends on their ability to reach the treatment target. Drugs that exert their effect within cells are constrained by an inability to cross the cell membrane. Methods are being developed to overcome this barrier including biochemical and biophysical strategies. The application of ultrasound with microbubbles increases the permeability of cell membranes allowing molecules, which otherwise would be excluded, to enter the intracellular space of cells; a phenomenon known as sonoporation. This thesis describes studies aimed at improving our understanding of the mechanism underpinning sonoporation and of the exposure parameters affecting sonoporation efficiency. Cancer cells (KHT-C) in suspension were exposed to ultrasound and microbubbles – total of 97 exposure conditions. The effects on cells were assessed through uptake of cell-impermeable molecules (10 kDa to 2 MDa FITC-dextran), cell viability and microscopic observations of the plasma membrane using flow cytometry, colony assay and electron microscopy techniques. Sonoporation was a result of the interaction of ultrasound and microbubbles with the cell membrane. Disruptions (30-100 nm) were generated on the cell membrane allowing cell impermeable molecules to cross the membrane. Molecules up to 2 MDa in size were delivered at high efficiency (~70% permeabilisation). Sonoporation was short lived; cells re-established their barrier function within one minute, which allowed compounds to remain inside the cell. Following uptake, cells remained viable; ~50% of sonoporated cells proliferated. Sonoporation efficiency depended on ultrasound and microbubble exposure conditions. Microbubble disruption was a necessary but insufficient indicator of ultrasound-induced permeabilisation. The exposure conditions can be tailored to achieve a desired effect; cell permeability of ~70% with ~25% cell death versus permeability of ~35% with ~2% cell death. In addition, sonoporation depended on position in the cell cycle. Cells in later stages were more prone to being permeabilised and killed by ultrasound and microbubbles. This study indicated that sonoporation can be controlled through exposure parameters and that molecular size may not be a limiting factor. However, the transient nature may necessitate that the drug be in close vicinity to target cells in sonoporation-mediated therapies. Future work will extend the investigation into in vivo models.

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yes / The compression stiffness of a phospholipid microbubble was determined using force-spectroscopy as a function of temperature. The stiffness was found to decrease by approximately a factor of three from 0.08 N m 1, at 10 C, down to 0.03 N m 1 at 37 C. This temperature dependence indicates that the surface tension of lipid coating is the dominant contribution to the microbubble stiffness. The timedependent material properties, e.g. creep, increased non-linearly with temperature, showing a factor of two increase in creep-displacement, from 24 nm, at 10 C, to 50 nm, at 37 C. The standard linear solid model was used to extract the visco-elastic parameters and their determination at different temperatures allowed the first determination of the activation energy for creep, for a microbubble, to be determined. / EPSRC

The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (kLa) and subsequently increased the cell mass and protein production compared to conventional fermentation. Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. Maximum cell mass productivity in the conventional 350 rpm was only 0.37 g / (L·h), while the maximum value in MBD 350 rpm was 2.0 g / (L·h), which was similar to 2.2 g / (L·h) in the conventional 750 rpm. Biomass yield on glycerol Ys (g cell/ g glycerol) was 0.334 g / g in the conventional 350 rpm, 0.431 g / g in MBD 350 rpm and 0.438 g / g in the conventional 750 rpm. Protein production in MBD 350 rpm was 7.3 times higher than that in the conventional 350 rpm, but similar to the conventional 750 rpm. Maximum protein productivity in the conventional 350 rpm was 0.37 mg / (L·h), 2.8 mg / (L·h) in MBD 350 rpm, and 3.3 mg / (L·h) in the conventional 750 rpm. Protein yield on methanol Yp (mg protein / g methanol) was 1.57 mg /g in the conventional 350 rpm, 5.02 in MBD 350 rpm, and 5.21 in the conventional 750 rpm. The volumetric oxygen transfer coefficient kLa was 1011.9 h-1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h-1) but was similar to the conventional 750 rpm (1098 h-1). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging. Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. / Master of Science

In the large-scale production of <i>Saccharomyces cerevisiae</i> (baker's yeast), oxygen transfer, which is one of the major limiting factors, is improved by using high agitation rates. However, high agitation rates subject the microorganisms to high shear stress and caused high power consumption. A microbubble dispersion (MBD) method was investigated to improve oxygen transfer at low agitation rates and thus reduce power consumption and shear stress on the microorganisms. The experiments were conducted at the 1-liter level and subsequently scaled-up to 50-liters using a constant volumetric oxygen transfer coefficient (<i>k_La</i>) method for scaling. In comparison to a conventional air-sparged fermentation, the MBD method considerably improved the cell mass yield, growth rate and power consumption in the 50-liter fermentor. Cell mass production in the MBD system at agitation rate of 150 rpm was about the same as those obtained for a conventional air-sparged system agitatid at 500 rpm. Power consumption in the conventional air-sparged system was three-fold that required for the same biomass yield in the MBD system. However, at the 1-liter scale, the MBD system did not show any significant advantage over the air-sparged system because of the high power consumption. / Master of Science

Microbubble drag reduction (MBDR) is an effective method to improve the efficiency of fluid systems. MBDR is a field that has been extensively studied in the past, and experimental values of up to 80% to 90% drag reduction have been obtained. The effectiveness and simplicity of MBDR makes it a viable method for real world applications, particularly in naval applications where it can reduce the drag between the surface of ships and the surrounding water. A two dimensional single phase model was created in ANSYS Fluent to effectively model the behavior of bubble laden flow over a flat plate. This model was used to analyze the effectiveness of MBDR based on the following factors: Reynolds number, types of gas injected, upstream flow velocity, upstream fluid type, density ratio, flow rate of injected gas, using air as the upstream injected fluid.

It has been proposed that coated gas microbubbles, currently used as ultrasound contrast agents could also be used as microscale sensors due to the sensitivity of their acoustic response to changes in their environment. However, their behaviour is not fully understood and there remains considerable scope for improving their characterisation. The aim of this thesis is to improve the theoretical description of microbubble dynamics under ultrasound excitation with the ultimate aim of assessing the regimes in which they could be exploited most effectively as sensors. Previous theoretical and experimental work relating to the confinement and acoustic excitation of microbubbles is reviewed. Specifically, optical trapping as a method for the isolation and manipulation of individual bubbles is studied for use in developing a sensor. An assessment of the existing models’ validity is undertaken. This is followed by the development of models for optical trapping of single microbubbles, and the coupled radial and translational motion of a microbubble under ultrasound excitation, which includes time dependent phenomena. The latter model is used to perform a sensitivity analysis to determine the uncertainty associated with using microbubbles as sensors. The potential for uniquely characterising the shell of the microbubble from experimental data is also assessed. Subsequent chapters present the results from a combination of computer simulations and experimental data, used to develop and assess the validity of the new models for describing microbubble behaviour. Particularly, the model is used to simulate the response of a dilute suspension of microbubbles undergoing large amplitude oscillations and single microbubbles undergoing lipid shedding. The optimal regimes in which microbubbles may be utilised as sensors for liquid physical properties and local pressure variations are then assessed. Finally, a summary of the conclusions and areas for further work is presented.

Ultrasound contrast agents (CAs) are typically used in clinic for perfusion studies (blood flow through a specific region) and border delineating (differentiate borders between tissue structures) during cardiac imaging. The CAs used during ultrasound imaging usually consist of gas filled microbubbles (MBs) (diameter 1-5 μm) that are injected intravenously into the circulatory system. This thesis partially involves a novel polymer-shelled ultrasound CA that consists of air filled MBs stabilized by a polyvinyl alcohol (PVA) shell. These MBs could be coupled with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as a combined CA for ultrasound and magnetic resonance imaging. The first three papers (Paper A-C) in this thesis investigate the structural characteristic and the elimination process of the CA. In Paper A, two types (PVA Type A and PVA Type B) of the novel CA were analyzed using transmission electron microscopy (TEM) images of thin sectioned MBs. The images demonstrated that the SPIONs were either attached to the PVA shell surface (PVA Type A) or embedded in the shell (PVA Type B). The average shell thickness of the MBs was determined in Paper B by introducing a model that calculated the shell thickness from TEM images of cross-sectioned MBs. The shell thickness of PVA Type A was determined to 651 nm, whereas the shell thickness of PVA Type B was calculated to 637 nm. In Paper C, a prolonged blood elimination time was obtained for PVA-shelled MBs compared to the lipid-shelled CA SonoVue used in clinic. In addition, TEM analyzed tissue sections showed that the PVA-shelled MBs were recognized by the macrophage system. However, structurally intact MBs were still found in the circulation 24 h post injection. These studies illustrate that the PVA-shelled MBs are stable and offer large chemical variability, which make them suitable as CA for multimodal imaging. This thesis also involves studies (Paper D-E) of the molecular chaperones (Hsp21 and DNAJB6). The small heat shock protein Hsp21 effectively protects other proteins from unfolding and aggregation during stress. This chaperone ability requires oligomerization of the protein. In Paper D, cryo-electron microscopy together with complementary structural methods, obtained a structure model which showed that the Hsp21 dodecamer (12-mer) is kept together by paired C-terminal interactions.The human protein DNAJB6 functions as a very efficient suppressor of polyglutamine (polyQ) and amyloid-β42 (Aβ42) aggregation. Aggregation of these peptides are associated with development of Huntington’s (polyQ) and Alzheimer’s (Aβ42) disease. In Paper E, a reconstructed map of this highly dynamic protein is presented, showing an oligomer with two-fold symmetry, indicating that the oligomers are assembled by two subunits. / <p>QC 20160527</p>

The development of ultrasound contrast agents, or microbubbles, over the past 40 years has increased the possibilities for diagnostic imaging, although, more recently they have been proposed as a new vehicle for delivery of drugs and genes. However, there yet remains a considerable lack of fundamental understanding of microbubble behaviour under ultrasound excitation which has restricted their translation to therapeutic use. This project focussed on three key areas relating to the generation, observation, and bioeffects of microbubbles and the ultrasound used in their excitation. The experimental endeavour involved first, a full characterisation of the performance of a rotating mirror high-speed camera (Cordin 550-62) that was previously used by our group [and others] to investigate microbubble dynamics. Specifically, the investigation begins with an assessment of the frame-rate reporting accuracy of the system, a key aspect to the robustness of quantitative measurements extracted from recorded image sequences. This is then followed by the demonstration of a novel method of analysis for examining the image formation process in this type of camera, which facilitates a sensor-by-sensor assessment of performance that was not previously realised. Consolidating with previous work from within the group, this new analysis method was used to clarify previous data, and in the process suggested the presence of a temporal anomaly embedded within recorded images. In addition, the analysis also revealed empirical evidence for the mechanisms leading to this anomaly. Following on, a holographic optical tweezer system was developed for the purpose of exercising precise spatial control over microbubbles within their experimental environment. By positioning microbubbles in specific arrangements, interesting behaviours that were not previously achieved experimentally in the context of shelled microbubbles, were observed. Furthermore, by careful positioning of microbubbles within the imaging plane, it was possible to exploit the temporal anomaly present in the camera to greatly improve the integrity of data recorded, and to also operate in an enhanced imaging mode. Group aspirations to accelerate the development of therapeutic microbubbles had previously generated some early work on the in-house generation of bespoke bubble populations using microfluidic lab-on-a-chip techniques. In order to facilitate further development in this area, a finite-element computational model was herein developed to aid next generation chip design. Finally, in a slightly different context, considering not only the mechanical effect a microbubble may effect in a therapeutic treatment, a single biological cell assay was developed in order to probe any mechanical effects that were induced by the excitation ultrasound itself. Capitalising on the precise force control possible with atomic force spectroscopy, the elastic moduli of cells pre- and post-ultrasound insonation (sans microbubbles) were recorded. These new developments have extended the group capability and expertise in the areas of high-speed imaging, experimental observations of microbubble dynamics and with microfluidic generation of microbubbles. Additionally, the insights garnered have both served to consolidate the group's previous and as yet unpublished data, opening the way for circulation with absolute confidence in the integrity of that data.