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.

Purpose: To compare hemodynamic of malignant and benign SRMs on CT and CEUS. Method: Seventy biopsy proven SRM underwent CEUS. Sixty-three had CT. After injection of 0.2 ml of Definity, 3min and after 0.9 ml infusion, 30 sec of data were acquires. Lesion hemodynamics relative to the cortex was evaluated both qualitatively and quantitatively. Results: Considering 15 and 20 HU as enhancement threshold, 10% to 13% of patients did not enhance on CT, while all lesions enhanced on CEUS. Papillary RCCs showed hypovascularity with 100% specificity. In other RCCs, PI, WI slope 5 to45%, 50 to100%, 10 to 90%, WO slope 100 to 50%, 100 to 10%, WO intensity at peak+30 seconds were statistically higher than benign SRMs. Conclusion: All solid SRMs enhance on CEUS, while CT does not show vascularity in 10-13% of solid SRMs. CEUS can differentiate malignant from benign SRMs by evaluating their hemodynamics.

Purpose: To compare hemodynamic of malignant and benign SRMs on CT and CEUS. Method: Seventy biopsy proven SRM underwent CEUS. Sixty-three had CT. After injection of 0.2 ml of Definity, 3min and after 0.9 ml infusion, 30 sec of data were acquires. Lesion hemodynamics relative to the cortex was evaluated both qualitatively and quantitatively. Results: Considering 15 and 20 HU as enhancement threshold, 10% to 13% of patients did not enhance on CT, while all lesions enhanced on CEUS. Papillary RCCs showed hypovascularity with 100% specificity. In other RCCs, PI, WI slope 5 to45%, 50 to100%, 10 to 90%, WO slope 100 to 50%, 100 to 10%, WO intensity at peak+30 seconds were statistically higher than benign SRMs. Conclusion: All solid SRMs enhance on CEUS, while CT does not show vascularity in 10-13% of solid SRMs. CEUS can differentiate malignant from benign SRMs by evaluating their hemodynamics.