Ph.D. Dissertation Defense by Robyn Schlicher
Friday, November 18, 2005

(Dr. Mark R. Prausnitz, Chair)

"Mechanisms of Ultrasound-Mediated Bioeffects"

Abstract

The inability to transport molecules efficiently and easily into cells and across tissues is one of the major limitations of developing drug delivery systems. A novel approach to overcoming this problem could be the use of low-frequency ultrasound to make cell membranes and tissues more permeable. Studies show that normally impermeant molecules can be transported into cells exposed to ultrasonic waves, however, the mechanism by which this occurs is not well understood. It is our hypothesis that low frequency ultrasound can reversibly disrupt membrane structure, thus allowing diffusion-driven intracellular delivery of molecules through a breach in the cell membrane. The effects of ultrasound are not limited to uptake of molecules; there is also a significant loss of cell viability in sonicated samples, the reasons for which are also not well characterized. Therefore, the focus of this work is to determine the mechanisms by which molecular uptake and cell death occur from ultrasound exposure. The long-term goal of this work is to increase the number of viable cells that experience uptake by controlling the effects that cause cell death.

Data have shown that large molecules (up to r = 28 nm) can be taken into the cell after exposure to 24 kHz (10% duty cycle for 2 s of exposure time at 0.1 pulse length over a range of pressures) ultrasound and that uptake of these molecules can occur even after sonication ended. In experiments developed to isolate the mechanism(s) of uptake, a test to block endocytosis, potassium depletion, showed no effect on uptake of calcein, a small fluorescent molecule (MW = 623 Da) in DU145 prostate cancer cells. However, cells depleted of ATP energy showed no uptake of calcein, nor did sonicated lipid bilayers (red blood cell ghosts), suggesting that uptake does not occur via endocytosis or by the same mechanism as electroporation, but does require active mechanisms to occur in viable cells. Multiple types of microscopy, including electron and laser scanning confocal, showed evidence of large disruptions, extrusion of cytosol and ejection of nuclei which support evidence of cell death via both oncotic (uncontrolled) necrosis and programmed cell death, including non-caspase mediated apoptosis (type I PCD), autophagy (type II PCD) and controlled necrosis (type III) PCD after sonication.