chemotherapy; drug delivery; ultrasound; gene delivery
Ultrasound (US) is used to enhance and target delivery of drugs and genes to cancer tissues. The advantages of focused delivery to select tissues are manifold, but the exact mechanisms are largely unknown and need clarification to optimize delivery. The present study further defined the role of collapse cavitation in US-induced permeabilization of cell membranes and subsequent drug or gene uptake by the cell. Cavitation, defined as the collapse of micro-bubbles, produces high shear stresses and shock waves that may transiently puncture cell membranes and has been suggested as the cause of increased permeability. The hypothesis that collapsing bubbles permeabilize cells was tested by exposing rat colon cancer cells to US in the presence of excess pressure. Application of pressure suppresses cavitation activity of bubbles without changing the ultrasonic wave; thus if pressure reduces drug uptake, cell permeabilization is strongly linked to bubble cavitation activity. DHD/K12 cancer cells at a concentration of 1x106/ml in growth media supplemented with 10 ìM calcein were placed at the focal point of a 500 kHz transducer. Healthy cell membranes are normally impermeable to calcein, a fluorescent dye used as a model drug in this work. Cells were exposed to 12.8 W/cm2pulsed US (1:10 duty cycle) at various pressures and times in an isothermal chamber. The cells were washed to remove any extracellular calcein. Propidium iodide was then added at a concentration of 10 ìM. Cell fractions with reversible membrane damage (calcein uptake) and irreversible damage (PI uptake) were analyzed by flow cytometry. The results of these experiments show that increased membrane permeability is proportional to exposure time, and as the cell suspension was pressurized, the calcein uptake decreased as much as ten fold. Pressurization to 3 atm nearly eliminated the effect of US (Fig. 1). Theoretically US could produce thermal and mechanical effects on cells that may enhance drug uptake. These results rule out thermal enhancement of drug uptake and other types of direct US effects. This research shows US-mediated cell membrane permeability is linked to cavitation bubble activity and not thermal effects, thus advancing our knowledge of US-targeted chemotherapy.