Non-invasive assessment and control of ultrasound-mediated membrane permeabilization

Purpose. Ultrasound has been shown to transiently permeabilize biological membranes, thereby facilitating delivery of large compounds such as proteins and DNA into cells and across tissues such as skin. In this study, we sought to quantitatively determine the dependence of cell membrane permeabilization on ultrasound parameters and to identify acoustic signals which correlate with observed membrane permeabilization. Methods. Bovine red blood cells were exposed to ultrasound at 24 kHz over a range of controlled conditions. The degree of membrane permeabilization was measured by release of hemoglobin and was determined as a function of ultrasound parameters and measured acoustic signals. Results. These studies showed that permeabilization increased with incident ultrasound pressure, increased with total exposure time above a threshold of approximately 100 msec, showed a weak dependence on pulse length with a small maximum at 3 msec, and did not depend on duty cycle under the conditions examined. Using measured acoustic spectra we found that red blood cell membrane permeabilization correlated best with the pressure measured at half the driving frequency (f/ 2 = 12 kHz) and its ultraharmonics, less strongly with the broadband noise pressure measured between peaks, and least strongly with pressure measured at the driving frequency and its higher harmonics. Permeabilization caused by ultrasound applied at any set of conditions tested in this study could be well predicted by the parameter tau.P-f/2, which characterizes the total cavitational exposure. Conclusions. This study provides a quantitative guide to designing ultrasound protocols useful for drug delivery. The acoustic measurements support the hypothesis that ultrasonic cavitation is the mechanism by which membranes are permeabilized. They also suggest that measurable acoustic signals can provide noninvasive, real-time feedback about membrane permeabilization and drug delivery.