High-efficiency endovascular gene delivery via therapeutic ultrasound.

OBJECTIVES: We studied enhancement of local gene delivery to the arterial wall by using an endovascular catheter ultrasound (US). BACKGROUND: Ultrasound exposure is standard for enhancement of in vitro gene delivery. We postulate that in vivo endovascular applications can be safely developed. METHODS: We used a rabbit model of arterial mechanical overdilation injury. After arterial overdilation, US catheters were introduced in bilateral rabbit femoral arteries and perfused with plasmidor adenovirus-expressing blue fluorescent protein (BFP) or phosphate buffered saline. One side received endovascular US (2 MHz, 50 W/cm2, 16 min), and the contralateral artery did not. RESULTS: Relative to controls, US exposure enhanced BFP expression measured via fluorescence 12-fold for plasmid (1,502.1+/-927.3 vs. 18,053.9+/-11,612 microm2, p < 0.05) and 19-fold for adenovirus (877.1+/-577.7 vs. 17,213.15+/-3,892 microm2, p < 0.05) while increasing cell death for the adenovirus group only (26+/-5.78% vs. 13+/-2.55%, p < 0.012). CONCLUSIONS: Endovascular US enhanced vascular gene delivery and increased the efficiency of nonviral platforms to levels previously attained only by adenoviral strategies.

Intracellular drug delivery using low-frequency ultrasound: quantification of molecular uptake and cell viability.

PURPOSE: To determine the dependence on acoustic parameters of molecular uptake and viability of cells exposed to low-frequency ultrasound. METHODS: DU145 prostate cancer cells bathed in a solution of calcein were exposed to ultrasound at 24 kHz over a range of different acoustic pressures. exposure times, pulse lengths, and duty cycles. Flow cytometry was employed to quantify the number of calcein molecules delivered into each cell and levels of cell viability. RESULTS: Both molecular uptake and cell viability showed a strong dependence on acoustic pressure and exposure time, weak dependence on pulse length, and no significant dependence on duty cycle. When all of the data were pooled together, they exhibited good correlation with acoustic energy exposure. Although molecular uptake showed large cell-to-cell heterogeneity, up to approximately 15% of cells achieved an intracellular calcein concentration approximately equal to its extracellular concentration. CONCLUSIONS: Large numbers of molecules can be delivered intracellularly using low-frequency ultrasound. Both uptake and viability correlate with acoustic energy, which is useful for design and control of ultrasound protocols.

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 x Pf/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.