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 similar to 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.