Saving cells from ultrasound-induced apoptosis: quantification of cell death and uptake following sonication and effects of targeted calcium chelation.

Applications of ultrasound for noninvasive drug and gene delivery have been limited by associated cell death as a result of sonication. In this study, we sought to quantify the distribution of cellular bioeffects caused by low-frequency ultrasound (24 kHz) and test the hypothesis that Ca(2+) chelation after sonication can shift this distribution by saving cells from death by apoptosis. Using flow cytometry, we quantitatively categorized sonicated cells among four populations: (i) cells that appear largely unaffected, (ii) cells reversibly permeabilized, (iii) cells rendered nonviable during sonication and (iv) cells that appear to be viable shortly after sonication, but later undergo apoptosis and die. By monitoring cells for 6 h after ultrasound exposure, we found that up to 15% of intact cells fell into this final category. Those apoptotic cells initially had the highest levels of uptake of a marker compound, calcein; also had highly elevated levels of intracellular Ca(2+); and contained an estimated plasma membrane wound radius of 100-300 nm. Finally, we showed that chelation of intracellular Ca(2+) after sonication reduced apoptosis by up to 44%, thereby providing a strategy to save cells. We conclude that cells can be saved from ultrasound-induced death by appropriate selection of ultrasound conditions and Ca(2+) chelation after sonication.