No abstract

Acoustic cavitation-mediated wounding (i.e., sonoporation) has great potential to improve medical and laboratory applications requiring intracellular uptake of exogenous molecules; however, the field lacks detailed understanding of cavitation-induced morphological changes in cells and their relative importance. Here, we present an in-depth study of the effects of acoustic cavitation on cells using electron and confocal microscopy coupled with quantitative flow cytometry. High resolution images of treated cells show that morphologically different types of blebs can occur after wounding conditions caused by ultrasound exposure as well as by mechanical shear and strong laser ablation. In addition, these treatments caused wound-induced non-lytic necrotic death resulting in cell bodies we call wound-derived perikarya (WD-P). However, only cells exposed to acoustic cavitation experienced ejection of intact nuclei and nearly instant lytic necrosis. Quantitative analysis by flow cytometry indicates that wound-derived perikarya are the dominant morphology of nonviable cells, except at the strongest wounding conditions, where nuclear ejection accounts for a significant portion of cell death after ultrasound exposure.

Applications of ultrasound for non-invasive drug and gene delivery have been limited by associated cell death due to 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 Ca2+ 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: (1) cells that appear largely unaffected, (2) cells reversibly permeabilized, (3) cells rendered nonviable during sonication and (4) 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 Ca2+; and contained an estimated plasma membrane wound radius of 100 – 300 nm. Finally, we showed that chelation of intracellular Ca2+ 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 Ca2+ chelation after sonication.

Ultrasound can temporarily make cells and tissue more permeable, an effect that could be used for enhanced and targeted drug delivery. Increased permeability is believed to involve creation of transient disruptions in cell membranes. This study seeks to characterize these disruptions and the mechanisms by which they are created, reseal and permit intracellular transport. To achieve this, DU145 prostate cancer cells were exposed to 24 kHz ultrasound with 0.1 s pulse length and 10% duty cycle for 2 s total exposure at pressures from 0.36 to 0.89 MPa. Disruptions were estimated to be at least 50 nm in diameter with lifetimes of 1–2 min using a range of fluorescent molecules with known molecular radii studied using flow cytometry. Cell morphological effects were examined using scanning electron, transmission electron, and laser scanning confocal microscopies after rapid fixation (within seconds after exposure). Images indicate that cell death from ultrasound exposure occurs due to a combination of apoptosis, necrosis and mechanical fragmentation and uptake may occur through physical disruptions in cell membrane structure. Using red blood cell ghosts and ATP-depleted prostate cancer cells, it was found that molecular uptake into viable cells requires active cellular processes which infers that cell recovery is an energy-dependent process.