Contributions of mechanical and sonochemical effects to cell membrane damage induced by single-shot pulsed ultrasound with adjacent microbubbles

Abstract

The objective was to investigate the contributions of mechanical effects due to kinetic force induced by the dynamic behavior of microbubbles and sonochemical effects due to free radicals produced by inertial cavitation to cell membrane damage under sonoporation conditions in which cells with adjacent microbubbles were irradiated with single-shot pulsed ultrasound. The free radical scavenger cysteamine was used to control the occurrence of sonochemical effects, and the ratios of cells with membrane damage to intact cells were compared in the presence and absence of cysteamine. To determine the optimal dose of cysteamine, free radical production on exposure to burst pulse ultrasound was investigated using KI-starch solutions with different concentrations (0-5 mM) of cysteamine. High-speed observation of the dynamic behavior of Levovist microbubbles during ultrasound exposure was also carried out in the presence and absence of cysteamine, and the difference in the ratios of the maximum bubble diameter to the initial diameter was evaluated. Next, human prostate cancer cells with adjacent Levovist microbubbles were exposed to single-shot pulsed ultrasound with a center frequency of 1 MHz, a peak negative pressure of 1.1 MPa, and a pulse width of 3 μs, and the percentages of cells with membrane damage were evaluated by fluorescent microscopy using propidium iodide in the presence and absence of cysteamine. It was confirmed that cysteamine at a concentration of 5 mM completely suppressed sonochemical effects without causing a change in the dynamic response of microbubbles to pulsed ultrasound. The percentages of cells with membrane damage in the presence and absence of cysteamine (5 mM) were 10.3% ± 4.1% (n = 13) and 8.7% ± 3.9% (n = 9), respectively. No significant difference was found (P = 0.36). The results indicate that cell membrane damage induced by single-shot pulsed ultrasound with adjacent microbubbles was due mainly to mechanical effects, not to sonochemical effects.