Developing a new sonoporation treatment model using plants: cellular mechanism analysis

Abstract

Plant cells (BY-2 tobacco) are used in this work as a new cellular model for investigating the biological mechanisms behind sonoporation-induced cytotoxic effects. We show that sonoporation may lead to chronic depolarization of plasma membrane and depolarization of mitochondrial membrane: both of which are known to be linked to a cell’s programmed cell death protocol. These findings are obtained in-vitro from BY-2 cell suspensions that were exposed to pulsed ultrasound for 1 min. (1 MHz ultrasound frequency, 10% duty cycle, 1 kHz pulse repetition frequency, 0.4 or 0.9 MPa peak negative pressure, 1% v/v microbubbles). Two potentio-dependent fluorescence probes (DiBAC4(3) and TMRE) were used to analyze the changes in the plasma membrane and mitochondrial membrane potential of the sonoporated cells at four post-exposure time points (0 h, 2 h, 4 h, 6 h). These changes were monitored by confocal and fluorescence microscopy, and they were quantified using a spectrofluorometer. The membrane depolarization effects are generally found to be more significant at higher peak negative pressures (0.9 MPa in our case).