Heterogeneous reactive oxygen species responses in the sonoporated cells at the single-cell level

The aim of this study is to investigate how the intracellular ROS responds to different degrees of sonoporation. Using a customized platform for 1.5-MHz ultrasound exposure (13.33-μs duration and 0.70-MPa peak negative pressure) and observing the dynamics of sonoporation and intracellular ROS at the single-cell level, we quantified the exogenous molecular uptake and intracellular ROS indicator to evaluate the degree of sonoporation and ROS change, respectively. Our results revealed : 1) Within about 100 s post-exposure, which covered intervals from membrane perforation to the completion of membrane resealing, intracellular ROS rapidly decreased because of extracellular diffusion of dichlorofluorescein through the perforated membrane, similar to the extracellular PI influx. 2) In the following 270 s (120-390 s post-exposure), ROS generation in reversibly sonoporated cells gradually increased, and was positively correlated with the degree of the sonoporation. 3) The ROS level in irreversibly sonoporated cells continuously reduced to depletion. The ROS generation in reversibly sonoporated cells may affect their long-term fate. These results provide new insight into the biological response to sonoporation.