Generation of Reactive Oxygen Species in Heterogeneously Sonoporated Cells by Microbubbles with Single-Pulse Ultrasound

To develop and realize sonoporation-based macromolecule delivery, it is important to understand the underlying cellular bioeffects involved. It is known that an appropriate level of reactive oxygen species (ROS) is necessary to maintain normal physiologic function, but excessive ROS triggers adverse downstream bioeffects. However, it is still unclear whether a relationship exists between intracellular ROS levels and sonoporation. Using a customized platform for 1.5-MHz ultrasound exposure (13.33 µs duration and 0.70 MPa peak negative pressure) and imaging the dynamics of sonoporation and intracellular ROS at the single-cell level, we quantified the exogenous molecular uptake and the concentration of intracellular ROS indicator to evaluate the extent of sonoporation and ROS change, respectively. Our results revealed that the intracellular ROS level was correlated with the degree of the sonoporation. (i) Within ~120 s of the onset of ultrasound, during which membrane perforation and complete membrane resealing occurred, intracellular ROS rapidly decreased because of extracellular diffusion of dichlorofluorescein through the perforated membrane and positively correlated with the degree of the sonoporation. (ii) 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. (iii) The ROS level in irreversibly sonoporated cells reduced to depletion during this time interval. It is possible that ROS generation in reversibly sonoporated cells can impact their long-term fate. These results thus provide new insight into the biological response to sonoporation.