Abstract
This study presents a unique approach to understanding the biophysical mechanisms of ultrasound-triggered cell membrane disruption (i.e., sonoporation). We report direct correlations between ultrasound-stimulated encapsulated microbubble oscillation physics and the resulting cellular membrane permeability by simultaneous microscopy of these two processes over their intrinsic physical timescales (microseconds for microbubble dynamics and seconds to minutes for local macromolecule uptake and cell membrane reorganization). We show that there exists a microbubble oscillation-induced shear-stress threshold, on the order of kilopascals, beyond which endothelial cellular membrane permeability increases. The shear-stress threshold exhibits an inverse square-root relation to the number of oscillation cycles and an approximately linear dependence on ultrasound frequency from 0.5 to 2 MHz. Further, via real-time 3D confocal microscopy measurements, our data provide evidence that a sonoporation event directly results in the immediate generation of membrane pores through both apical and basal cell membrane layers that reseal along their lateral area (resealing time of ∼<2 min). Finally, we demonstrate the potential for sonoporation to indirectly initiate prolonged, intercellular gaps between adjacent, confluent cells (∼>30-60 min). This real-time microscopic approach has provided insight into both the physical, cavitation-based mechanisms of sonoporation and the biophysical, cell-membrane-based mechanisms by which microbubble acoustic behaviors cause acute and sustained enhancement of cellular and vascular permeability.