Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and microbubbles

• Molecular uptake levels were quantified to characterize the degree of sonoporation.
• Localized collapse of small bubbles led to predictably heterogeneous sonoporation.
• Degrees of small-bubble-induced sonoporation depended on bubble-to-cell distance.
• Translation motion of large bubbles caused unpredictable degrees of sonoporation.
• The number of bubbles correlated positively with the extent of sonoporation.

Sonoporation—transient plasma membrane perforation elicited by the interaction of ultrasound waves with microbubbles—has shown great potential for drug delivery and gene therapy. However, the heterogeneity of sonoporation introduces complexities and challenges in the realization of controllable and predictable drug delivery. The aim of this investigation was to understand how non-acoustic parameters (bubble related and bubble-cell interaction parameters) affect sonoporation. Using a customized ultrasound-exposure and fluorescence-imaging platform, we observed sonoporation dynamics at the single-cell level and quantified exogenous molecular uptake levels to characterize the degree of sonoporation. Sonovue microbubbles were introduced to passively regulate microbubble-to-cell distance and number, and bubble size. 1 MHz ultrasound with 10-cycle pulse duration and 0.6 MPa peak negative pressure were applied to trigger the inertial collapse of microbubbles. Our data revealed the impact of non-acoustic parameters on the heterogeneity of sonoporation. (i) The localized collapse of relatively small bubbles (diameter, D < 5.5 μm) led to predictable sonoporation, the degree of which depended on the bubble-to-cell distance (d). No sonoporation was observed when d/D > 1, whereas reversible sonoporation occurred when d/D < 1. (ii) Large bubbles (D > 5.5 μm) exhibited translational movement over large distances, resulting in unpredictable sonoporation. Translation towards the cell surface led to variable reversible sonoporation or irreversible sonoporation, and translation away from the cell caused either no or reversible sonoporation. (iii) The number of bubbles correlated positively with the degree of sonoporation when D < 5.5 μm and d/D < 1. Localized collapse of two to three bubbles mainly resulted in reversible sonoporation, whereas irreversible sonoporation was more likely following the collapse of four or more bubbles. These findings offer useful insight into the relationship between non-acoustic parameters and the degree of sonoporation.