Direct Observation of Post-Sonoporation Membrane Resealing in the Giant Unilamellar Vesicle Model

Abstract

Sonoporation is a microbubble (MB)-mediated ultrasonic cavitation, which has attracted much attention in recent years due to its high efficiency and precision in non-invasive drug and gene delivery (Bouakaz et al., 2016; Helfield et al., 2016). However, the cell spontaneous repair process of the membrane after sonoporation, especially the dynamic mechanism of membrane resealing, has not been fully elucidated (Rich et al., 2022). Among them, a key mechanistic question is whether biophysical forces (such as membrane tension) play an important role in the membrane resealing process. However, this issue remains challenging due to the large number of biological processes in cells that may affect the dynamic behavior of membranes, such as cytoskeletal rearrangement (Chen et al., 2014) and endocytosis (Delalande et al., 2015; Zeghimi et al., 2015). To address this issue, this study innovatively used giant unilamellar vesicles (GUVs) composed only of phospholipid bilayers as a minimally simplified membrane research model to explore the membrane reseal mechanism in sonoporation. In the methodology, GUVs were successfully prepared by electroforming. Combined with a coupled ultrasound platform of fluorescence microscopy and high- speed camera, the GUV and microbubble mixed system was exposed in a precise controllable ultrasound field (ultrasonic frequency: 1 MHz, 10% duty cycle, 1Vpp) to simulate the sonoporation. The experimental results showed that GUVs were able to spontaneously reseal their membrane pore after sonoporation, indicating that the phospholipid bilayer has the ability to self-reseal even without the contribution of cell-mediated physiological processes. Further statistical analysis showed that the size of the microbubble had a significant effect on the GUV self reseal results: smaller microbubbles (2–3 μm in diameter) usually formed reversible and easily healed micropores, while larger microbubbles (>8 μm) often led to severe holes that were difficult to bridge (p < 0.01), ultimately destroying the stability of the vesicles. This discovery demonstrated for the first time the key role of biophysical forces in the membrane reseal stage after sonoporation. This study not only provides a new experimental model and platform based on GUVs, laying the foundation for future studies of the biophysical mechanism of sonoporation, but also provides a new perspective for a deeper understanding of the biophysical nature of membrane reseal.