Ultrasound and microbubble mediated plasmid DNA uptake: A fast, global and multi-mechanisms involved process.

Ultrasound application combined with microbubbles has shown great potential for intracellular gene delivery. However, the fundamental mechanistic question of how plasmid DNA enters the intracellular space mediated by ultrasound and microbubble has not been fully explored and understood. The goal of this study is to unveil the detailed intracellular uptake process of plasmid DNA stimulated by ultrasound and microbubbles, uniquely highlighting the role of microbubbles play in this process. The usage of targeted microbubbles pinpointed the subcellular membrane site, where ultrasound exerted acoustic force onto the cell membrane. With the combination of high-speed video microscopy and 3D confocal fluorescence microscopy, we show the spatiotemporal correlation between the microbubble dynamics and intracellular plasmid DNA distribution. Two ultrasound modes (high pressure short pulse and low pressure long pulse) were chosen to trigger different plasmid DNA uptake routes. We found that reversible cell membrane disruption, induced by high pressure short pulse ultrasound, permitted plasmid DNA passage across cell membrane, but not in an exclusive way. Under both ultrasound modes, with or without cell membrane disruption, global plasmid DNA internalization, even nuclear-localization, was observed immediately post ultrasound application. Our results show that plasmid DNA uptake evoked by localized acoustically excited microbubbles is a fast (<2min), global (not limited to the site where microbubbles were attached), and multi-mechanisms involved process.