Ultrasound and microbubble-assisted gene delivery: insights for intracellular mechanism
Résumé
Microbubbles are capable to oscillate under specific ultrasound settings that can lead to a permeabilisation of surrounding cells and this phenomenon is called sonoporation. The sonoporation technique has been used to deliver drugs in vitro and in vivo for gene or chemotherapeutic drug delivery applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. We have used a luciferase reporter plasmid to identify the optimal parameters for an efficient gene delivery in human cancer HeLa cells. The optimal parameters found at 1 MHz were 150 kPa, 40% duty cycle, 60 sec sonication time using 0.3% of microbubbles. The interactions between microbubbles and cells were investigated by high-speed imaging during sonoporation. Specifically at 150 kPa, microbubbles entry into cells has been observed during sonoporation. These results strongly suggest that the microbubble entry could be closely correlated to the plasmid DNA entry into the cell. The intracellular kinetic of the plasmid delivery was followed by confocal microscopy using fluorescent molecules and specific cell markers for late endosomes and nuclear envelope. Most of plasmids were located in late endosomes 3 hours after sonoporation and some could reach the nucleus 3 hours later. The gene transfer efficiency was almost totally inhibited when depleting the cell ATP and in presence of chlorpromazine suggesting that plasmid uptake after sonoporation was an active mechanism mostly involving the clathrin-mediated pathway.
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