Transient transmembrane release of green fluorescent proteins with sonoporation

Microbubbles under ultrasound (US) activation are assumed to induce pore formation in the plasma membrane, causing its permeabilization and hence molecule incorporation from the extracellular environment. In this study, we investigated whether this permeabilization also engenders a transient release of small molecules from the cytosol of mammalian eukaryotic cells under the combined action of US and microbubbles. Using Hela cells stably expressing the enhanced green fluorescent protein (EGFP) gene, the release of EGFP was evaluated by flow cytometry in terms of the percentage of EGFP-positive cells (EGFP + cells) and the mean cell fluorescence intensity (MFI). Sonoporation was performed at 1 MHz, with peak negative pressures ranging from 0.2 to 0.6 MPa, duty cycles of 40% and 75% and a repetition rate of 10 kHz. The results showed that the insonation of Hela-EGFP cells at the peak negative pressure 400 kPa and the 75% duty cycle for 2 min in the presence of microbubbles induced a 60% decrease in both EGFP+ cells percentage and MFI. Our results demonstrate that the reduction of cell fluorescence is attributed to the EGFP release. Most importantly, this EGFP release was not due to lethal effects of sonoporation because the EGFP expression was significantly recovered by 48-h post-insonation. In conclusion, this study demonstrates for the first time a transient release of intracellular molecules produced by the sonoporation process. This controlled release showed the possibility of extracting molecules from the cell cytoplasm through the membrane while preserving cell viability. Taken together, the results obtained in this study reinforce the hypothesis of the transient pore formation mechanism induced by sonoporation.