8B-3 Transmembrane Extraction of Fluorescent Proteins with Ultrasound and Microbubbles

Abstract

Recent studies have shown that microbubbles under ultrasound (US) activation permeabilize plasma membrane. But the mechanism of permeabilization is not completely understood. The recurrent hypothesis assumes the formation of pores in the cell membrane allowing molecule delivery into cells. The aim of our study is to comfort this assumption by investigating whether US and microbubbles facilitate outward transport of molecules across the plasma membrane. Stably transfected Hela-GFP cells (GFP + cells) and propidium iodide (PI) were used to follow the US- microbubbles mediated permeabilization. To correlate these observations with molecular incorporation, US waves were generated from a 1 MHz unfocused transducer with pressures from 200 kPa to 600 kPa and duty cycles of 40% and 75%. Exposure time was 2 min with or without BR14 microbubbles (Bracco Research, Geneva). Immediately after insonation, the cells were immersed in a 4degC bath to interrupt cell activity. The percentage of GFP positive cells and the mean cell fluorescence intensity (FI) were measured by flow cytometry to evaluate the GFP release. We observed a significant decrease of FI and the percentage of GFP+ cells using US in presence of BR14 microbubbles. These effects were maximal at 400 kPa, 75% of duty cycle and 2 minutes insonation compared to control conditions. In parallel, the number of GFP+ cells that incorporated PI was higher than 50% for the same acoustic parameters. US alone had a slight impact on cell membrane permeability. These observations were also correlated to an increase of FITC-dextran incorporation that varied also vs acoustic parameters in presence of BR14 microbubbles. This result demonstrates the possibility to incorporate extracellular molecules into Hela cells throw probably pore formation. All observations would be explained by pore formation occurring under activation of microbubbles with ultrasound. This phenomenon leads to probably gradient dependant transport through pore opening during insonation.