Direct imaging of siRNA electrotransfer at the single-cell level

Short interfering RNAs (siRNAs) represent new potential therapeutic tools owing to their capacity to induce strong, sequence-specific, gene silencing in cells. Electropulsation is one of the physical methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is proved to be effective for silencing gene expression by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the electro-delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time-lapse fluorescence microscopy and was correlated with the silencing of EGFP expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. The oligonucleotide then freely diffused across the cytosol. Therefore, we show that the electric field pulse acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The mechanism involved was clearly specific for the physicochemical properties of the electrotransferred molecule and was different from that observed with the electro-transfer of small molecules or plasmid DNA.