Dye Transport through Bilayers Agrees with Lipid Electropore Molecular Dynamics

Although transport of molecules into cells via electroporation is a common biomedical procedure, its protocols are often based on trial and error. Despite a long history of theoretical effort, the underlying mechanisms of cell membrane electroporation are not sufficiently elucidated, in part, because of the number of independent fitting parameters needed to link theory to experiment. Here, we ask if the electroporation behavior of a reduced cell membrane is consistent with time-resolved, atomistic, molecular dynamics (MD) simulations of phospholipid bilayers responding to electric fields. To avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 10–50-ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes.