Nanoelectropulse-driven membrane perturbation and permeabilization

Abstract

Alterations in the asymmetric arrangement of phospholipids in the plasma membrane serve as both a semaphore and a structural component in platelet activation, lymphocyte signal transduction, and the phagocytotic clearance of apoptotic cells and aging erythrocytes. Nanosecond, megavolt-per-meter electric pulses - high instantaneous power but low total energy - induce phosphatidylserine (PS) externalization without physically disrupting the membrane, providing a means for manipulating a key biomolecular structure in living cells through a remote, non-contact agent. Effects of nanoelectropulse exposure include not only phospholipid translocation but also, with doses appropriate for the cell type and the physical and chemical environment, intracellular calcium release, chromatin structural modifications, and apoptosis. Experimental evidence is consistent with electrophysical models that predict the penetration of electrical pulses with durations shorter than the charging time of the cell membrane (<100 ns) into the cell interior. Real-time fluorescence microscopic observations and molecular dynamics (MD) simulations associate membrane perturbation directly and immediately with nanoelectropulse exposure and support the hypothesis that PS externalization is driven by the potential that develops across the lipid bilayer as the membrane capacitance charges during a pulse. Threshold pulsed field amplitudes correspond to the transmembrane potential (ges0.5 V) that causes increased conductance when much longer (microsecond) pulses are applied (conventional electroporation), suggesting that some membrane poration may be associated with these pulse doses. Nanometer-diameter hydrophilic pores form within nanoseconds of the application of megavolt-per-meter electric fields normal to the lipid bilayer in MD simulations, and PS migrates electrophoretically through the pores in the direction of the anode, consistent with imaging analysis of PS externalization using the fluorescent dye FM1-43. Influx of YO-PRO-1, a fluorochrome that binds to nucleic acids, and which is also used as a sensitive indicator of early apoptosis and of activation of P2X <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">7</sub> receptor channels, is in fact observed after delivery of sufficiently large numbers of nanosecond pulses to sensitive cells. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher nanoelectropulse counts