Electroporation of mammalian cells by nanosecond electric field oscillations and its inhibition by the electric field reversal

The present study compared electroporation efficiency of bipolar and unipolar nanosecond electric field oscillations (NEFO). Bipolar NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2-4 decreased to 35%, 12%, and 7% of the first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4. Membrane permeabilization was quantified in CHO and GH3 cells by uptake of a membrane integrity marker dye YO-PRO-1 (YP) and by the membrane conductance increase measured by patch clamp. For treatments with 1-20 unipolar NEFO, at 9.6-24 kV/cm, 10 Hz, the rate and amount of YP uptake were consistently 2-3-fold higher than after bipolar NEFO treatments, despite delivering less energy. However, the threshold amplitude was about 7 kV/cm for both NEFO waveforms. A single 14.4 kV/cm unipolar NEFO caused a 1.5-2 times greater increase in membrane conductance (p<0.05) than bipolar NEFO, along with a longer and less frequent recovery. The lower efficiency of bipolar NEFO was preserved in Ca2+-free conditions and thus cannot be explained by the reversal of electrophoretic flows of Ca2+. Instead, the data indicate that the electric field polarity reversals reduced the pore yield.