Electropermeabilization of cells by closely spaced paired nanosecond-range pulses.

Decreasing the time gap between two identical electric pulses is expected to render bioeffects similar to those of a single pulse of equivalent total duration. In this study, we show that it is not necessarily true, and that the effects vary for different permeabilization markers. We exposed individual CHO or NG108 cells to one 300-ns pulse (3.7-11.6 kV/cm), or a pair of such pulses (0.4-1000 µs interval), or to a single 600-ns pulse of the same amplitude. Electropermeabilization was evaluated (a) by the uptake of YO-PRO-1 (YP) dye; (b) by the amplitude of elicited Ca2+ transients, and (c) by the entry of Tl+ ions. For YP uptake, applying a 600-ns pulse or a pair of 300-ns pulses doubled the effect of a single 300-ns pulse; this additive effect did not depend on the time interval between pulses or the electric field, indicating that already permeabilized cells are as susceptible to electropermeabilization as naïve cells. In contrast, Ca2+ transients and Tl+ uptake increased in a supra-additive fashion when two pulses were delivered instead of one. Paired pulses at 3.7 kV/cm with minimal separation (0.4 and 1 µs) elicited 50-100% larger Ca2+ transients than either a single 600-ns pulse or paired pulses with longer separation (10-1000 µs). This paradoxically high efficiency of the closest spaced pulses was emphasized when Ca2+ transients were elicited in a Ca2+-free solution (when the endoplasmic reticulum (ER) was the sole significant source of Ca2+), but was eliminated by Ca2+ depletion from the ER and was not observed for Tl+ entry through the electropermeabilized membrane. We conclude that closely spaced paired pulses specifically target ER, by either permeabilizing it to a greater extent than a single double-duration pulse thus causing more Ca2+ leak, or by amplifying Ca2+-induced Ca2+ release by an unknown mechanism.

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.