Multiple nanosecond electric pulses increase the number but not the size of long-lived nanopores in the cell membrane

Abstract

Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr), in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at < 4 µM), and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60 ns, 13.2 kV/cm, 10 Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3 min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.

Asymmetric Waveforms Decrease Lethal Thresholds in High Frequency Irreversible Electroporation Therapies

“…Previous studies on sub-microsecond pulses showed that increasing the number of monopolar pulses increased the number, but not the size of nanopores formed in the membrane41. A similar phenomena may be occurring here in which continued delivery of pulses 1 μs or shorter leads to the formation of numerous smaller pores rather than rapid cell membrane destruction caused by pore expansion and any lethal effects are due to mechanisms which occur post-treatment.…”

“…However, we hypothesize that the rapid charging and discharging of the cell membrane by the symmetric pulses inhibits pore expansion and instead results in an increase in the number, but not size of the pores formed41. However, it is unclear if these symmetric waveforms will be capable of transporting molecules across biological membranes efficiently.…”

Diffuse, non-polar electropermeabilization and reduced propidium uptake distinguish the effect of nanosecond electric pulses

“…At the same time, previous studies as well as in silico models found that shorter electric pulses produce fewer large-diameter pores [12, 14, 30, 36] and should be less damaging. Measuring the electroporative entry of a small cation such as Ca 2+ does not necessarily reveal the difference in pore size, hence it was probed with a larger Pr cation [15, 54]. …”

“…The technique of electric field delivery to individual cells was the same as reported previously [4, 13–15, 20, 55]. Trapezoidal pulses of up to 20 kV amplitude and approximately 10 ns duration (at 50% height) were produced by a model FPG 20–1NM pulse generator (FID GmbH, Burbach, Germany).…”

“…Pulses were triggered externally and synchronized with image acquisition and bath buffer exchanges by a TTL pulse protocol using Digidata 1440A board and Clampex v. 10.2 software (Molecular Devices, Sunnyvale, CA). The electric field at the location of the cells was determined by a 3D simulation with a finite-element Maxwell equation solver Amaze 3D (Field Precision, Albuquerque, NM) as described earlier[15]. …”