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

Gianulis EC, Lee J, Jiang C, Xiao S, Ibey BL, Pakhomov AG - Sci Rep (2015)

Bottom Line: 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.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.Instead, the data indicate that the electric field polarity reversals reduced the pore yield.

View Article: PubMed Central - PubMed

Affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Unipolar and bipolar nanosecond electric field oscillations (A) have different potency to electroporate CHO cells (B–D).(A) The two types of NEFO have the same shape and amplitude of positive-going phases, with the first phase duration of 140 ns at 50% height. The unipolar NEFO lacks negative-going phases 2 and 4. (B–D) Electroporation is revealed by the time-lapse imaging of YP dye uptake. Cells were exposed to either 1 (A), 5 (B), or 10 (D) unipolar () or bipolar () NEFO (24 kV/cm, 10 Hz) at 28 s into the experiment (vertical dashed line). The exposure parameters are also provided in panel legends. Mean ± SE for 9–23 cells in each group. Higher dye uptake in cells exposed to unipolar NEFO was significant in all groups (p < 0.01). A common control group subjected to sham exposure (plotted in panel (B) only) showed no appreciable fluorescence gain.
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f1: Unipolar and bipolar nanosecond electric field oscillations (A) have different potency to electroporate CHO cells (B–D).(A) The two types of NEFO have the same shape and amplitude of positive-going phases, with the first phase duration of 140 ns at 50% height. The unipolar NEFO lacks negative-going phases 2 and 4. (B–D) Electroporation is revealed by the time-lapse imaging of YP dye uptake. Cells were exposed to either 1 (A), 5 (B), or 10 (D) unipolar () or bipolar () NEFO (24 kV/cm, 10 Hz) at 28 s into the experiment (vertical dashed line). The exposure parameters are also provided in panel legends. Mean ± SE for 9–23 cells in each group. Higher dye uptake in cells exposed to unipolar NEFO was significant in all groups (p < 0.01). A common control group subjected to sham exposure (plotted in panel (B) only) showed no appreciable fluorescence gain.

Mentions: A concurrent aim of this study was to extend the observation of bipolar cancellation from individual rectangular pulses to a different type of stimuli, namely to nanosecond electric field oscillations (NEFO). An individual bipolar NEFO (Fig. 1A) is essentially a damped sine wave which can serve as a laboratory surrogate of high power electromagnetic pulse and ultra-wide band emissions, two environmental factors which have been intensely studied for physiological and health effects4243444546. We hypothesized that the bipolar cancellation effect could underlie the experimentally established inefficiency of these emissions as biological stimuli. However, NEFO are substantially different from previously studied rectangular nanosecond pulses (by a slow risetime, the lack of a plateau at the high voltage, sharply reduced amplitude of the reverse polarity phase, and by a fast repetition of several polarity reversals), thus making extrapolation of the earlier findings to NEFO questionable. Finally, we also aimed to shed light on mechanisms underlying the bipolar cancellation by analyzing how the electroporation by bipolar NEFO and subsequent recovery depend on the electric field amplitude and delivery protocols. For comparison, we used a unipolar NEFO which had no polarity reversals to evoke the bipolar cancellation. The unipolar NEFO was produced by rectification of the bipolar one and therefore had essentially the same peak amplitude and duration (Fig. 1A).


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

Gianulis EC, Lee J, Jiang C, Xiao S, Ibey BL, Pakhomov AG - Sci Rep (2015)

Unipolar and bipolar nanosecond electric field oscillations (A) have different potency to electroporate CHO cells (B–D).(A) The two types of NEFO have the same shape and amplitude of positive-going phases, with the first phase duration of 140 ns at 50% height. The unipolar NEFO lacks negative-going phases 2 and 4. (B–D) Electroporation is revealed by the time-lapse imaging of YP dye uptake. Cells were exposed to either 1 (A), 5 (B), or 10 (D) unipolar () or bipolar () NEFO (24 kV/cm, 10 Hz) at 28 s into the experiment (vertical dashed line). The exposure parameters are also provided in panel legends. Mean ± SE for 9–23 cells in each group. Higher dye uptake in cells exposed to unipolar NEFO was significant in all groups (p < 0.01). A common control group subjected to sham exposure (plotted in panel (B) only) showed no appreciable fluorescence gain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562301&req=5

f1: Unipolar and bipolar nanosecond electric field oscillations (A) have different potency to electroporate CHO cells (B–D).(A) The two types of NEFO have the same shape and amplitude of positive-going phases, with the first phase duration of 140 ns at 50% height. The unipolar NEFO lacks negative-going phases 2 and 4. (B–D) Electroporation is revealed by the time-lapse imaging of YP dye uptake. Cells were exposed to either 1 (A), 5 (B), or 10 (D) unipolar () or bipolar () NEFO (24 kV/cm, 10 Hz) at 28 s into the experiment (vertical dashed line). The exposure parameters are also provided in panel legends. Mean ± SE for 9–23 cells in each group. Higher dye uptake in cells exposed to unipolar NEFO was significant in all groups (p < 0.01). A common control group subjected to sham exposure (plotted in panel (B) only) showed no appreciable fluorescence gain.
Mentions: A concurrent aim of this study was to extend the observation of bipolar cancellation from individual rectangular pulses to a different type of stimuli, namely to nanosecond electric field oscillations (NEFO). An individual bipolar NEFO (Fig. 1A) is essentially a damped sine wave which can serve as a laboratory surrogate of high power electromagnetic pulse and ultra-wide band emissions, two environmental factors which have been intensely studied for physiological and health effects4243444546. We hypothesized that the bipolar cancellation effect could underlie the experimentally established inefficiency of these emissions as biological stimuli. However, NEFO are substantially different from previously studied rectangular nanosecond pulses (by a slow risetime, the lack of a plateau at the high voltage, sharply reduced amplitude of the reverse polarity phase, and by a fast repetition of several polarity reversals), thus making extrapolation of the earlier findings to NEFO questionable. Finally, we also aimed to shed light on mechanisms underlying the bipolar cancellation by analyzing how the electroporation by bipolar NEFO and subsequent recovery depend on the electric field amplitude and delivery protocols. For comparison, we used a unipolar NEFO which had no polarity reversals to evoke the bipolar cancellation. The unipolar NEFO was produced by rectification of the bipolar one and therefore had essentially the same peak amplitude and duration (Fig. 1A).

Bottom Line: 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.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.Instead, the data indicate that the electric field polarity reversals reduced the pore yield.

View Article: PubMed Central - PubMed

Affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus