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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

Reduced membrane permeabilization by bipolar NEFO is revealed by whole-cell electrical conductance measurements.(A) Current-voltage dependence in CHO cells at indicated times before (−20 s) and after exposure (10 to 60 s) to a single unipolar () or bipolar () NEFO at 14.4 kV/cm. Note the different vertical scale in the first panel. (B) Traces of the whole-cell current in a representative cell, 20 s before (top) and 10 s after (center) the exposure to one bipolar NEFO. The bottom traces are the command voltage steps, from −100 to + 40 mV in 10-mV increments; the holding level is at −80 mV. Calibration bars: 100 μs and 250 pA. (C) The time course of the whole cell conductance in the experiments from panels (A–E). Mean ± SE for 11 cells in each group. Vertical dashed line denotes the time of NEFO application. Significant differences (p < 0.05) are labeled with asterisk in panel (C) only.
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f5: Reduced membrane permeabilization by bipolar NEFO is revealed by whole-cell electrical conductance measurements.(A) Current-voltage dependence in CHO cells at indicated times before (−20 s) and after exposure (10 to 60 s) to a single unipolar () or bipolar () NEFO at 14.4 kV/cm. Note the different vertical scale in the first panel. (B) Traces of the whole-cell current in a representative cell, 20 s before (top) and 10 s after (center) the exposure to one bipolar NEFO. The bottom traces are the command voltage steps, from −100 to + 40 mV in 10-mV increments; the holding level is at −80 mV. Calibration bars: 100 μs and 250 pA. (C) The time course of the whole cell conductance in the experiments from panels (A–E). Mean ± SE for 11 cells in each group. Vertical dashed line denotes the time of NEFO application. Significant differences (p < 0.05) are labeled with asterisk in panel (C) only.

Mentions: The first series of experiments (Fig. 5) was performed in the presence of 2 mM extracellular Ca2+, but using CHO cells which lack any voltage-gated Ca2+ channels. Pipette solution was buffered with EGTA, thereby excluding the impact of Ca2+ which could potentially be released from the endoplasmic reticulum1251. The whole-cell configuration was established 1–2 min prior to the delivery of a single unipolar or bipolar NEFO at 14.4 kV/cm. The membrane conductance was measured in a voltage clamp mode, by applying a voltage-step protocol at 10 s prior to NEFO and then at 10, 20, 30, and 60 s after it.


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)

Reduced membrane permeabilization by bipolar NEFO is revealed by whole-cell electrical conductance measurements.(A) Current-voltage dependence in CHO cells at indicated times before (−20 s) and after exposure (10 to 60 s) to a single unipolar () or bipolar () NEFO at 14.4 kV/cm. Note the different vertical scale in the first panel. (B) Traces of the whole-cell current in a representative cell, 20 s before (top) and 10 s after (center) the exposure to one bipolar NEFO. The bottom traces are the command voltage steps, from −100 to + 40 mV in 10-mV increments; the holding level is at −80 mV. Calibration bars: 100 μs and 250 pA. (C) The time course of the whole cell conductance in the experiments from panels (A–E). Mean ± SE for 11 cells in each group. Vertical dashed line denotes the time of NEFO application. Significant differences (p < 0.05) are labeled with asterisk in panel (C) only.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Reduced membrane permeabilization by bipolar NEFO is revealed by whole-cell electrical conductance measurements.(A) Current-voltage dependence in CHO cells at indicated times before (−20 s) and after exposure (10 to 60 s) to a single unipolar () or bipolar () NEFO at 14.4 kV/cm. Note the different vertical scale in the first panel. (B) Traces of the whole-cell current in a representative cell, 20 s before (top) and 10 s after (center) the exposure to one bipolar NEFO. The bottom traces are the command voltage steps, from −100 to + 40 mV in 10-mV increments; the holding level is at −80 mV. Calibration bars: 100 μs and 250 pA. (C) The time course of the whole cell conductance in the experiments from panels (A–E). Mean ± SE for 11 cells in each group. Vertical dashed line denotes the time of NEFO application. Significant differences (p < 0.05) are labeled with asterisk in panel (C) only.
Mentions: The first series of experiments (Fig. 5) was performed in the presence of 2 mM extracellular Ca2+, but using CHO cells which lack any voltage-gated Ca2+ channels. Pipette solution was buffered with EGTA, thereby excluding the impact of Ca2+ which could potentially be released from the endoplasmic reticulum1251. The whole-cell configuration was established 1–2 min prior to the delivery of a single unipolar or bipolar NEFO at 14.4 kV/cm. The membrane conductance was measured in a voltage clamp mode, by applying a voltage-step protocol at 10 s prior to NEFO and then at 10, 20, 30, and 60 s after it.

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