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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.


The reduced electroporation efficiency of bipolar NEFO is preserved in a Ca2+-free solution (no added Ca2+, 2 mM EGTA).Cells were exposed to a train of 20 unipolar () or bipolar () NEFO (10 Hz, 24 kV/cm), delivered starting at 28 s. Mean +/− SE for 12–17 cells in each group. See Fig. 1 for other details.
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f4: The reduced electroporation efficiency of bipolar NEFO is preserved in a Ca2+-free solution (no added Ca2+, 2 mM EGTA).Cells were exposed to a train of 20 unipolar () or bipolar () NEFO (10 Hz, 24 kV/cm), delivered starting at 28 s. Mean +/− SE for 12–17 cells in each group. See Fig. 1 for other details.

Mentions: One of mechanistic hypotheses aimed at explaining the bipolar cancellation phenomenon points to the reversal of electrophoretic entry of Ca2+ when the electric field polarity is reversed3341. This hypothesis considers various long-lasting effects of nanosecond pulses (such as YP uptake and cell death) as consequences of cell overload with Ca2+ during the electric pulse. In the case of a bipolar pulse, the first phase of the pulse facilitates the entry of Ca2+ into the electroporated cell, whereas the next phase moves it out and thereby decreases the net Ca2+ uptake and its consequences. To test this idea, we repeated the experiments shown in Fig. 2D in a Ca2+-free buffer. Contrary to theoretical predictions, unipolar NEFO still caused a 3-fold greater YP uptake (Fig. 4). Thus, the reversal of Ca2+ flow across the plasma membrane was ruled out as a reason for the reduced effect of bipolar stimuli. With that said, nanosecond stimuli can permeabilize the cytoplasmic reticulum to elevate the cytosolic Ca2+ level121851, so the possible impact of the reverse Ca2+ drift across the reticulum membrane has not been excluded.


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)

The reduced electroporation efficiency of bipolar NEFO is preserved in a Ca2+-free solution (no added Ca2+, 2 mM EGTA).Cells were exposed to a train of 20 unipolar () or bipolar () NEFO (10 Hz, 24 kV/cm), delivered starting at 28 s. Mean +/− SE for 12–17 cells in each group. See Fig. 1 for other details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The reduced electroporation efficiency of bipolar NEFO is preserved in a Ca2+-free solution (no added Ca2+, 2 mM EGTA).Cells were exposed to a train of 20 unipolar () or bipolar () NEFO (10 Hz, 24 kV/cm), delivered starting at 28 s. Mean +/− SE for 12–17 cells in each group. See Fig. 1 for other details.
Mentions: One of mechanistic hypotheses aimed at explaining the bipolar cancellation phenomenon points to the reversal of electrophoretic entry of Ca2+ when the electric field polarity is reversed3341. This hypothesis considers various long-lasting effects of nanosecond pulses (such as YP uptake and cell death) as consequences of cell overload with Ca2+ during the electric pulse. In the case of a bipolar pulse, the first phase of the pulse facilitates the entry of Ca2+ into the electroporated cell, whereas the next phase moves it out and thereby decreases the net Ca2+ uptake and its consequences. To test this idea, we repeated the experiments shown in Fig. 2D in a Ca2+-free buffer. Contrary to theoretical predictions, unipolar NEFO still caused a 3-fold greater YP uptake (Fig. 4). Thus, the reversal of Ca2+ flow across the plasma membrane was ruled out as a reason for the reduced effect of bipolar stimuli. With that said, nanosecond stimuli can permeabilize the cytoplasmic reticulum to elevate the cytosolic Ca2+ level121851, so the possible impact of the reverse Ca2+ drift across the reticulum membrane has not been excluded.

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.