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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

Increased electric field augments electroporation (A–D) but has little impact on the relative efficiency of uni- and bipolar NEFO (F).(A–D) YP dye uptake triggered by cell permeabilization with a train of 20 unipolar () or bipolar () NEFO, delivered at 10 Hz starting at 28 s. The peak amplitude of NEFO is indicated in legends. Mean ± SE for 19–29 cells in each group. A control group subjected to sham exposure is plotted in (D) only. See Fig. 1 for more details. (E) Representative differential-interference contrast (DIC; top) and YP fluorescence images (bottom) before exposure and at 300 s after 20 unipolar or bipolar NEFO at 24 kV/cm, 10 Hz (left and right panels, respectively). Scale bars are 10 μm. Note greater YP uptake and morphological changes (swelling, blebbing, and cytoplasm granulation) after unipolar NEFO. (F) The effect of NEFO amplitude on YP emission reached by 300 s (top plot) and the ratio of emission values after uni- and bipolar NEFO (Eu/Eb, bottom). Both linear fits (top) cross the zero fluorescence level (dashed line) at ~7 kV/cm, indicating a common threshold for electroporation by either NEFO waveform. The unipolar NEFO caused 2–3 fold greater YP uptake with no apparent dependence on the peak electric field (bottom).
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f2: Increased electric field augments electroporation (A–D) but has little impact on the relative efficiency of uni- and bipolar NEFO (F).(A–D) YP dye uptake triggered by cell permeabilization with a train of 20 unipolar () or bipolar () NEFO, delivered at 10 Hz starting at 28 s. The peak amplitude of NEFO is indicated in legends. Mean ± SE for 19–29 cells in each group. A control group subjected to sham exposure is plotted in (D) only. See Fig. 1 for more details. (E) Representative differential-interference contrast (DIC; top) and YP fluorescence images (bottom) before exposure and at 300 s after 20 unipolar or bipolar NEFO at 24 kV/cm, 10 Hz (left and right panels, respectively). Scale bars are 10 μm. Note greater YP uptake and morphological changes (swelling, blebbing, and cytoplasm granulation) after unipolar NEFO. (F) The effect of NEFO amplitude on YP emission reached by 300 s (top plot) and the ratio of emission values after uni- and bipolar NEFO (Eu/Eb, bottom). Both linear fits (top) cross the zero fluorescence level (dashed line) at ~7 kV/cm, indicating a common threshold for electroporation by either NEFO waveform. The unipolar NEFO caused 2–3 fold greater YP uptake with no apparent dependence on the peak electric field (bottom).

Mentions: We further checked if the degree of cancellation can be controlled by the stimulus intensity. A brief 10 Hz train of 20 NEFO applied at 28 s into the experiment triggered immediate YP uptake at pulse amplitudes from 9.6 to 24 kV/cm (Fig. 2A–D), but not at 4.8 kV/cm (data not shown). Within the electroporating range of amplitudes, the dye uptake (as measured by the end of the experiment) increased linearly with increasing the electric field for both uni- and bipolar NEFO, whereas the latter consistently was 2–3 fold less effective (Fig. 2F). The linear fits for uni- and bipolar NEFO crossed zero at the same extrapolated electric field strength of 7 kV/cm, which can be regarded as a threshold for both waveform types. This value is remarkably close to 6 kV/cm electroporation threshold for 60-ns rectangular pulses, as measured by patch clamp49, arguably the most sensitive method to detect electroporation50. In contrast to long (micro- and millisecond) electric pulses, electroporation by NEFO may rely on dielectric stacking rather than Maxwell-Wagner polarization50 thus being less dependent on the cell size or shape51; hence the above measurements of the electroporation threshold can hold true for a variety of cells. The greater extent of cell membrane disruption by unipolar NEFO is also manifested by morphological changes (cell swelling, blebbing, and cytoplasm granulation), whereas cells exposed to bipolar NEFO showed little or no change (Fig. 2E).


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)

Increased electric field augments electroporation (A–D) but has little impact on the relative efficiency of uni- and bipolar NEFO (F).(A–D) YP dye uptake triggered by cell permeabilization with a train of 20 unipolar () or bipolar () NEFO, delivered at 10 Hz starting at 28 s. The peak amplitude of NEFO is indicated in legends. Mean ± SE for 19–29 cells in each group. A control group subjected to sham exposure is plotted in (D) only. See Fig. 1 for more details. (E) Representative differential-interference contrast (DIC; top) and YP fluorescence images (bottom) before exposure and at 300 s after 20 unipolar or bipolar NEFO at 24 kV/cm, 10 Hz (left and right panels, respectively). Scale bars are 10 μm. Note greater YP uptake and morphological changes (swelling, blebbing, and cytoplasm granulation) after unipolar NEFO. (F) The effect of NEFO amplitude on YP emission reached by 300 s (top plot) and the ratio of emission values after uni- and bipolar NEFO (Eu/Eb, bottom). Both linear fits (top) cross the zero fluorescence level (dashed line) at ~7 kV/cm, indicating a common threshold for electroporation by either NEFO waveform. The unipolar NEFO caused 2–3 fold greater YP uptake with no apparent dependence on the peak electric field (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Increased electric field augments electroporation (A–D) but has little impact on the relative efficiency of uni- and bipolar NEFO (F).(A–D) YP dye uptake triggered by cell permeabilization with a train of 20 unipolar () or bipolar () NEFO, delivered at 10 Hz starting at 28 s. The peak amplitude of NEFO is indicated in legends. Mean ± SE for 19–29 cells in each group. A control group subjected to sham exposure is plotted in (D) only. See Fig. 1 for more details. (E) Representative differential-interference contrast (DIC; top) and YP fluorescence images (bottom) before exposure and at 300 s after 20 unipolar or bipolar NEFO at 24 kV/cm, 10 Hz (left and right panels, respectively). Scale bars are 10 μm. Note greater YP uptake and morphological changes (swelling, blebbing, and cytoplasm granulation) after unipolar NEFO. (F) The effect of NEFO amplitude on YP emission reached by 300 s (top plot) and the ratio of emission values after uni- and bipolar NEFO (Eu/Eb, bottom). Both linear fits (top) cross the zero fluorescence level (dashed line) at ~7 kV/cm, indicating a common threshold for electroporation by either NEFO waveform. The unipolar NEFO caused 2–3 fold greater YP uptake with no apparent dependence on the peak electric field (bottom).
Mentions: We further checked if the degree of cancellation can be controlled by the stimulus intensity. A brief 10 Hz train of 20 NEFO applied at 28 s into the experiment triggered immediate YP uptake at pulse amplitudes from 9.6 to 24 kV/cm (Fig. 2A–D), but not at 4.8 kV/cm (data not shown). Within the electroporating range of amplitudes, the dye uptake (as measured by the end of the experiment) increased linearly with increasing the electric field for both uni- and bipolar NEFO, whereas the latter consistently was 2–3 fold less effective (Fig. 2F). The linear fits for uni- and bipolar NEFO crossed zero at the same extrapolated electric field strength of 7 kV/cm, which can be regarded as a threshold for both waveform types. This value is remarkably close to 6 kV/cm electroporation threshold for 60-ns rectangular pulses, as measured by patch clamp49, arguably the most sensitive method to detect electroporation50. In contrast to long (micro- and millisecond) electric pulses, electroporation by NEFO may rely on dielectric stacking rather than Maxwell-Wagner polarization50 thus being less dependent on the cell size or shape51; hence the above measurements of the electroporation threshold can hold true for a variety of cells. The greater extent of cell membrane disruption by unipolar NEFO is also manifested by morphological changes (cell swelling, blebbing, and cytoplasm granulation), whereas cells exposed to bipolar NEFO showed little or no change (Fig. 2E).

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