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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 resealing kinetics of electropermeabilized cells does not depend on the type of NEFO or its amplitude.Panels (A–D) (top row) show the change in YP fluorescence between the sequential images (i.e., emission gain per 10-s intervals). In the respective bottom row plots, the data are normalized to the maximum emission gain values for each plot. Note the essentially identical time course of the plots for uni- and bipolar NEFO. All data are from the experiments presented in Fig. 2. (E) All eight normalized curves from the above panels plotted together to show no apparent impact of either shape or amplitude of NEFO. Error bars are omitted for clarity. (F) The effect of NEFO amplitude on the maximum YP emission gain. See Fig. 2 and text for more details.
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f3: The resealing kinetics of electropermeabilized cells does not depend on the type of NEFO or its amplitude.Panels (A–D) (top row) show the change in YP fluorescence between the sequential images (i.e., emission gain per 10-s intervals). In the respective bottom row plots, the data are normalized to the maximum emission gain values for each plot. Note the essentially identical time course of the plots for uni- and bipolar NEFO. All data are from the experiments presented in Fig. 2. (E) All eight normalized curves from the above panels plotted together to show no apparent impact of either shape or amplitude of NEFO. Error bars are omitted for clarity. (F) The effect of NEFO amplitude on the maximum YP emission gain. See Fig. 2 and text for more details.

Mentions: The rate of YP uptake gradually decreased with time after exposure, reflecting the shrinkage and/or resealing of NEFO-opened membrane pores (Fig. 3). This figure shows the difference in YP fluorescence between the sequential images (i.e., the gain in fluorescence per 10-s interval between the images). For all studied NEFO amplitudes, the maximum YP uptake rate was 2-3-fold lower for bipolar NEFO (Fig. 3F); however, the pore resealing kinetics for uni- and bipolar NEFO showed no difference. When the dye uptake rates were normalized to the peak value for the respective treatment, the dynamics of the rate reduction after both types of NEFO were the same (Fig. 3A–D, lower panels). Moreover, there was no difference in the rate reduction kinetics for NEFO applied at different electric field amplitudes (Fig. 3E). Consistent with this observation, fitting the reduction of the YP uptake rate after uni- and bipolar NEFO treatments with a double-exponential function showed no difference in either fast or slow time constants. For example, for 24 kV/cm uni- and bipolar NEFO, respectively, the fast time constants were 7.9 +/− 0.6 s and 7.6 +/− 1.5 s, and the slow time constants were 93 +/− 15 s and 97 +/− 21 s. The similarity of the pore resealing/shrinkage kinetics suggests that uni- and bipolar NEFO opened membrane pores with similar properties which sealed at the same rate. Therefore, the increased YP uptake after unipolar NEFO should be attributed to the increased number of membrane pores formed, rather than to the formation of qualitatively different (e.g., larger) pores.


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 resealing kinetics of electropermeabilized cells does not depend on the type of NEFO or its amplitude.Panels (A–D) (top row) show the change in YP fluorescence between the sequential images (i.e., emission gain per 10-s intervals). In the respective bottom row plots, the data are normalized to the maximum emission gain values for each plot. Note the essentially identical time course of the plots for uni- and bipolar NEFO. All data are from the experiments presented in Fig. 2. (E) All eight normalized curves from the above panels plotted together to show no apparent impact of either shape or amplitude of NEFO. Error bars are omitted for clarity. (F) The effect of NEFO amplitude on the maximum YP emission gain. See Fig. 2 and text for more details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The resealing kinetics of electropermeabilized cells does not depend on the type of NEFO or its amplitude.Panels (A–D) (top row) show the change in YP fluorescence between the sequential images (i.e., emission gain per 10-s intervals). In the respective bottom row plots, the data are normalized to the maximum emission gain values for each plot. Note the essentially identical time course of the plots for uni- and bipolar NEFO. All data are from the experiments presented in Fig. 2. (E) All eight normalized curves from the above panels plotted together to show no apparent impact of either shape or amplitude of NEFO. Error bars are omitted for clarity. (F) The effect of NEFO amplitude on the maximum YP emission gain. See Fig. 2 and text for more details.
Mentions: The rate of YP uptake gradually decreased with time after exposure, reflecting the shrinkage and/or resealing of NEFO-opened membrane pores (Fig. 3). This figure shows the difference in YP fluorescence between the sequential images (i.e., the gain in fluorescence per 10-s interval between the images). For all studied NEFO amplitudes, the maximum YP uptake rate was 2-3-fold lower for bipolar NEFO (Fig. 3F); however, the pore resealing kinetics for uni- and bipolar NEFO showed no difference. When the dye uptake rates were normalized to the peak value for the respective treatment, the dynamics of the rate reduction after both types of NEFO were the same (Fig. 3A–D, lower panels). Moreover, there was no difference in the rate reduction kinetics for NEFO applied at different electric field amplitudes (Fig. 3E). Consistent with this observation, fitting the reduction of the YP uptake rate after uni- and bipolar NEFO treatments with a double-exponential function showed no difference in either fast or slow time constants. For example, for 24 kV/cm uni- and bipolar NEFO, respectively, the fast time constants were 7.9 +/− 0.6 s and 7.6 +/− 1.5 s, and the slow time constants were 93 +/− 15 s and 97 +/− 21 s. The similarity of the pore resealing/shrinkage kinetics suggests that uni- and bipolar NEFO opened membrane pores with similar properties which sealed at the same rate. Therefore, the increased YP uptake after unipolar NEFO should be attributed to the increased number of membrane pores formed, rather than to the formation of qualitatively different (e.g., larger) pores.

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.