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. 2009 Nov 23;4(11):e7966.
doi: 10.1371/journal.pone.0007966.

Electroporating fields target oxidatively damaged areas in the cell membrane

P Thomas Vernier  1 , Zachary A Levine, Yu-Hsuan Wu, Vanessa Joubert, Matthew J Ziegler, Lluis M Mir, D Peter Tieleman
Affiliations

Electroporating fields target oxidatively damaged areas in the cell membrane

P Thomas Vernier et al. PLoS One. .

Abstract

Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Oxidized and unoxidized phospholipid conformations change over time.
Composite snapshots (21 images captured at 0.5 ns intervals over a 10 ns period) of PLPC and two oxidized variants, oxPLPC (12-al) and oxPLPC (13-tc), showing their conformations in molecular dynamics simulations of PLPC with 11% oxPLPC bilayers in a 360 mV/nm field. The spheres near the end of the lipid tails mark the location of the introduced oxygens or C-13 of PLPC. Structures of the individual lipid molecules are shown below the corresponding composite. Teal – C, red – O, gold – P, blue – N, gray – C-13.
Figure 2
Figure 2. Poration of an oxidized phospholipid bilayer.
Snapshots of a PLPC system with an 11% concentration of 12-al oxPLPC before (a) and after (b) an electropore is formed (separated by about 2 ns.) Only water (small red and white "v's") and the head groups and sn-2 tails of the oxPLPC molecules are shown. The large red spheres near the ends of the tails are aldehyde oxygens, which appear to facilitate the entry of water into the bilayer interior. Dimensions of the simulation box are approximately 5 nm×5 nm×7 nm.
Figure 3
Figure 3. Quilted PLPC:oxPLPC bilayer.
The simulated system, bounded by the black square in panel A, is divided (dashed lines) into two regions of approximately 100% PLPC (light gray) and two regions of approximately 50% oxPLPC (12-al) (dark blue) and 50% PLPC, as described in the text. To show more clearly where poration occurs after application of an external electric field, copies of the simulated system are tiled to make a periodic 2×2 system (approximately 10 nm×10 nm×7 nm). Preferential electroporation of the PLPC bilayer in regions of high oxidized lipid content is demonstrated in panel B, which shows the system 1 ns after applying a 360 mV/nm field normal to the bilayer. The bilayer is in the plane of the page.
Figure 4
Figure 4. Electropermeabilization enhancement by treatment with peroxidation agents.
(A). Fluorescence images of Jurkat cells showing pulse-induced (30 ns, 3 MV/m, 50 Hz) YO-PRO-1 influx into control and peroxidized cells after 10 min exposure to 500 µM H2O2+1000 µM FeSO4. (B). Integrated YO-PRO-1 fluorescence intensity from more than 300 individual cells from three independent experiments for each condition. Error bars are standard error of the mean.
Figure 5
Figure 5. Enhanced electropermeabilization of peroxidized cells with both ultra-short and conventional electric pulse treatments.
(A). The fluorescence of calcein-loaded DC-3F (Chinese hamster lung fibroblast) cells exposed to 1000 10 ns, 2.5 MV/m pulses with a 10 Hz repetition rate decreases after 10 min, indicating membrane permeabilization. The effect is much greater in peroxidized cells than in untreated control cells. (B). Peroxidized cells treated with a single 100 µs, 50 kV/m pulse show a similar increased susceptibility to electropermeabilization. Increasing the 100 µs pulse amplitude to 60 kV/m results in significant permeabilization of control cells, indicating that these doses are near the threshold for a detectable response under these conditions. Peroxidized cells treated with a single 100 µs, 60 kV/m pulse show a strong decrease of fluorescence, indicating substantial membrane permeabilization. Bars are the mean, and error bars are the standard deviation. * p<0.05; ** p<0.01; *** p<0.001.
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