Research Article: Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane

Date Published: November 23, 2009

Publisher: Public Library of Science

Author(s): P. Thomas Vernier, Zachary A. Levine, Yu-Hsuan Wu, Vanessa Joubert, Matthew J. Ziegler, Lluis M. Mir, D. Peter Tieleman, Boris Rubinsky.

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.

Partial Text: External electric fields of sufficient strength and duration cause a rapid increase in the electrical conductance of biological membranes, with an associated increased permeability to ions and small and large molecules [1]–[3]. If the dose is limited, cells can survive the treatment. Electropermeabilization (also called electroporation) technology is widely used in laboratories to facilitate transfection in cells and tissues [4]–[6] and recently has appeared in the clinic as a component of systems for electrochemotherapy [7] and tumor killing and ablation [8]–[11]. Although the physical and electrochemical fundamentals of electric field-induced permeabilization of lipid membranes are well known [12]–[14], the mechanistic details of the membrane restructuring that follows electric field exposure in living cells have not been definitively established. Electrical measurements [15], flow cytometry [16], and fluorescence microscopy [17] indicate that permeabilization can occur in less than 10 ns, implying a direct rearrangement of membrane components, but real-time analysis of any kind at this time scale is difficult [18]–[19]. Observations with living cells [20], artificial membranes and lipid vesicles [21]–[24], continuum electrophysical models [25]–[26], and molecular dynamics (MD) simulations of phospholipid bilayers [27]–[31] provide a valuable perspective, but significant gaps remain between these model systems and the complexity of the tissue in an electropermeabilized tumor.

Molecular dynamics simulations of lipid bilayers and laboratory studies of model membranes and cells in electric fields are consistent with the stochastic pore hypothesis for electropermeabilization [44]–[47]. Here we have shown that it is possible to bias pore formation at the molecular level by oxidatively modifying the properties of the membrane in situ, so that we have now a new method for lowering the energy barrier to poration and a mechanism that might be used to localize where in the membrane poration occurs. This demonstration of the increased sensitivity of oxidatively damaged cells to electropermeabilization has practical implications for the laboratory and the clinic.



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