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Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Electropermeabilization is a physical method that uses electric field pulses to deliver molecules into cells and tissues. Despite its increasing interest in clinics, little is known about plasma membrane destabilization process occurring during electropermeabilization. In this work, we took advantage of atomic force microscopy to directly visualize the consequences of electropermeabilization in terms of membrane reorganization and to locally measure the membrane elasticity. We visualized transient rippling of membrane surface and measured a decrease in membrane elasticity by 40%. Our results obtained both on fixed and living CHO cells give evidence of an inner effect affecting the entire cell surface that may be related to cytoskeleton destabilization. Thus, AFM appears as a useful tool to investigate basic process of electroporation on living cells in absence of any staining or cell preparation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Electroporation is a physical method to induce the uptake of therapeutic drugs and DNA, by eukaryotic cells and tissues. The phenomena behind electro-mediated membrane permeabilization to plasmid DNA have been shown to be significantly more complex than those for small molecules. Small molecules cross the permeabilized membrane by diffusion whereas plasmid DNA first interacts with the electropermeabilized part of the cell surface, forming localized aggregates. The dynamics of this process is still poorly understood because direct observations have been limited to scales of the order of seconds. Here, cells are electropermeabilized in the presence of plasmid DNA and monitored with a temporal resolution of 2 ms. This allows us to show that during the first pulse application, plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane. FRAP measurements show that the positions of these sites are remarkably immobile during the application of further pluses. A theoretical model is proposed to explain the appearance of distinct interaction sites, the quantitative increase in DNA and also their immobility leading to a tentative explanation for the success of electro-mediated gene delivery., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Electropermeabilization designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. A position-dependent reversible local membrane permeabilization is induced leading to an exchange of hydrophilic molecules across the membrane. This permeabilized state can be used to load cells with therapeutic molecules. In the case of small molecules, such as anticancer drugs, transfer occurs through simple diffusion. In the case of DNA, transfer occurs through a multi-step mechanism, a process that involves the electrophoretically driven association of the DNA molecule with the destabilised membrane and then its passage.

Electropermeabilization designates the use of electric pulses to overcome the barrier of the cell membrane. This physical method is used to transfer anticancer drugs or genes into living cells. Its mechanism remains to be elucidated. A position-dependent modulation of the membrane potential difference is induced, leading to a transient and reversible local membrane alteration. Electropermeabilization allows a fast exchange of small hydrophilic molecules across the membrane. It occurs at the positions of the cell facing the two electrodes on an asymmetrical way. In the case of DNA transfer, a complex process is present, involving a key step of electrophoretically driven association of DNA only with the destabilized membrane facing the cathode. We report here at the membrane level, by using fluorescence microscopy, the visualization of the effect of the polarity and the orientation of electric pulses on membrane permeabilization and gene transfer. Membrane permeabilization depends on electric field orientation. Moreover, at a given electric field orientation, it becomes symmetrical for pulses of reversed polarities. The area of cell membrane where DNA interacts is increased by applying electric pulses with different orientations and polarities, leading to an increase in gene expression. Interestingly, under reversed polarity conditions, part of the DNA associated with the membrane can be removed, showing some evidence for two states of DNA in interaction with the membrane: DNA reversibly associated and DNA irreversibly inserted.

Cell electropulsation is routinely used in cell Biology for protein, RNA or DNA transfer. Its clinical applications are under development for targeted drug delivery and gene therapy. Nevertheless, the molecular mechanisms supporting the induction of permeabilizing defects in the membrane assemblies remain poorly understood. This minireview describes the present state of the investigations concerning the different steps in the reversible electropermeabilization process. The different hypotheses, which were proposed to give a molecular description of the membrane events, are critically discussed. Other possibilities are then given. The need for more basic research on the associated loss of cohesion of the membrane appears as a conclusion.

Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure, composition, and equal molecular weight, not changed during the evolution. An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3, and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells were observed. Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria were in "high-energy" state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in apoptosis.

Electropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells.

Electropermeabilization is a promising nonviral method for gene therapy. However, despite the fact that it is widely used to transfer genes into living cells, the steps that limit DNA transfer remain to be determined. Here, we report the effect of cell synchronization on membrane permeabilization and gene delivery by electric fields. Chinese hamster ovary (CHO) cells were synchronized by aphidicolin or butyrate treatment. Electro-mediated transfection of these cells was evaluated under electric field conditions leading to the same level of membrane permeabilization. Aphidicolin cell synchronization in G2/M phase leads to a slight increase in plasma membrane permeabilization but to a three-fold increase in percentage of transfected cells and to an eight-fold increase in gene expression. This increase in cell transfection is specifically due to the G2/M synchronization process. Indeed, cell synchronization in G1 phase by sodium butyrate has no effect on cell permeabilization and transfection. Our results suggest that the enhanced transfection level in G2/M phase is not simply due to enhanced permeabilization, but reinforce the statement that the melting of the nuclear membrane facilitates direct access of plasmid DNA to the nucleus.

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivo results have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown. Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we report here the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability, gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. By acting as a protector of cell viability, serum indeed increases gene transfer and expression.

Chinese hamster ovary (CHO) cells and human erythrocytes were pulsed by using square-wave electric-field pulses. This treatment induced their permeabilization. This phenomenon appears to be a three-step process of creation, expansion and annihilation of permeated structures. Altering the cell cytoskeleton, either with drugs, such as colchicine, known to depolymerise the microtubules in CHO cells, or by high temperature shock to affect the spectrin-actin network in erythrocytes, induced no modification on the first two steps of the electropermeabilization process, but was associated with a dramatic decrease in the stability of the electro-induced permeated structures. These experimental observations support the hypothesis of an implication of cytoskeleton in electropermeabilization in agreement with thermodynamic conclusions.

Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.

Mammalian cells were shown to fuse after direct electric pulsation of the plated cells in culture. The extent of fusion was controlled by the duration of the post-pulse incubation. Formation of polynucleated cells was slow, even at 37 degrees C. Pre-pulse incubation with colchicine increased the fusion yield slightly. Cytoskeletal organization during the post-pulse incubation was observed using immunofluorescence techniques. Microfilaments were unaffected, but microtubules disappeared during the first minutes following the pulse, and then reformed on subsequent incubation.