Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon.

Transient membrane permeabilization by application of high electric field intensity pulses on cells (electropermeabilization) depends on several physical parameters associated with the technique (pulse intensity, number, and duration). In the present study, electropermeabilization is studied in terms of flow of diffusing molecules between cells and external medium. Direct quantification of the phenomenon shows that electric field intensity is a critical parameter in the induction of permeabilization. Electric field intensity must be higher than a critical threshold to make the membrane permeable. This critical threshold depends on the cell size. Extent of permeabilization (i.e., the flow rate across the membrane) is then controlled by both pulse number and duration. Increasing electric field intensity above the critical threshold needed for permeabilization results in an increase membrane area able to be permeabilized but not due to an increase in the specific permeability of the field alterated area. The electroinduced permeabilization is transient and disappears progressively after the application of the electric field pulses. Its life time is under the control of the electric field parameters. The rate constant of the annealing phase is shown to be dependent on both pulse duration and number, but is independent of electric field intensity which creates the permeabilization. The phenomenon is described in terms of membrane organization transition between the natural impermeable state and the electro-induced permeable state, phenomenon only locally induced for electric field intensities above a critical threshold and expanding in relation to both pulse number and duration.     

Electropermeabilization of dense cell suspensions

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 10⁶ cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 10⁶ to 400 × 10⁶ cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 10⁶ and 400 × 10⁶ cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.     

Dependence of Electroporation Detection Threshold on Cell Radius: An Explanation to Observations Non Compatible with Schwan’s Equation Model

It is widely accepted that electroporation occurs when the cell transmembrane voltage induced by an external applied electric field reaches a threshold. Under this assumption, in order to trigger electroporation in a spherical cell, Schwan’s equation leads to an inversely proportional relationship between the cell radius and the minimum magnitude of the applied electric field. And, indeed, several publications report experimental evidences of an inverse relationship between the cell size and the field required to achieve electroporation. However, this dependence is not always observed or is not as steep as predicted by Schwan’s equation. The present numerical study attempts to explain these observations that do not fit Schwan’s equation on the basis of the interplay between cell membrane conductivity, permeability, and transmembrane voltage. For that, a single cell in suspension was modeled and the electric field necessary to achieve electroporation with a single pulse was determined according to two effectiveness criteria: a specific permeabilization level, understood as the relative area occupied by the pores during the pulse, and a final intracellular concentration of a molecule due to uptake by diffusion after the pulse, during membrane resealing. The results indicate that plausible model parameters can lead to divergent dependencies of the electric field threshold on the cell radius. These divergent dependencies were obtained through both criteria and using two different permeabilization models. This suggests that the interplay between cell membrane conductivity, permeability, and transmembrane voltage might be the cause of results which are noncompatible with the Schwan’s equation model.     

Electroporation in dense cell suspension--theoretical and experimental analysis of ion diffusion and cell permeabilization

Electroporation is a process where increased permeability of cells exposed to an electric field is observed. It is used in many biomedical applications including electrogene transfection and electrochemotherapy. Although the increased permeability of the membrane is believed to be the result of pores due to an induced transmembrane voltage U(m), the exact molecular mechanisms are not fully explained. In this study we analyze transient conductivity changes during the electric pulses and increased membrane permeability for ions and molecules after the pulses in order to determine which parameters affect stabilization of pores, and to analyze the relation between transient pores and long-lived transport pores. By quantifying ion diffusion, fraction of transport pores f(per) was obtained. A simple model, which assumes a quadratic dependence of f(per) on E in the area where U(m)>U(c) very accurately describes experimental values, suggesting that f(per) increases with higher electric field due to larger permeabilized area and due to higher energy available for pore formation. The fraction of transport pores increases also with the number of pulses N, which suggest that each pulse contributes to formation of more and/or larger stable transport pores, whereas the number of transient pores does not depend on N.     

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion.     

The influence of divalent cations on the dynamic properties of actin filaments: a spectroscopic study.

Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.     

Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane.

Interaction of two stains (propidium iodide and ethidium bromide) with electropermeabilized living Chinese hamster ovary cells is observed using an ultrafast fluorescence image acquisition system. The computing process is linked to an ultra-low-light intensifying camera working with a very short time resolution (3.33 ms per image). Altered parts of the cell membrane were identified via the enhancement in fluorescence intensity of the dyes. They reflect the electropermeabilized part of the membrane in which free flow of dye occurred. Images of the fluorescence interaction patterns of the two dyes, in a maximum 20-ms time lag after pulsation, reveal asymmetrical permeabilization of the cell membrane. For electric field intensities higher than a first threshold value, permeabilization is always observed on the anode-facing side of the cell. For electric field intensities over a second higher threshold value, the two electrode-facing hemispheres of the cell are permeabilized, the hemisphere facing the anode being most permeable. These data support the conclusion that electropermeabilization of living cell membrane is affected by its resting potential. The asymmetrical pattern of the dye interaction is not dependent on the nature or concentration of the dye, the ionic strength of the pulsing buffer, or the duration of the pulse. The field intensity determines the fraction of the membrane in which molecular alterations can occur. The extent of alteration in this localized region is determined by the duration of the pulse when a single pulse in the millisecond time range is applied.     

Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of the outer-cell membrane, could substantially increase intracellular fields to achieve selective manipulations of intracellular organelles. The underlying principle of this earlier work is further developed and applied to the systems studied here. Under appropriate conditions, we show preferential permeabilization of one vesicle population in a mixed preparation of vesicles of similar size distribution. It is further shown that large endocytosed vacuoles in COS-7 cells can be selectively permeabilized with little effect on the integrity of outer cell membrane.     

The Role of Electrophoresis in Gene Electrotransfer

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.     

Electroporation of DC-3F Cells Is a Dual Process

Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and −231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere patch-clamp artifact. In short, the response of DC-3F cells to strong pulsed electric fields was separated into a transient electroporation and a persistent permeabilization. The latter dominates postpulse membrane properties but to date has not been addressed by electroporation theory or MD simulations.     

Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure

Cells can be transiently permeabilized by application of high electric pulses of short duration. A direct consequence of this treatment is to induce a fusogenic state in the pulsed membrane. The molecular events underlying these phenomena remain to be explained. During our work, we investigated the effects of pulsing buffer osmotic pressure on both electric field induced permeabilization and fusion of Chinese hamster ovary cells growing either in monolayers or in suspension. Osmotic pressure has no effect on the induction step of permeabilization, but its increase was shown to inhibit the expansion step and to decrease the efficiency of the resealing phase. Fusion efficiency was greatly affected by the osmotic pressure and by the physiological state of the cells. When cells were grown plated and when intercellular contacts were spontaneous and present during pulsation, increasing the osmotic pressure resulted in an increase in the fusion index. The opposite effect was observed for cells growing in suspension and brought into contact after pulsation. These results were tentatively explained in terms of the effect of the osmotic pressure on the membrane organization and cell-cell contact quality.     

Inactivation of Pseudomonas putida by Pulsed Electric Field Treatment: A Study on the Correlation of Treatment Parameters and Inactivation Efficiency in the Short-Pulse Range

An important issue for an economic application of the pulsed electric field treatment for bacterial decontamination of wastewater is the specific treatment energy needed for effective reduction of bacterial populations. The present experimental study performed in a field amplitude range of 40 > E > 200 kV/cm and for a suspension conductivity of 0.01 = κ _e > 0.2 S/m focusses on the application of short pulses, 25 ns > T > 10 μs, of rectangular, bipolar and exponential shape and was made on Pseudomonas putida, which is a typical and widespread wastewater microorganism. The comparison of inactivation results with calculations of the temporal and azimuthal membrane charging dynamics using the model of Pauly and Schwan revealed that for efficient inactivation, membrane segments at the cell equator have to be charged quickly and to a sufficiently high value, on the order of 0.5 V. After fulfilling this basic condition by an appropriate choice of pulse field strength and duration, the log rate of inactivation for a given suspension conductivity of 0.2 S/m was found to be independent of the duration of individual pulses for constant treatment energy expenditure. Moreover, experimental results suggest that even pulse shape plays a minor role in inactivation efficiency. The variation of the suspension conductivity resulted in comparable inactivation performance of identical pulse parameters if the product of pulse duration and number of pulses was the same, i.e., required treatment energy can be linearly downscaled for lower conductivities, provided that pulse amplitude and duration are selected for entire membrane surface permeabilization.     

Calcium influx determines the muscular response to electrotransfer

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca(2+)-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca(2+) influx. Larger Ca(2+) influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca(2+) influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca(2+) may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.     

Electric Field Orientation for Gene Delivery Using High-Voltage and Low-Voltage Pulses

Electropermeabilization is a biological physical process in response to the presence of an applied electric field that is used for the transfer of hydrophilic molecules such as anticancer drugs or DNA across the plasma membranes of living cells. The molecular processes that support the transfer are poorly known. The aim of our study was to investigate the effect of high-voltage and low-voltage (HVLV) pulses in vitro with different orientations on cell permeabilization, viability and gene transfection. We monitored the permeabilization with unipolar and bipolar HVLV pulses with different train repetition pulses, showing that HVLV pulses increase cell permeabilization and cell viability. Gene transfer was also observed by measuring green fluorescent protein (GFP) expression. The expression was the same for HVLV pulses and electrogenotherapy pulses for in vitro experimentation. As the viability was better preserved for HVLV-pulsed cells, we managed to increase the number of GFP-expressing cells by up to 65?% under this condition. The use of bipolar HVLV train pulses increased gene expression to a higher extent, probably by affecting a larger part of the cell surface.     

An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization.

When applied on intact cell suspension, electric field pulses are known to induce membrane permeabilization (electropermeabilization) and fusion (electrofusion). These effects are triggered through a modulation of the membrane potential difference. Due to the vectorial character of the electric field effects, this modulation, which is superimposed on the resting membrane potential difference, is position-dependent on the cell surface. This explains the difference between the experimentally observed critical field strengths requested to trigger the processes of permeabilization and fusion. The critical membrane potential difference which induces membrane permeabilization can be calculated from these experimental observations. It is observed that its value is always about 200 mV for many different cell systems as we previously reported in the case of pure lipid vesicles. This is much less than assumed in most previous studies.     

Rhabdomyolysis due to pulsed electric fields

High-voltage electrical trauma frequently results in extensive and scattered destruction of skeletal muscle along the current path. The damage is commonly believed to be mediated by heating. Recent experimental and theoretical evidence suggests, however, that the rhabdomyolysis and secondary myoglobin release that occur also can result from electroporation, a purely nonthermal mechanism. Based on the results of a computer simulation of a typical high-voltage electric shock, we have postulated that electroporation contributes substantially to skeletal muscle damage and could be the primary mechanism of damage in some cases of electrical injury. In this study, we determined the threshold field strength and exposure duration required to produce rhabdomyolysis by the electroporation mechanism. The change in the electrical impedance of intact skeletal muscle tissue following the application of short-duration, high-intensity electric field pulses is used as an indicator of membrane damage. Our experiments show that a decrease in impedance magnitude occurs following electric field pulses that exceed threshold values of 60 V/cm magnitude and 1-ms duration. The field strength, pulse duration, and number of pulses are factors that determine the extent of damage. The effect does not depend on excitation-contraction coupling. Electron micrographs confirm structural defects created in the membranes by the applied electric field pulses, and these represent the first clear demonstration of rhabdomyolysis in intact muscle due to electroporation. These results provide compelling evidence in support of our postulate.     

Electropermeabilization, a physical method for the delivery of therapeutic molecules into cells

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, a physical method for the delivery of therapeutic molecules into cells

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.