Electroporation: A general phenomenon for manipulating cells and tissues

Electroporation is a fascinating cell membrane phenomenon with several existing biological applications and others likely. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for (1) introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; (2) selective biochemical loading of one size cell in the presence of many smaller cells; (3) introduction of virus and other particles; (4) cell killing under nontoxic conditions; and (5) insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including (1) enhanced cancer tumor chemotherapy, (2) gene therapy, (3) transdermal drug delivery, and (4) noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cell and artificial planar bilayer membranes. For short pulses (μs to ms), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5–1.5 V. In the case of isolated cells, the pulse magnitude is 10³–10⁴ V/cm. These pulses cause reversible electrical breakdown (REB), accompanied by a tremendous increase molecular transport across the membrane. REB results in a rapid membrane discharge, with the elevated U(t) returning to low values within a few microseconds of the pulse. However, membrane recovery can be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death. Basic phenomena, present understanding of mechanism, and the existing and potential applications are briefly reviewed.     

Active mechanisms are needed to describe cell responses to submicrosecond, megavolt-per-meter pulses: cell models for ultrashort pulses

Intracellular effects of submicrosecond, megavolt-per-meter pulses imply changes in a cell's plasma membrane (PM) and organelle membranes. The maximum reported PM transmembrane voltage is only 1.6 V and phosphatidylserine is translocated to the outer membrane leaflet of the PM. Passive membrane models involve only displacement currents and predict excessive PM voltages (∼25 V). Here we use a cell system model with nonconcentric circular PM and organelle membranes to demonstrate fundamental differences between active (nonlinear) and passive (linear) models. We assign active or passive interactions to local membrane regions. The resulting cell system model involves a large number of interconnected local models that individually represent the 1), passive conductive and dielectric properties of aqueous electrolytes and membranes; 2), resting potential source; and 3), asymptotic membrane electroporation model. Systems with passive interactions cannot account for key experimental observations. Our active models exhibit supra-electroporation of the PM and organelle membranes, some key features of the transmembrane voltage, high densities of small pores in the PM and organelle membranes, and a global postpulse perturbation in which cell membranes are depolarized on the timescale of pore lifetimes.     

Modeling electroporation in a single cell

Electroporation uses electric pulses to promote delivery of DNA and drugs into cells. This study presents a model of electroporation in a spherical cell exposed to an electric field. The model determines transmembrane potential, number of pores, and distribution of pore radii as functions of time and position on the cell surface. For a 1-ms, 40 kV/m pulse, electroporation consists of three stages: charging of the cell membrane (0-0.51 micros), creation of pores (0.51-1.43 micros), and evolution of pore radii (1.43 micros to 1 ms). This pulse creates approximately 341,000 pores, of which 97.8% are small ( approximately 1 nm radius) and 2.2% are large. The average radius of large pores is 22.8 +/- 18.7 nm, although some pores grow to 419 nm. The highest pore density occurs on the depolarized and hyperpolarized poles but the largest pores are on the border of the electroporated regions of the cell. Despite their much smaller number, large pores comprise 95.3% of the total pore area and contribute 66% to the increased cell conductance. For stronger pulses, pore area and cell conductance increase, but these increases are due to the creation of small pores; the number and size of large pores do not increase.     

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.     

Transmembrane molecular transport during versus after extremely large, nanosecond electric pulses

Recently there has been intense and growing interest in the non-thermal biological effects of nanosecond electric pulses, particularly apoptosis induction. These effects have been hypothesized to result from the widespread creation of small, lipidic pores in the plasma and organelle membranes of cells (supra-electroporation) and, more specifically, ionic and molecular transport through these pores. Here we show that transport occurs overwhelmingly after pulsing. First, we show that the electrical drift distance for typical charged solutes during nanosecond pulses (up to 100 ns), even those with very large magnitudes (up to 10 MV/m), ranges from only a fraction of the membrane thickness (5 nm) to several membrane thicknesses. This is much smaller than the diameter of a typical cell (∼16 μm), which implies that molecular drift transport during nanosecond pulses is necessarily minimal. This implication is not dependent on assumptions about pore density or the molecular flux through pores. Second, we show that molecular transport resulting from post-pulse diffusion through minimum-size pores is orders of magnitude larger than electrical drift-driven transport during nanosecond pulses. While field-assisted charge entry and the magnitude of flux favor transport during nanosecond pulses, these effects are too small to overcome the orders of magnitude more time available for post-pulse transport. Therefore, the basic conclusion that essentially all transmembrane molecular transport occurs post-pulse holds across the plausible range of relevant parameters. Our analysis shows that a primary direct consequence of nanosecond electric pulses is the creation (or maintenance) of large populations of small pores in cell membranes that govern post-pulse transmembrane transport of small ions and molecules.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

The resealing process of lipid bilayers after reversible electrical breakdown

The resealing process of lipid bilayer membranes after reversible electrical breakdown was investigated using two voltage pulses switched on together. Electrical breakdown of the membranes was induced with a voltage pulse of high intensity and short duration. The time course of the change in membrane conductance after the application of the high (short) voltage pulse was measured with a longer voltage pulse of low amplitude. The decrease in membrane conductance during the resealing process could be fitted to a single exponential curve with a time constant of 10-2 μs in the temperature range between 2 and 20°C. The activation energy for this exponential decay process was found to be about 50 kJ/mol, which might indicate a diffusion process. Above 25°C the resealing process is controlled by two exponential processes.The data obtained for the time course of the resealing process can be explained in terms of pore formation in the membranes in response to the high electrical field strength. A radius of about 4 nm is calculated for the initial pore size. From the assumed exponential change of the pore area with progressive resealing time a diffusion constant of 10^?8 cm²/s for lateral lipid diffusion can be estimated.     

Tissue electroporation. Observation of reversible electrical breakdown in viable frog skin.

Experiments by others have used isolated cell or bilayer membrane preparations to study the dramatic phenomena associated with electroporation. The present study observes electroporation behavior in an intact tissue. Viable samples of frog skin (Rana pipiens) were exposed to short electrical pulses of varying width and magnitude under "charge injection" conditions. After a pulse, the transtissue potential decayed with two distinct time constants, one short (tau approximately 0.3 ms) and the other longer (tau L approximately 2 ms). Above thresholds for the pulse magnitude and for the pulse width tau L decreased significantly, with progressively smaller tau L as the pulse magnitude and width increased. The postpulse potential, delta Utissue (t), and resistance, Rtissue, also decreased progressively. The tissue subsequently recovered to its original resistance and open circuit potential, delta U tissue,oc, within 2-3 min after a pulse. At that time another pulse experiment could be carried out, demonstrating repeatability and reversibility. No significant permanent changes in Rtissue and delta Utissue,oc were found. This is interpreted as avoidance of significant tissue damage. Taken together, these dramatic phenomena are characteristic of the reversible electrical breakdown previously observed in charge injection experiments with artificial planar bilayer membranes and with isolated cell membranes by similar very short pulses. The present experiments therefore demonstrate that electroporation can be repeatedly caused and observed in a viable tissue without apparent damage.     

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.     

Stochastic model for electric field-induced membrane pores. Electroporation

Electric impulses (1-20 kV cm-1, 1-5 microseconds) cause transient structural changes in biological membranes and lipid bilayers, leading to apparently reversible pore formation ( electroporation ) with cross-membrane material flow and, if two membranes are in contact, to irreversible membrane fusion ( electrofusion ). The fundamental process operative in electroporation and electrofusion is treated in terms of a periodic lipid block model, a block being a nearest-neighbour pair of lipid molecules in either of two states: (i) the polar head group in the bilayer plane or (ii) facing the centre of a pore (or defect site). The number of blocks in the pore wall is the stochastic variable of the model describing pore size and stability. The Helmholtz free energy function characterizing the transition probabilities of the various pore states contains the surface energies of the pore wall and the planar bilayer and, if an electric field is present, also a dielectric polarization term (dominated by the polarization of the water layer adjacent to the pore wall). Assuming a Poisson process the average number of blocks in a pore wall is given by the solution of a non-linear differential equation. At subcritical electric fields the average pore size is stationary and very small. At supercritical field strengths the pore radius increases and, reaching a critical pore size, the membrane ruptures (dielectric breakdown). If, however, the electric field is switched off, before the critical pore radius is reached, the pore apparently completely reseals to the closed bilayer configuration (reversible electroporation ).     

Model of creation and evolution of stable electropores for DNA delivery

Electroporation, in which electric pulses create transient pores in the cell membrane, is becoming an important technique for gene therapy. To enable entry of supercoiled DNA into cells, the pores should have sufficiently large radii (>10 nm), remain open long enough for the DNA chain to enter the cell (milliseconds), and should not cause membrane rupture. This study presents a model that can predict such macropores. The distinctive features of this model are the coupling of individual pores through membrane tension and the electrical force on the pores, which is applicable to pores of any size. The model is used to explore the process of pore creation and evolution and to determine the number and size of pores as a function of the pulse magnitude and duration. Next, our electroporation model is combined with a heuristic model of DNA uptake and used to predict the dependence of DNA uptake on pulsing parameters. Finally, the model is used to examine the mechanism of a two-pulse protocol, which was proposed specifically for gene delivery. The comparison between experimental results and the model suggests that this model is well-suited for the investigation of electroporation-mediated DNA delivery.     

Breakdown of lipid bilayer membranes in an electric field

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO₂²⁺-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO₂²⁺-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.     

A patch clamp study on the electro-permeabilization of higher plant cells: Supra-physiological voltages induce a high-conductance, K+ selective state of the plasma membrane

Permeabilization of biological membranes by pulsed electric fields ("electroporation") is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current-voltage relations of protoplasts derived from the tobacco culture cell line "Bright yellow-2". Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1 nS/pF) to a high-conductance state (~5 nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around -250 to -300 mV and +200 to +250 mV, respectively. Current-voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5-10 times more permeable to K+ than to gluconate. The K+ channel blocker tetraethylammonium (25 mM) did not affect currents elicited by 10 ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K+ channels. Supra-physiological voltage pulses even reduced "regular" K+ channel activity, probably due to an increase of cytosolic Ca2+ that is known to inhibit outward-rectifying K+ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages.     

The exocytotic fusion pore interface: a model of the site of neurotransmitter release

infrastructurel techniques have shown that an early event in the exocytotic fusion of a secretory vesicle is the formation of a narrow, water-filled pore spanning both the vesicle and plasma membranes and connecting the lumen of the secretory vesicle to the extracellular environment. Smaller precursors of the exocytotic fusion pore have been detected using electrophysio-logical techniques, which reveal a dynamic fusion pore that quickly expands to the size of the pores seen with electron microscopy. While it is clear that in the latter stages of expansion, when the size of the fusion pore is several orders of magnitude bigger than any known macromolecule, the fusion pore must be mainly made of lipids, the structure of the smaller precursors is unknown. Patch-clamp measurements of the activity of individual fusion pores in mast cells have shown that the fusion pore has some unusual and unexpected properties, namely that there is a large flux of lipid through the pore and the rate of pore closure has a discontinuous temperature dependency, suggesting a purely lipidic fusion pore. Moreover, comparisons of experimental data with theoretical fusion pores and with breakdown pores support the view that the fusion pore is initially a pore through a single bilayer, as would be expected for membrane fusion proceeding through a hemifusion mechanism. Based on these observations we present a model where the fusion pore is initially a pore through a single bilayer. Fusion pore formation is regulated by a macromolecular scaffold of proteins that is responsible for bringing the plasma membrane into a highly curved dimple very close to a tense secretory granule membrane, creating the architecture where the strongly attractive hydrophobic force causes the membranes to form a ‘hemifusion’ intermediate. Membrane fusion is completed by the formation of an aqueous pore after rupture of the shared bilayer. We also propose that the microenvironment of the interface when the pore first opens, dominated by the charged groups on the secretory vesicle matrix and phospholipids, will greatly influence the release of secretory products.     

Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation

Extremely large but very short (20 kV/cm, 300 ns) electric field pulses were reported recently to non-thermally destroy melanoma tumors. The stated mechanism for field penetration into cells is pulse characteristic times faster than charge redistribution (displacement currents). Here we use a multicellular model with irregularly shaped, closely spaced cells to show that instead overwhelming pore creation (supra-electroporation) is dominant, with field penetration due to pores (ionic conduction currents) during most of the pulse. Moreover, the model's maximum membrane potential (about 1.2 V) is consistent with recent experimental observations on isolated cells. We also use the model to show that conventional electroporation resulting from 100 microsecond, 1 kV/cm pulses yields a spatially heterogeneous electroporation distribution. In contrast, the melanoma-destroying pulses cause nearly homogeneous electroporation of cells and their nuclear membranes. Electropores can persist for times much longer than the pulses, and are likely to be an important mechanism contributing to cell death.     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

Electroporation of curved lipid membranes in ionic strength gradients

A thermodynamic theory for the membrane electroporation of curved membranes such as those of lipid vesicles and cylindrical membrane tubes has been developed. The theory covers in particular the observation that electric pore formation and shape deformation of vesicles and cells are dependent on the salt concentration of the suspending solvent. It is shown that transmembrane salt gradients can appreciably modify the electrostatic part of Helfrich's spontaneous curvature, elastic bending rigidity and Gaussian curvature modulus of charged membranes. The Gibbs reaction energy of membrane electroporation can be explicitely expressed in terms of salt gradient-dependent contributions of bending, the ionic double layers and electric surface potentials and dielectric polarisation of aqueous pores. In order to cover the various physical contribution to the chemical process of electroporation-resealing, we have introduced a generalised chemophysical potential covering all generalised forces and generalised displacements in terms of a transformed Gibbs energy formalism. Comparison with, and analysis of, the data of electrooptical relaxation kinetic studies show that the Gibbs reaction energy terms can be directly determined from turbidity dichroism (Planck's conservative dichroism). The approach also quantifies the electroporative cross-membrane material exchange such as electrolyte release, electrohaemolysis of red blood cells or uptake of drugs and dyes and finally gene DNA by membrane electroporation.     

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.     

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.     

Effect of actin cytoskeleton disruption on electric pulse‐induced apoptosis and electroporation in tumour cells

Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse‐induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.