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Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

This article was invited in order to summarize for the special edition of a Plenary Review presentation at the IEEE ICOPS/PPC meeting, June 23 2019 by M. Gundersen. It is a review of research conducted into primarily academic environments in nanosecond pulsed power, applications of the pulsed power research to bioelectrics, and combustion and ignition over a period of $\approx 45$ years. Research was conducted primarily at the University of Southern California (USC), at Texas Tech University (TTU), and through collaborations with other laboratories and personnel. Pulsed power, applied on a nanosecond time scale, was found to enable a range of new applications; several examples are detailed below. The range of applications for research based in short-pulse, or nanosecond pulsed power, and applied plasma science, strongly encourage defining and supporting the extension of programmatic research goals to beyond the academic laboratories and into the commercial environment.

Nanosecond bipolar pulse cancellation, a recently discovered phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (>100 μs) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). Here we report 2 ns bipolar pulse cancellation, extending the scale of previously published results down to the time required to construct the permeabilizing lipid electropores observed in molecular simulations. We add new cancellation endpoints, and we describe new bipolar pulse effects that are distinct from cancellation. This new data, which includes transport of cationic and anionic permeability indicators, fluorescence of membrane labels, and patterns of entry into permeabilized cells, is not readily explained by the accelerated discharge mechanism. We suggest that multi-step processes that involve first charged species movement and then responses of cellular homeostasis and repair mechanisms are more likely to explain the broad range of reported results.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca(2+)-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-β-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca(2+)-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca(2+). In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca(2+)-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

Exposures to short-duration, strong electric field pulses have been utilized for stimulation, ablation, and the delivery of molecules into cells. Ultrashort, nanosecond duration pulses have shown unique benefits, but they require higher field strengths. One way to overcome this requirement is to use trains of nanosecond pulses with high repetition rates, up to the MHz range. Here we present a theoretical model to describe the effects of pulse trains on the plasma membrane and intracellular membranes modeled as resistively charged capacitors. We derive the induced membrane potential and the stimulation threshold as functions of pulse number, pulse duration, and repetition rate. This derivation provides a straightforward method to calculate the membrane charging time constant from experimental data. The derived excitation threshold agrees with nerve stimulation experiments, indicating that nanosecond pulses are not more effective than longer pulses in charging nerve fibers. The derived excitation threshold does not, however, correctly predict the nanosecond stimulation of cardiomyocytes. We show that a better agreement is possible if multiple charging time constants are considered. Finally, we expand the model to intracellular membranes and show that pulse trains do not lead to charge buildup, but can create significant oscillations of the intracellular membrane potential.

Although transport of molecules into cells via electroporation is a common biomedical procedure, its protocols are often based on trial and error. Despite a long history of theoretical effort, the underlying mechanisms of cell membrane electroporation are not sufficiently elucidated, in part, because of the number of independent fitting parameters needed to link theory to experiment. Here, we ask if the electroporation behavior of a reduced cell membrane is consistent with time-resolved, atomistic, molecular dynamics (MD) simulations of phospholipid bilayers responding to electric fields. To avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 10–50-ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes.

Delivery of molecules to cells via electropermeabilization (electroporation) is a common procedure in laboratories and clinics. However, despite a long history of theoretical effort, electroporation protocols are still based on trial and error because the biomolecular structures and mechanisms underlying this phenomenon have not been established. Electroporation models, developed to explain observations of electrical breakdown of lipid membranes, describe the electric field-driven formation of pores in lipid bilayers. These transient pore models are consistent with molecular dynamics simulations, where field-stabilized lipid pores form within a few nanoseconds and collapse within tens of nanoseconds after the field is removed. Here we experimentally validate this nanoscale restructuring of bio-membranes by measuring the kinetics of transport of the impermeant fluorescent dye calcein into lipid vesicles exposed to ultrashort electric fields (6 ns and 2 ns), and by comparing these results to molecular simulations. Molecular transport after vesicle permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns, while the additive property of transport is no longer observed when the interval is reduced to 0 ns, consistent with the lifetimes of lipid electropores in molecular simulations. These results show that lipid vesicle responses to pulsed electric fields are significantly different from those of living cells where, for similar pulse properties, the uptake of fluorescent dye continues for several minutes.Graphical abstract

Background Applications of electric-field-induced permeabilization of cells range from cancer therapy to wastewater treatment. A unified understanding of the underlying mechanisms of membrane electropermeabilization, however, has not been achieved. Protocols are empirical, and models are descriptive rather than predictive, which hampers the optimization and expansion of electroporation-based technologies. A common feature of existing models is the assumption that the permeabilized membrane is passive, and that transport through it is entirely diffusive. To demonstrate the necessity to go beyond that assumption, we present here a quantitative analysis of the post-permeabilization transport of three small molecules commonly used in electroporation research — YO-PRO-1, propidium, and calcein — after exposure of cells to minimally perturbing, 6 ns electric pulses. Results Influx of YO-PRO-1 from the external medium into the cell exceeds that of propidium, consistent with many published studies. Both are much greater than the influx of calcein. In contrast, the normalized molar efflux of calcein from pre-loaded cells into the medium after electropermeabilization is roughly equivalent to the influx of YO-PRO-1 and propidium. These relative transport rates are correlated not with molecular size or cross-section, but rather with molecular charge polarity. Conclusions This comparison of the kinetics of molecular transport of three small, charged molecules across electropermeabilized cell membranes reveals a component of the mechanism of electroporation that is customarily taken into account only for the time during electric pulse delivery. The large differences between the influx rates of propidium and YO-PRO-1 (cations) and calcein (anion), and between the influx and efflux of calcein, suggest a significant role for the post-pulse transmembrane potential in the migration of ions and charged small molecules across permeabilized cell membranes, which has been largely neglected in models of electroporation. Electronic supplementary material The online version of this article (10.1186/s13628-018-0044-2) contains supplementary material, which is available to authorized users.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca2+-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-β-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca2+-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca2+. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca2+-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

Imaging of fluorescent small molecule transport into electropermeabilized cells reveals polarized patterns of entry, which must reflect in some way the mechanisms of the migration of these molecules across the compromised membrane barrier. In some reports, transport occurs primarily across the areas of the membrane nearest the positive electrode (anode), but in others cathode-facing entry dominates. Here we compare YO-PRO-1, propidium, and calcein uptake into U-937 cells after nanosecond (6 ns) and microsecond (220 µs) electric pulse exposures. Each of the three dyes exhibits a different pattern. Calcein shows no preference for anode- or cathode-facing entry that is detectable with our measurement system. Immediately after a microsecond pulse, YO-PRO-1 and propidium enter the cell roughly equally from the positive and negative poles, but transport through the cathode-facing side dominates in less than 1 s. After nanosecond pulse permeabilization, YO-PRO-1 and propidium enter primarily on the anode-facing side of the cell. Electronic supplementary material The online version of this article (doi:10.1007/s00232-017-9962-1) contains supplementary material, which is available to authorized users.

Plasma membrane vesicles can be effective, non- toxic carriers for microscale material transport, provide a convenient model for probing membrane- related processes, since intracellular biochemical processes are eliminated. We describe here a fine- tuned protocol for isolating ghost plasma membrane vesicles from the unicellular alga Dunaliella tertiolecta, and preliminary characterization of their structural features and permeability properties, with comparisons to giant unilamellar phospholipid vesicles. The complexity of the algal ghost membrane vesicles reconstructed from the native membrane material released after hypoosmotic stress lies between that of phospholipid vesicles and of cells. AFM structural characterization of reconstructed vesicles shows a thick envelope and a nearly empty vesicle interior. The surface of the envelope contains a heterogeneous distribution of densely packed, nanometer-scale globules and pore-like structures which may be derived from surface coat proteins. Confocal fluorescence imaging reveals the highly pigmented photosynthetic apparatus located within the thylakoid membrane and retained in the vesicle membrane. Transport of the fluorescent dye calcein into ghost and giant unilamellar vesicles reveals significant differences in permeability. Expanded knowledge of this unique membrane system will contribute to the design of marine bio-inspired carriers for advanced biotechnological applications.

Using a combination of molecular dynamics simulations and experiments, we have examined the interactions of alkanes and phospholipids at charged interfaces in order to understand how interfacial charge densities affect the association of these two representative molecules with electrodes. Consistent with theory and experiment, these model systems reveal interfacial associations mediated through a combination of Coulombic and van der Waals forces. Van der Waals forces, in particular, mediate rapid binding of decane to neutral electrodes. No decane binding was observed at high surface charge densities because of interfacial water polarization, which screens hydrophobic attractions. The positively charged choline moiety of the phospholipid palmitoyloleoylphosphatidylcholine (POPC) is primarily responsible for POPC attraction by a moderately negatively-charged electrode. The hydrocarbon tails of POPC interact with the hydrophobic electrode interface similarly to decane. Previously reported electrochemical results confirm these findings by demonstrating bipolar displacement currents from PC vesicles adhering to moderately negatively-charged interfaces, originating from the choline interactions observed in simulations. At more negatively-charged interfaces, choline-to-surface binding was stronger. In both simulations and experiments the maximal interaction of anionic PS occurs with a positively charged interface, provided that the electrostatic forces outweigh local Lennard-Jones interactions. Direct comparisons between the binding affinities measured in experiments and those obtained in simulations reveal previously unobserved atomic interactions that facilitate lipid vesicle adhesion to charged interfaces. Moreover, the implementation of a charged interface in molecular dynamics simulations provides an alternative method for the generation of large electric fields across phospholipid bilayers, especially for systems with periodic boundary conditions, and may be useful for simulations of membrane electropermeabilization.

Reversible electroporation is used to increase the uptake of chemotherapeutic drugs in local tumor treatment (electrochemotherapy) by applying the pulsing protocol (8 rectangular pulses, 1000 V/cm, 100 µs) standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy multicenter trial. Currently, new electrochemotherapy strategies are under development to extend its applicability to tumors with different histology. Electrical parameters and drug type are critical factors. A possible approach is to test pulse parameters different from European Standard Operating Procedure on Electrochemotherapy but with comparable electroporation yield (European Standard Operating Procedure on Electrochemotherapy-equivalent protocols). Moreover, the use of non-toxic drugs combined with electroporation represents the new frontier for electrochemotherapy applications; calcium electroporation has been recently proposed as a simple tool for anticancer therapy. In vitro investigations facilitate the optimization of electrical parameters and drugs for in vivo and clinical testing. In this optimization study, new pulsing protocols have been tested by increasing the pulse number and reducing the electric field with respect to the standard. European Standard Operating Procedure on Electrochemotherapy-equivalent protocols have been identified in HL-60 and A431 cancer cell models, and a higher sensitivity in terms of electroporation yield has been recorded in HL-60 cells. Moreover, cell killing efficacy of European Standard Operating Procedure on Electrochemotherapy-equivalent protocols has been demonstrated in the presence of increasing calcium concentrations on both cell lines. Equivalent European Standard Operating Procedure on Electrochemotherapy protocols can be used to optimize the therapeutic effects in the clinic, where different regions of the same cancer tissue, with different electrical properties, might result in a differential electroporation yield of the standard protocol over the same tissue, or, eventually, in an override of the operational limits of the instrument. Moreover, using calcium can help overcome the drawbacks of standard drugs (side effects, high costs, difficult handling, preparation, and storage procedures). These results support the possibility of new treatment options in both standard electrochemotherapy and calcium electroporation, with clear advantages in the clinic.

Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr), in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at < 4 µM), and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60 ns, 13.2 kV/cm, 10 Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3 min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.

Nonthermal probing and stimulation with subnanosecond electric pulses and terahertz electromagnetic radiation may lead to new, minimally invasive diagnostic and therapeutic procedures and to methods for remote monitoring and analysis of biological systems, including plants, animals, and humans. To effectively engineer these still-emerging tools, we need an understanding of the biophysical mechanisms underlying the responses that have been reported to these novel stimuli. We show here that subnanosecond (≤ 500 ps) electric pulses induce action potentials in neurons and cause calcium transients in neuroblastoma-glioma hybrid cells, and we report complementary molecular dynamics simulations of phospholipid bilayers in electric fields in which membrane permeabilization occurs in less than 1 ns. Water dipoles in the interior of these model membranes respond in less than 1 ps to permeabilizing electric potentials by aligning in the direction of the field, and they re-orient at terahertz frequencies to field reversals. The mechanism for subnanosecond lipid electropore formation is similar to that observed on longer time scales — energy-minimizing intrusions of interfacial water into the membrane interior and subsequent reorganization of the bilayer into hydrophilic, conductive structures.

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated transport and with molecular simulations of transport across electropores in a phospholipid bilayer. The results challenge the “drift and diffusion through a pore” model that dominates conventional explanatory schemes for the electroporative transfer of small molecules into cells and point to the necessity for a more complex model.

A simple prediction method for the bipolar pulse cancellation effect is proposed, based on analysis of single-cell transmembrane potential frequency spectra for a variety of unipolar and bipolar input signals in the nanosecond and microsecond time scales. Our evaluations are in good agreement with experimental results for electropermeabilization-induced Ca2+ influx using 300 ns, 750 kV/m pulses. © 2017 IEEE.

Pulsed electric fields are used to permeabilize cell membranes in biotechnology and the clinic. Although molecular and continuum models provide compelling representations of the mechanisms underlying this phenomenon, a clear structural link between the biomolecular transformations displayed in molecular dynamics (MD) simulations and the micro- and macroscale cellular responses observed in the laboratory has not been established. In this paper, plasma membrane electropermeabilization is characterized by exposing Jurkat T lymphoblasts to pulsed electric fields less than 10 ns long (including single pulse exposures), and by monitoring the resulting osmotically driven cell swelling as a function of pulse number and pulse repetition rate. In this way, we reduce the complexity of the experimental system and lay a foundation for gauging the correspondence between measured and simulated values for water and ion transport through electropermeabilized membranes. We find that a single 10 MV/m pulse of 5 ns duration produces measurable swelling of Jurkat T lymphoblasts in growth medium, and we estimate from the swelling kinetics the ion and water flux that follows the electropermeabilization of the membrane. From these observations we set boundaries on the net conductance of the permeabilized membrane, and we show how this is consistent with model predictions for the conductance and areal density of nanoelectropulse-induced lipid nanopores.

In this paper a simple prediction method for the bipolar pulse cancellation effect is proposed, based on the frequency analysis of the TMP spectra of a single cell and the computed relative global spectral content up to a defined frequency threshold. We present a spectral analysis of pulses applied in experiments, and we extract the induced TMP from a microdosimetric model of the cell. The induced TMP computation is carried out on a hemispherical multi-layered cell model in the time domain. The analysis is presented for a variety of unipolar and bipolar input signals in the nanosecond and the microsecond time scales. Our evaluations are in good agreement with experimental results for bipolar pulse cancellation of electropermeabilization-induced Ca2+ influx using 300 ns, 750 kV/m pulses and with other results reported in recent literature. © 2017 Elsevier B.V.

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. This work was supported by the Ministry of Economy and Competitiveness of Spain through Grant TEC2014-52383-C3-2-R. PTV received support from the Old Dominion University Frank Reidy Research Center for Bioelectrics and the Air Force Office of Scientific Research (FA9550-15-1-0517, FA9550-14-1-0123).

International audience; Pulsed electric fields of sufficient magnitude and duration trigger functional responses and modifications in biological cells. Transient nanometer-sized pores are believed to form within nanoseconds in cell membranes exposed to high-intensity (MV/m) nanosecond pulsed electric fields (nsPEFs), and while it is clear that polar water molecules play a key role in electroporation, no signature for pore initiation has yet been identified. To address this, we combine molecular dynamics simulations and quasi-static 3-D finite-difference analysis to investigate the electrostatic interactions that drive pore formation in homogenous lipid bilayers exposed to intense nsPEFs. The developed methodology uniquely enables the extraction of 3-D spatiotemporal profiles of electric potentials, electric fields, and electric field gradients in biological membranes with atomistic detail and sub-nanosecond resolution. As a result, this study captures and elucidates several dynamic phenomena observed experimentally and provides a fundamental framework for further development.

Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in cell membranes without the permeabilization associated with longer, lower-field pulses. A single 30 ns, 2.5 MV/m pulse produces perturbations consistent with phosphatidylserine (PS) externalization in Jurkat T lymphoblasts within milliseconds, polarized in the direction of the applied field, indicating an immediate interaction between membrane components and the electric field. This disturbance occurs only at the anode pole of the cell, supporting the hypothesis that the pulsed field drives the negatively charged PS head group toward the positive electrode, directly providing the energy for crossing the membrane dielectric barrier.

Pulsed electric fields can induce transport of normally impermeant ions and small molecules across the cell membrane, a process called electroporation or electropermeabilization. This phenomenon is often studied by using microscopic imaging to monitor changes in the intracellular concentration of fluorescent dyes. Most of these studies, however, report their findings in relative or arbitrary units. To produce truly quantitative measurements of electroporative transport we employ calibrated confocal microscopy to track YO-PRO-1 entry into cells after a single 6 ns, 20 MV/m pulse, and we correlate these observations with molecular dynamics simulations of YO-PRO-1 transport through lipid electropores. These baseline studies have been extended to quantitative observations of transport of calcein (an anionic fluorescent small molecule) and propidium (a cationic dye like YO-PRO-1 that is fluorescent only after binding to polynucleotides). Our results challenge the common assumption that small molecule transport through electropermeabilized membranes is dominated by simple diffusion through electropores. Ongoing experimental studies into the potential role of the post-pulse transmembrane voltage in the migration of charged small molecules across permeabilized cell membranes will also be discussed.

Science increasingly involves complex modeling. Here we describe a model for cell electroporation in which membrane properties are dynamically modified by poration. Spatial scales range from cell membrane thickness (5 nm) to a typical mammalian cell radius (10 μ m), and can be used with idealized and experimental pulse waveforms. The model consists of traditional passive components and additional active components representing nonequilibrium processes. Model responses include measurable quantities: transmembrane voltage, membrane electrical conductance, and solute transport rates and amounts for the representative “long” and “short” pulses. The long pulse—1.5 kV/cm, 100 μ s—evolves two pore subpopulations with a valley at ∼ 5 nm, which separates the subpopulations that have peaks at ∼ 1.5 and ∼ 12 nm radius. Such pulses are widely used in biological research, biotechnology, and medicine, including cancer therapy by drug delivery and nonthermal physical tumor ablation by causing necrosis. The short pulse—40 kV/cm, 10 ns—creates 80-fold more pores, all small ( < 3 nm; ∼ 1 nm peak). These nanosecond pulses ablate tumors by apoptosis. We demonstrate the model’s responses by illustrative electrical and poration behavior, and transport of calcein and propidium. We then identify extensions for expanding modeling capability. Structure-function results from MD can allow extrapolations that bring response specificity to cell membranes based on their lipid composition. After a pulse, changes in pore energy landscape can be included over seconds to minutes, by mechanisms such as cell swelling and pulse-induced chemical reactions that slowly alter pore behavior., Harvard University--MIT Division of Health Sciences and Technology (Fellowship), National Science Foundation (U.S.) (Fellowship), National Institutes of Health (U.S.) (NIH grant GM063857), United States. Air Force Office of Scientific Research, University of Southern California. Center for High Performance Computing and Communications

This special issue of the Journal of Membrane Biology contains reports on recent developments in the field of electroporation by participants in the International Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments held in November 2014 in Ljubljana. This was the eighth session of what is now an annual event, first organized in 2003. The 66 participants— students, young scientists, invited lecturers, faculty members, and special guests—came from 15 countries (Algeria, Belgium, China, Croatia, France, Germany, Ireland, Israel, Italy, Lithuania, Macedonia, Netherlands, Slovenia, Spain, and United States). In addition to lectures on the physics, electrical engineering, and biology of electroporation, this year’s sessions included topical talks on electrochemical processes associated with pulsed electric field exposures, electroporation in food and biomass processing, systemic electrochemotherapy, and electrochemotherapy of colorectal liver metastases. One of the features of this week-long workshop course is the practical training offered each afternoon. Organized and led by associates of the Laboratory of Biocybernetics of the Faculty of Electrical Engineering at the University of Ljubljana, and by visiting researchers, these laboratory sessions provide a unique opportunity for hands-on experience guided by experts, with activities including gene electrotransfer, ratiometric fluorescence microscopy, microbial inactivation, electrical characterization of artificial membranes, treatment planning for electrochemotherapy, and molecular dynamics simulations. The school is now an established and unique platform for the acquisition of theoretical and practical knowledge of electroporation mechanisms and applications. The workshop and postgraduate course were conducted within the scope and with the support of the European Associated Laboratory on Pulsed Electric Fields Applications in Biology and Medicine (LEA EBAM) and co-organized by COST TD1104 Action (http://www. electroporation.net/). The peer-reviewed selection of articles in this issue provides a cross section of the ongoing electroporationrelated research being carried out by participants at the Ljubljana meeting. We are grateful to the contributors for their efforts in presenting their recent results, which will challenge readers, whether new or old in the field, to evaluate assumptions and consider new ideas, and we are especially grateful to our scientific colleagues who reviewed the manuscripts. Finally, we acknowledge the indispensable support of the agencies, societies, and companies who sponsor the school, in particular, the Slovenian Research Agency, the Centre National de la Recherche Scientifique (CNRS), and the Bioelectrochemical Society, which have sponsored the school from its very beginning, and also IGEA (Italy), Mediline (Slovenia), Iskra Medical (Slovenia), BIA Separations (Slovenia), LTFE (Slovenia), and Betatech (France), who made it possible to increase student participation through reduced fees and lodging expenses. & P. Thomas Vernier pvernier@odu.edu

Nanosecond, megavolt-per-meter electric pulses applied to biological cells can target subcellular structures with minimal loss of plasma membrane integrity, opening up new perspectives for intracellular manipulations. Experimentally observed effects of intense nanopulses include intracellular calcium release, externalization of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane, and non-thermal cell death by apoptosis. Molecular dynamics (MD) simulations have shown that PS re-distribution occurs after the electric-field-driven formation of nanometer-sized pores in the plasma membrane and is facilitated by electrophoresis of PS along the pore walls. Nanopulse-induced pore creation occurs on a nanosecond time scale, but the underlying molecular mechanisms are not yet clear. Experimental observations of the process of pore formation are challenging because of the time and spatial scales required.In this study, we combine MD simulations and a quasi-static approach using a custom implementation of 3D finite-difference analysis to investigate the physical mechanisms of electropore creation. First, MD simulations of pore formation in phospholipid bilayers in external electric fields are performed at nanoscopic scale. From these simulations we extract the charge densities across the electroporated bilayer. Second, the charge densities are injected into a new, custom, quasi-static algorithm based on the Poisson equation. The software computes 3D nanoscopic profiles of the transmembrane potential, electric field, and electric field gradient. The goal of the two-step simulation is to establish whether and how electric field gradients, water and phospholipid head group dipole moments, and the site of initial water intrusion in pore initiation are correlated.

Molecular dynamics (MD) simulations of electrophoretic transport of monovalent ions through field-stabilized electropores in POPC lipid bilayers permit systematic characterization of the conductive properties of lipid nanopores. The radius of the electropore can be controlled by the magnitude of the applied sustaining external electric field, which also drives the transport of ions through the pore. We examined pore conductances for two monovalent salts, NaCl and KCl, at physiological concentrations. Na(+) conductance is significantly less than K(+) and Cl(-) conductance and is a nonlinear function of pore radius over the range of pore radii investigated. The single pore electrical conductance of KCl obtained from MD simulation is comparable to experimental values measured by chronopotentiometry.

Electroporation is the formation of permeabilizing structures in the cell membrane under the influence of an externally imposed electric field. The resulting increased permeability of the membrane enables a wide range of biological applications, including the delivery of normally excluded substances into cells. While electroporation is used extensively in biology, biotechnology, and medicine, its molecular mechanism is not well understood. This lack of knowledge limits the ability to control and fine-tune the process. In this article we propose a novel molecular mechanism for the electroporation of a lipid bilayer based on energetics analysis. Using molecular dynamics simulations we demonstrate that pore formation is driven by the reorganization of the interfacial water molecules. Our energetics analysis and comparisons of simulations with and without the lipid bilayer show that the process of poration is driven by field-induced reorganization of water dipoles at the water-lipid or water-vacuum interfaces into more energetically favorable configurations, with their molecular dipoles oriented in the external field. Although the contributing role of water in electroporation has been noted previously, here we propose that interfacial water molecules are the main players in the process, its initiators and drivers. The role of the lipid layer, to a first-order approximation, is then reduced to a relatively passive barrier. This new view of electroporation simplifies the study of the problem, and opens up new opportunities in both theoretical modeling of the process and experimental research to better control or to use it in new, innovative ways.

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7-30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.

International audience; Intracellular structures of biological cells can be disturbed by exposure to nanosecond pulsed electric field (nsPEF). A microchamber-based delivery system mounted on a microscope setup for real-time exposure to nsPEF is studied in this paper. A numerical and experimental characterization of the delivery system is performed both in frequency and time domains. The microchamber delivery system presents a high impedance compared to classical 50 Ω loads. Its frequency behavior and limits are investigated using an in-house finite-difference time-domain (FDTD) simulator and through experimental measurements. High-voltage measurements for two nsPEF generators are carried out. The applied pulse voltage measured across the microchamber electrodes is ~1 kV, corresponding to ~10 MV/m electric fields in the microchamber. Depending on the nsPEF generator used, the measured pulse durations are equal to 3.0 and 4.2 ns, respectively. The voltage distribution provided by FDTD simulations indicates a good level of homogeneity across the microchamber electrodes. Experimental results include permeabilization of biological cells exposed to 3.0-ns, 10-MV/m PEFs.

Molecular-scale details of the mechanism of electric field-driven pore formation in phospholipid bilayers are not well understood, in part because the nanoscopic domain at which individual pore formation occurs is not readily accessible to experimental observation. Analytical and numerical models can help to fill this void. Previous studies using molecular dynamics (MD) simulations defined the stages in the creation and annihilation of an electropore as a function of the externally applied electric field, from the formation of an initial water defect to the restoration of the intact bilayer [1]. Here we vary the temperature at which electropermeabilization occurs, and we extract heat capacity and energy profiles for each system. Results will be compared to and, to the extent possible at this time, reconciled with existing mathematical models of electroporation, presenting a more unified and complete framework for future studies.[1] Levine, Z. A. and P. T. Vernier. 2010. Life Cycle of an Electropore: Field-Dependent and Field-Independent Steps in Pore Creation and Annihilation. Journal of Membrane Biology 236:27–36.

Electropermeabilization (electroporation) occurs when a supraphysiological electric field is applied across the cell membrane, but this phenomenon is not yet accessible to direct experimental observation. Molecular dynamics simulations suggest that interfacial waters located at the water:lipid interface are key determinants of electric field-driven pore formation in phospholipid bilayers [1]. We compared poration dynamics in water:vacuum:water and water:lipid:water systems at varying external electric fields. Both systems form water columns bridging the vacuum or lipid-filled gaps that are similar in structure and energetic signature. We identify these columns as electropores and study their structure and dynamics at the emerging and mature stages in their evolution. The electropores in both systems appear to originate from structurally similar water intrusions. We report pore formation times for each system as a function of electric field, and dipole-dipole interaction energies between interfacial water molecules.[1] Tieleman, D.P., The molecular basis of electroporation. BMC Biochemistry, 5:10, 2004.

When a cell is exposed to a sufficiently large electric field, even for a very short time, its plasma membrane is changed so that molecules that normally cross the membrane only in minute amounts can more readily pass through the barrier. This phenomenon was first described by Neumann and Rosenheck (1972) in this journal. A decade later gene transfer was achieved by means of electroporation (Neumann et al. 1982). This technique is now widely used in laboratories around the world and is gaining even more attention in recent years as a method for introducing foreign genes into cells in vivo (Mir 2009), with good prospects for use in a clinical setting (Daud et al. 2008). Electroporation has been used in the last decade also for improving cancer drug delivery to cells. Preclinical investigations in the late 1980s (Orlowski et al. 1988; Mir et al. 1991) were followed by the first clinical trial in 1991 (Mir et al. 1991; Belehradek et al. 1993). Electrochemotherapy as an effective and safe local treatment for cutaneous and subcutaneous tumors (Marty et al. 2006; Sersa and Miklavcic 2008) is now accepted in a number of countries, is routinely employed in more than 60 cancer treatment centers and is being further developed for treating more deep-seated tumors (Miklavcic et al. 2010). Electroporation can be used in all kinds of isolated cells as well as in tissues. The electric field to which one exposes the target cell has to be of sufficient strength and the exposure of sufficient duration. The magnitude of the electric field to be used depends on cell type, size, orientation and density; pulse duration; and number of pulses. The selection of pulse parameters is influenced also by the size and type of molecule to be internalized. Depending on the location and size of the targeted tissue, electric pulses will be delivered via appropriate electrodes chosen among a number of different types. Geometry and positioning of electrodes affect electric field distribution, which is important for effective in vivo electropermeabilization (Miklavcic et al. 1998, 2000). Strategies for the treatment of malignancies based on direct effects of pulsed electric fields on cells, independent of the induced influx of pharmacological or genetic material, are also under development. These include tumor ablation by irreversible electroporation (Al-Sakere et al. 2007) and by the application of intense (MV/m), nanosecond-duration pulses, which induce apoptosis and destruction of the tumor vascularization (Nuccitelli et al. 2006). This special issue of the Journal of Membrane Biology features selected peer-reviewed reports from investigators currently active in electroporation-related research who attended the Fourth International Scientific Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments in Ljubljana, Slovenia, in November 2009. This unique working conference at the University of Ljubljana has been organized every 2 years since the first one in 2003. More than 200 participants from 21 countries D. Miklavcic Faculty of Electrical Engineering, University of Ljubljana, Tržaska 25, 1000 Ljubljana, Slovenia e-mail: damijan.miklavcic@fe.uni-lj.si

Negative electrophoretic mobilities of oil in water are widely interpreted in terms of adsorption of hydroxide leading to negative surface charge. Challenging this traditional view, an increasing body of evidence suggests surface depletion of hydroxide and surface accumulation of hydronium leading to a positive surface charge. We present results from molecular dynamics (MD) simulations showing electrophoretic mobilities of oil in water with the same sign and magnitude as in experiment but in the absence of ions. The underlying mechanism involves interfacial roughness leading to gradients in dielectric permittivity in field direction and, thus, local elevation of the applied electric field. Although all molecules have zero net charge, their partial charges are distributed non-uniformly such that oil exhibits negative and water positive excess charge in regions of high field intensities; this induces a net force on the oil or the water against or in field direction, respectively. Our results indicate that deducing net charges from electrophoretic mobilities as widely done can be misleading. Our findings suggest that pH dependent electrophoretic mobilities in experiment being negative above and positive below pH 2.5 arise from a competition between the negative mobility of the ion-free interface and the positive mobility from adsorbed hydronium ions.