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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. [Copyright &y& Elsevier]

An introduction to the journal is presented, in which the editor discusses electropolation, as well as, reports from investiagtors currently active in electropolation-related research who attended the Fourth International Scientific Workshop and Postgraduate Course on Electroporation-Based Technologies and Treatments in Ljubljana, Slovenia.

Abstract: Exposing chromaffin cells to a single 5ns, 5 MV/m pulse causes Ca2+ influx and a rapid, transient rise in intracellular calcium concentration ([Ca2+]i). A comparison of responses at room temperature versus 37°C revealed no effect of temperature on the magnitude of the increase in [Ca2+]i. The Ca2+ transient, however, was shortened in duration almost twofold at 37°C, indicating that the rate of recovery was temperature-sensitive. Temperature also affected the interval required for a second pulse to elicit another maximal rise in [Ca2+]i, which was shorter at the higher temperature. In addition, a second pulse applied 5s after the first pulse was sufficient to cause cells at room temperature to become refractory to subsequent stimulation. At 37°C, cells became refractory after 5 pulses regardless of whether pulse delivery was at low (1 and 10Hz) or high (1kHz) rates. When refractory, cells showed no signs of swelling or uptake of the impermeant dye YO-PRO-1. These results demonstrate that temperature plays a role in determining how chromaffin cells respond to and become refractory to nanoelectropulses. They also indicate that despite the ultra-short duration of the pulses, pronounced effects on cell excitability result from the application of only very few pulses. [Copyright &y& Elsevier]

Although electric field-induced cell membrane permeabilization (electroporation) is used in a wide range of clinical applications from cancer therapy to cardiac ablation, the cellular- and molecular-level details of the processes that determine the success or failure of these treatments are poorly understood. Nanosecond pulsed electric field (nsPEF)-based tumor therapies are known to have an immune component, but whether and how immune cells sense the electroporative damage and respond to it have not been demonstrated. Damage- and pathogen-associated stresses drive inflammation via activation of cytosolic multiprotein platforms known as inflammasomes. The assembly of inflammasome complexes triggers caspase-1-dependent secretion of IL-1β and in many settings a form of cell death called pyroptosis. In this study we tested the hypothesis that the nsPEF damage is sensed intracellularly by the NLRP3 inflammasome. We found that 200-ns PEFs induced aggregation of the inflammasome adaptor protein ASC, activation of caspase-1, and triggered IL-1β release in multiple innate immune cell types (J774A.1 macrophages, bone marrow-derived macrophages, and dendritic cells) and in vivo in mouse skin. Efflux of potassium from the permeabilized cell plasma membrane was partially responsible for nsPEF-induced inflammasome activation. Based on results from experiments using both the NRLP3-specific inhibitor MCC950 and NLRP3 knockout cells, we propose that the damage created by nsPEFs generates a set of stimuli for the inflammasome and that more than one sensor can drive IL-1β release in response to electrical pulse stimulation. This study shows, to our knowledge, for the first time, that PEFs activate the inflammasome, suggesting that this pathway alarms the immune system after treatment., (Copyright © 2024 by The American Association of Immunologists, Inc.)

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 Ca 2+ levels due to Ca 2+ 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 Ca 2+ 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 Ca 2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

High intensity, nanosecond duration electric pulses (NEPs) permeabilize plasma membranes causing osmotic cell swelling that can elicit a wide variety of cellular effects. This study examined the possibility that cell swelling is the mechanism by which 5 ns NEPs trigger the release of catecholamines from neuroendocrine adrenal chromaffin cells. Swelling was assessed by comparing measurements of cell area obtained from bright field images of the cells before and at 10s intervals following exposure of the cells to 5 ns pulses at a field intensity of 5-6 MV/m. The results indicated that chromaffin cells do not swell in response to a single pulse or a train of ten pulses delivered at repetition frequencies of 10 Hz and 1 kHz. Swelling was also not observed in response to a train of 50 pulses whereas Jurkat T lymphoblast cell area increased 15% on average under the same NEP exposure conditions. These results demonstrating that chromaffin cells do not undergo swelling when exposed to 5 ns NEPs have important implications regarding the mechanism by which these pulses stimulate the release of catecholamines from these cells, namely that catecholamine secretion is most likely not caused by cell swelling., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 μm), 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 μs--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.

Formation of a water bridge across the lipid bilayer is the first stage of pore formation in molecular dynamic (MD) simulations of electroporation, suggesting that the intrusion of individual water molecules into the membrane interior is the initiation event in a sequence that leads to the formation of a conductive membrane pore. To delineate more clearly the role of water in membrane permeabilization, we conducted extensive MD simulations of water bridge formation, stabilization, and collapse in palmitoyloleoylphosphatidylcholine bilayers and in water-vacuum-water systems, in which two groups of water molecules are separated by a 2.8 nm vacuum gap, a simple analog of a phospholipid bilayer. Certain features, such as the exponential decrease in water bridge initiation time with increased external electric field, are similar in both systems. Other features, such as the relationship between water bridge lifetime and the diameter of the water bridge, are quite different between the two systems. Data such as these contribute to a better and more quantitative understanding of the relative roles of water and lipid in membrane electropore creation and annihilation, facilitating a mechanism-driven development of electroporation protocols. These methods can be extended to more complex, heterogeneous systems that include membrane proteins and intracellular and extracellular membrane attachments, leading to more accurate models of living cells in electric fields.

Molecular dynamics simulations of electroporation of homogeneous phospholipid bilayers show that the pore creation time is strongly dependent on the magnitude of the applied electric field. Here, we investigated whether heterogeneous bilayers containing phospholipids with zwitterionic and anionic headgroups exhibit a similar dependence. To facilitate this analysis we divide the life cycle of an electropore into several stages, marking the sequence of steps for pore creation and pore annihilation (restoration of the bilayer after removal of the electric field). We also report simulations of calcium binding isotherms and the effects of calcium ions on the electroporation of heterogeneous lipid bilayers. Calcium binding simulations are consistent with experimental data using a 1:2 Langmuir binding isotherm. We find that calcium ions and phosphatidylserine increase pore creation time and decrease pore annihilation time. For all systems tested, pore creation time was inversely proportional to the bilayer internal electric field.

Exposing chromaffin cells to a single 5 ns, 5 MV/m pulse causes Ca(2+) influx and a rapid, transient rise in intracellular calcium concentration ([Ca(2+)](i)). A comparison of responses at room temperature versus 37°C revealed no effect of temperature on the magnitude of the increase in [Ca(2+)](i). The Ca(2+) transient, however, was shortened in duration almost twofold at 37°C, indicating that the rate of recovery was temperature-sensitive. Temperature also affected the interval required for a second pulse to elicit another maximal rise in [Ca(2+)](i), which was shorter at the higher temperature. In addition, a second pulse applied 5s after the first pulse was sufficient to cause cells at room temperature to become refractory to subsequent stimulation. At 37°C, cells became refractory after 5 pulses regardless of whether pulse delivery was at low (1 and 10 Hz) or high (1 kHz) rates. When refractory, cells showed no signs of swelling or uptake of the impermeant dye YO-PRO-1. These results demonstrate that temperature plays a role in determining how chromaffin cells respond to and become refractory to nanoelectropulses. They also indicate that despite the ultra-short duration of the pulses, pronounced effects on cell excitability result from the application of only very few pulses., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nanosecond, high-voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP-induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators-rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt-quenched calcein-we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO-PRO-1 influx) was detected in addition to mitochondrial membrane permeabilization., (© 2011 Wiley Periodicals, Inc.)

In this paper, a versatile electrode assembly for cell electroporation is proposed. For validation of the delivery system, biological cell electroporation experiments using 2.5 ns and 5 ns, 10 MV/m pulsed electric fields have been conducted. Electromagnetic, time domain, and frequency analyses demonstrate the broadband behavior of the delivery system.

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.

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.

Ultra-short, high-field electric pulses permeabilize plasma and intracellular membranes. We report here nanosecond pulse-induced permeabilization of mitochondrial membranes in living cells. Using four independent methods based on fluorescent dyes--JC-1, rhodamine 123, tetramethyl rhodamine ethyl ester, and cobalt-quenched calcein--we show that as few as five, 4 ns, 10 MV/m pulses delivered at 1 kHz cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. The most likely interpretation of these results is a pulse-induced permeabilization of the inner mitochondrial membrane.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca(2+) entry into the cells via L-type voltage-gated Ca(2+) channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca(2+) level ([Ca(2+)](i)) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca(2+) entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca(2+)](i) elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca(2+)](i) response of the cells, suggesting Ca(2+) influx occurs only via VGCCs. Lowering extracellular K(+) concentration from 5 to 2 mM or pulsing cells in Na(+)-free medium suppressed the pulse-induced rise in [Ca(2+)](i) in the majority of cells. Thus, both membrane potential and Na(+) entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca(2+) influx. However, activation of voltage-gated Na(+) channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca(2+)](i). These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na(+) transport across the plasma membrane. Whether Na(+) transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.

Electropermeabilization, an electric field-induced modification of the barrier functions of the cell membrane, is widely used in laboratories and increasingly in the clinic; but the mechanisms and physical structures associated with the electromanipulation of membrane permeability have not been definitively characterized. Indirect experimental observations of electrical conductance and small molecule transport as well as molecular dynamics simulations have led to models in which hydrophilic pores form in phospholipid bilayers with increased probability in the presence of an electric field. Presently available methods do not permit the direct, nanoscale examination of electroporated membranes that would confirm the existence of these structures. To facilitate the reconciliation of poration models with the observed properties of electropermeabilized lipid bilayers and cell membranes, we propose a scheme for characterizing the stages of electropore formation and resealing. This electropore life cycle, based on molecular dynamics simulations of phospholipid bilayers, defines a sequence of discrete steps in the electric field-driven restructuring of the membrane that leads to the formation of a head group-lined, aqueous pore and then, after the field is removed, to the dismantling of the pore and reassembly of the intact bilayer. Utilizing this scheme we can systematically analyze the interactions between the electric field and the bilayer components involved in pore initiation, construction and resealing. We find that the pore creation time depends strongly on the electric field gradient across the membrane interface and that the pore annihilation time is at least weakly dependent on the magnitude of the pore-initiating electric field and, in general, much longer than the pore creation time.

Manipulating the plasma membrane, the gateway to the cell interior, with chemical and physical agents for genetic and pharmacological therapy, and understanding the interactions of lipid membrane components with proteins and other structural and functional elements of the cell, require a detailed biomolecular membrane model. We report here progress along one path toward such a model: molecular dynamics simulations of mixed, zwitterionic-anionic, asymmetric phospholipid bilayers with monovalent and divalent cations. With phosphatidylcholine/phosphatidylserine systems, we identify temporal and concentration boundaries for equilibration of calcium with the bilayer and saturation of the calcium capacity of the membrane, we demonstrate the electrostatic- and entropic-driven associations of calcium and sodium ions with polar groups in the bilayer interface region, expressed in spatial distribution profiles and in changes in the orientation of the phospholipid head groups, and we describe for the first time simulations of dynamic, calcium-mediated adjustments in the conformation of mixed phospholipid species coresident in the same leaflet of the bilayer. The results are consistent with experimental observations and point the way to further refinement and increased realism of these molecular models.

This paper presents a combined physical and electrical post-fabrication method for determining the thicknesses of the various layers in a commercial 1.5 μm complementary-metal-oxide-semiconductor (CMOS) foundry process available through MOSIS. Forty-two thickness values are obtained from physical step-height measurements performed on thickness test structures and from electrical measurements of capacitances, sheet resistances, and resistivities. Appropriate expressions, numeric values, and uncertainties for each layer of thickness are presented, along with a systematic nomenclature for interconnect and dielectric thicknesses. However, apparent inconsistencies between several of the physical and electrical results for film thickness suggest that further uncertainty analysis is required and the effects of several assumptions need to be quantified.

Nanosecond pulsed electric fields can pass through the external membrane of biological cells and disturb fast-responding intracellular structures and processes. To enable real-time imaging and investigation of these phenomena, a microchamber with integral electrodes and optical path for observing individual cells exposed to ultrashort electric pulses was designed and fabricated utilizing photolithographic and microelectronic methods. SU-8 photoresist was patterned to form straight sidewalls from 10 to 30 microm in height, with gold film deposited on the top and sidewalls for conductive, nonreactive electrodes and a uniform electric field. Channel dimensions (10-30 microm x 100 microm x 12 000 microm) are suitable for observations of mammalian cells during nanosecond, megavolt-per-meter pulsed electric field exposure. Experimental studies utilizing the electrode microchamber include live-cell imaging of nanoelectropulse-induced intracellular calcium bursts and membrane phospholipid translocation.

Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in the plasma membrane, release intracellular calcium, trigger cardiomyocyte activity, and induce apoptosis in mammalian cancer cells, without the permeabilizing effects associated with longer, lower-field pulses. Dose dependencies with respect to pulse width, amplitude, and repetition rate, and total pulse count are observed for all of these phenomena. Sensitivities vary among cell types; cells of lymphoid origin growing in suspension are more susceptible to nanoelectropulse exposure than solid tumor lines. Simple electrical models of the cell are useful for first-order explanations, but more sophisticated treatments will be required for analysis and prediction at both biomolecular and tissue levels.

Nanosecond, megavolt-per-meter, pulsed electric fields induce phosphatidylserine (PS) externalization, intracellular calcium redistribution, and apoptosis in Jurkat T-lymphoblasts, without causing immediately apparent physical damage to the cells. Intracellular calcium mobilization occurs within milliseconds of pulse exposure, and membrane phospholipid translocation is observed within minutes. Pulsed cells maintain cytoplasmic membrane integrity, blocking propidium iodide and Trypan blue. Indicators of apoptosis-caspase activation and loss of mitochondrial membrane potential-appear in nanoelectropulsed cells at later times. Although a theoretical framework has been established, specific mechanisms through which external nanosecond pulsed electric fields trigger intracellular responses in actively growing cells have not yet been experimentally characterized. This report focuses on the membrane phospholipid rearrangement that appears after ultrashort pulse exposure. We present evidence that the minimum field strength required for PS externalization in actively metabolizing Jurkat cells with 7-ns pulses produces transmembrane potentials associated with increased membrane conductance when pulse widths are microseconds rather than nanoseconds. We also show that nanoelectropulse trains delivered at repetition rates from 2 to 2000 Hz have similar effects, that nanoelectropulse-induced PS externalization does not require calcium in the external medium, and that the pulse regimens used in these experiments do not cause significant intra- or extracellular Joule heating.

We report here real-time imaging of calcium bursts in human lymphocytes exposed to nanosecond, megavolt-per-meter pulsed electric fields. Ultra-short (less than 30 ns), high-field (greater than 1 MV/m), electric pulses induce increases in cytosolic calcium concentration and translocation of phosphatidylserine (PS) to the outer layer of the plasma membrane in Jurkat T lymphoblasts. Pulse-induced calcium bursts occur within milliseconds and PS externalization within minutes. Caspase activation and other indicators of apoptosis follow these initial symptoms of nanosecond pulse exposure. Pulse-induced PS translocation is observed even in the presence of caspase inhibitors. Ultra-short, high-field, electroperturbative pulse effects differ substantially from those associated with electroporation, where pulses of a few tens of kilovolts-per-meter lasting a few tens of microseconds open pores in the cytoplasmic membrane. Nanosecond pulsed electric fields, because their duration is less than the plasma membrane charging time, develop voltages across intracellular structures without porating the cell.