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The quest for safe and effective ablation resulted in the development of nanosecond pulsed electric fields (nsPEF) technology for tumor treatment. For future applications of nsPEF in urothelial cancer treatment, we evaluated the effect of urine presence at the ablation site. We prepared artificial urine (AU) with compounds commonly present in the healthy human urine at physiological concentrations. We compared nsPEF cytotoxicity for cancerous (T24) and non-cancerous (SV-HUC-1) human urothelial cell lines pulsed either in the AU or in a physiological solution (PS). Cell monolayers were exposed to trains of 300-ns, 10-Hz pulses using a two-needle electrode assembly placed orthogonal to the monolayer. The assembly produced the electric field gradually weakening with the distance from the electrodes. The electric field which killed 50 % of cells (LD50) was measured by staining with propidium iodide and matching the stained area with the simulated electric field strength. nsPEF exposure in PS was more cytotoxic to cancer cells. The AU protected both healthy and cancer urothelial cells, increasing their LD50 1.4 and 1.6 times, respectively. Omitting urea from the AU reduced the LD50 for healthy and cancer urothelial cells. Testing the role of other AU components, we found that it was the high concentration of phosphates what also rendered the protective effect of the AU. Our findings suggest that the nsPEF ablation of bladder cancer will be less efficient if the bladder is filled with urine.

The reversal of the electric field direction inhibits various biological effects of nanosecond electric pulses (nsEP). This feature, known as "bipolar cancellation," enables interference targeting of nsEP bioeffects remotely from stimulating electrodes, for prospective applications such as precise cancer ablation and non-invasive deep brain stimulation. This study was undertaken to achieve the maximum cancellation of electroporation, by quantifying the impact of the pulse shape, duration, number, and repetition rate across a broad range of electric field strengths. Monolayers of endothelial cells (BPAE) were electroporated in a non-uniform electric field. Cell membrane permeabilization was quantified by YO-PRO-1 (YP) dye uptake and correlated to local electric field strength. For most conditions tested, adding an opposite polarity phase reduced YP uptake by 50-80 %. The strongest cancellation, which reduced YP uptake by 95-97 %, was accomplished by adding a 50 % second phase to 600-ns pulses delivered at a high repetition rate of 833 kHz. Strobe photography of nanosecond kinetics of membrane potential in single CHO cells revealed the temporal summation of polarization by individual unipolar nsEP applied at sub-MHz rate, leading to enhanced electroporation. In contrast, there was no summation for bipolar pulses, and increasing their repetition rate suppressed electroporation. These new findings are discussed in the context of bipolar cancellation mechanisms and remote focusing applications.

The purpose of this review article is to summarize our current understanding of the efficacy and safety of cardiac defibrillation with nanosecond shocks. Experiments in isolated hearts, using optical mapping of the electrical activity, have demonstrated that nanosecond shocks can defibrillate with lower energies than conventional millisecond shocks. Single defibrillation strength nanosecond shocks do not cause obvious damage, but repeated stimulation leads to deterioration of the hearts. In isolated myocytes, nanosecond pulses can also stimulate at lower energies than at longer pulses and cause less electroporation (propidium uptake). The mechanism is likely electroporation of the plasma membrane. Repeated stimulation leads to distorted calcium gradients.

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20-200 kHz, with up to 20-30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-μs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3-5 %. nsEP bursts were compared with single "long" pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2-3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-μs pulse decreased to 57 ± 8 V/cm for a 100-μs burst of 100-ns, 0.25-MHz pulses (p 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage.

Aims Recently, a new defibrillation modality using nanosecond pulses was shown to be effective at much lower energies than conventional 10 millisecond monophasic shocks in ex vivo experiments. Here we compare the safety factors of 300 nanosecond and 10 millisecond shocks to assess the safety of nanosecond defibrillation. Methods and results The safety factor, i.e. the ratio of median effective doses (ED50) for electroporative damage and defibrillation, was assessed for nanosecond and conventional (millisecond) defibrillation shocks in Langendorff-perfused New Zealand white rabbit hearts. In order to allow for multiple shock applications in a single heart, a pair of needle electrodes was used to apply shocks of varying voltage. Propidium iodide (PI) staining at the surface of the heart showed that nanosecond shocks had a slightly lower safety factor (6.50) than millisecond shocks (8.69), p = 0.02; while PI staining cross-sections in the electrode plane showed no significant difference (5.38 for 300 ns shocks and 6.29 for 10 ms shocks, p = 0.22). Conclusions In Langendorff-perfused rabbit hearts, nanosecond defibrillation has a similar safety factor as millisecond defibrillation, between 5 and 9, suggesting that nanosecond defibrillation can be performed safely.

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca

Exposure of living cells to intense nanosecond pulsed electric field (nsPEF) increases membrane permeability to small solutes, presumably by the formation of nanometer-size membrane lesions. Mechanisms responsible for the restoration of membrane integrity over the course of minutes after nsPEF have not been identified. This study explored if ESCRT-III and Annexin V calcium-dependent repair mechanisms, which play critical role in resealing large membrane lesions, are also activated by electroporation and contribute to the membrane resealing. The extent of membrane damage and the time course of resealing were monitored by the time-lapse imaging of propidium (Pr) uptake in human cervical carcinoma (HeLa) cells exposed to trains of 300-ns PEF. The removal of the extracellular Ca2+ slowed down the resealing, although did not prevent it. Recruitment of CHMP4B protein, a component of ESCRT-III complex, to the electroporated plasma membrane was not observed, thus providing no evidence for possible contribution of the macro-vesicle shedding mechanism. In contrast, silencing the AnxA5 gene impaired resealing and reduced the viability of nsPEF-treated cells. We conclude that Annexin V but not ESCRT-III was involved in the repair of HeLa cells permeabilized by 300-ns stimuli, but it was not the only and perhaps not the main repair mechanism.

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.

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca2+]. We explored the impact of Ca2+ on the kinetics of membrane resealing by monitoring the entry of a YO-PRO-1 dye (YP) in BPAE and HEK cells. Ca2+ was promptly removed or added after the electric pulse (EP) by a fast-step perfusion. YP entry increased sharply after the EP and gradually slowed down following either a single- or a double-exponential function. In BPAE cells permeabilized by a single 300- or 600-ns EP at 14 kV/cm in a Ca2+-free medium, perfusion with 2 mM of external Ca2+ advanced the 90% resealing and reduced the dye uptake about twofold. Membrane restoration was accomplished by a combination of fast, Ca2+-independent resealing (τ = 13–15 s) and slow, Ca2+-dependent processes (τ ~70 s with Ca2+ and ~ 110 s or more without it). These time constants did not change when the membrane damage was doubled by increasing EP duration from 300 to 600 ns. However, injury by microsecond-range EP (300 and 600 μs) took longer to recover even when the membrane initially was less damaged, presumably because of the larger size of pores made in the membrane. Full membrane recovery was not prevented by blocking both extra- and intracellular Ca2+ (by loading cells with BAPTA or after Ca2+ depletion from the reticulum), suggesting the recruitment of unknown Ca2+-independent repair mechanisms.

Irreversible Electroporation (IRE) has gained significant interest as an alternative treatment for atrial fibrillation (AF). Typically, to terminate AF, thermal energy is delivered to the arrhythmic tissue causing rare but serious damage to surrounding areas. IRE is a non-thermal approach that has the potential to be faster yet more targeted. We are developing a novel, species relevant, standard laboratory test to evaluate IRE cardiac ablation using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model. hiPSC-CMs were cultured in monolayers. Pulses were delivered by a AVOZ D7B generator through contact electrodes. Electric field (E) distribution maps were obtained with numerical simulations, Sim4Life v3.2. Electroporation (EP) was quantified by florescence with YO-PRO-1 10 minutes after exposure. hiPSC-CMs were exposed to a range of electrical parameters including 120 unipolar pulses of 5 μs duration, at 1 kHz frequency (Fig. 1). We found that the EP threshold was ~1.5 kV/cm in line with previous reports on single adult ventricular mouse cardiomyocytes (Semenov et al. 2018). The treated area increased with the voltage (V) applied. These data suggest that this assay can delineate the extent of EP. In this work we will vary different pulse parameters (durations, amplitude, shape, repetition rate and number) to evaluate their effects on different human cell lines anatomically adjacent to the heart. Fig.1: (A) Representation of the EP threshold determined by overlaying the E map (B) with a fluorescence image of pulse-treated hiPSC-CMs (C). To define the E threshold the distributions values obtained by modeling 1 V applied was scaled by the experimental V.

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within 7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing., This article belongs to the Section Molecular Biophysics, The study was supported by a 2015 AFOSR MURI grant A9550-15-1-0517 (to A.G.P.), by a scholarship from the China Scholarship Council (to W.B.), and by a grant S-MIP-19-13 from the Research Council of Lithuania (to V.N.).

Bipolar cancellation refers to a phenomenon when applying a second electric pulse reduces (“cancels”) cell membrane damage by a preceding electric pulse of the opposite polarity. Bipolar cancellation is a reason why bipolar nanosecond electric pulses (nsEP) cause weaker electroporation than just a single unipolar phase of the same pulse. This study was undertaken to explore the dependence of bipolar cancellation on nsEP parameters, with emphasis on the amplitude ratio of two opposite polarity phases of a bipolar pulse. Individual cells (CHO, U937, or adult mouse ventricular cardiomyocytes (VCM)) were exposed to either uni- or bipolar trapezoidal nsEP, or to nanosecond electric field oscillations (NEFO). The membrane injury was evaluated by time-lapse confocal imaging of the uptake of propidium (Pr) or YO-PRO-1 (YP) dyes and by phosphatidylserine (PS) externalization. Within studied limits, bipolar cancellation showed little or no dependence on the electric field intensity, pulse repetition rate, chosen endpoint, or cell type. However, cancellation could increase for larger pulse numbers and/or for longer pulses. The sole most critical parameter which determines bipolar cancellation was the phase ratio: maximum cancellation was observed with the 2(nd) phase of about 50% of the first one, whereas a larger 2(nd) phase could add a damaging effect of its own. “Swapping” the two phases, i.e., delivering the smaller phase before the larger one, reduced or eliminated cancellation. These findings are discussed in the context of hypothetical mechanisms of bipolar cancellation and electroporation by nsEP.

Decreasing the time gap between two identical electric pulses is expected to render bioeffects similar to those of a single pulse of equivalent total duration. In this study, we show that it is not necessarily true, and that the effects vary for different permeabilization markers. We exposed individual CHO or NG108 cells to one 300-ns pulse (3.7–11.6 kV/cm), or a pair of such pulses (0.4–1000 μs interval), or to a single 600-ns pulse of the same amplitude. Electropermeabilization was evaluated (a) by the uptake of YO-PRO-1 (YP) dye; (b) by the amplitude of elicited Ca(2+) transients, and (c) by the entry of Tl(+) ions. For YP uptake, applying a 600-ns pulse or a pair of 300-ns pulses doubled the effect of a single 300-ns pulse; this additive effect did not depend on the time interval between pulses or the electric field, indicating that already permeabilized cells are as susceptible to electropermeabilization as naïve cells. In contrast, Ca(2+) transients and Tl(+) uptake increased in a supra-additive fashion when two pulses were delivered instead of one. Paired pulses at 3.7 kV/cm with minimal separation (0.4 and 1 μs) elicited 50–100% larger Ca(2+) transients than either a single 600-ns pulse or paired pulses with longer separation (10–1000 μs). This paradoxically high efficiency of the closest spaced pulses was emphasized when Ca(2+) transients were elicited in a Ca(2+)-free solution (when the endoplasmic reticulum (ER) was the sole significant source of Ca(2+)), but was eliminated by Ca(2+) depletion from the ER and was not observed for Tl(+) entry through the electropermeabilized membrane. We conclude that closely spaced paired pulses specifically target ER, by either permeabilizing it to a greater extent than a single double-duration pulse thus causing more Ca(2+) leak, or by amplifying Ca(2+)-induced Ca(2+) release by an unknown mechanism.

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid.

Stimulation and electroporation by nanosecond electric pulses (nsEP) are distinguished by a phenomenon of bipolar cancellation, which stands for a reduced efficiency of bipolar pulses compared to unipolar ones. When two pairs of stimulating electrodes are arrayed in a quadrupole, bipolar cancellation inhibits nsEP effects near the electrodes, where the electric field is the strongest. Two properly shaped and synchronized bipolar nsEP overlay into a unipolar pulse towards the center of the electrode array, thus canceling the bipolar cancellation (a "CANCAN effect"). High efficiency of the re-created unipolar nsEP outweighs the weakening of the electric field with distance and focuses nsEP effects to the center. In monolayers of CHO, BPAE, and HEK cells, CANCAN effect achieved by the interference of two bipolar nsEP enhanced electroporation up to tenfold, with a peak at the quadrupole center. Introducing a time interval between bipolar nsEP prevented the formation of a unipolar pulse and eliminated the CANCAN effect. Strong electroporation by CANCAN stimuli killed cells over the entire area encompassed by the electrodes, whereas the time-separated pulses caused ablation only in the strongest electric field near the electrodes. The CANCAN approach is promising for uniform tumor ablation and stimulation targeting away from electrodes.

Aims Reliable defibrillation with reduced energy deposition has long been the focus of defibrillation research. We studied the efficacy of single shocks of 300 ns duration in defibrillating rabbit hearts as well as the tissue damage they may cause. Methods and results New Zealand white rabbit hearts were Langendorff-perfused and two planar electrodes were placed on either side of the heart. Shocks of 300 ns duration and 0.3-3 kV amplitude were generated with a transmission line generator. Single nanosecond shocks consistently induced waves of electrical activation, with a stimulation threshold of 0.9 kV (over 3 cm) and consistent activation for shock amplitudes of 1.2 kV or higher (9/9 successful attempts). We induced fibrillation (35 episodes in 12 hearts) and found that single shock nanosecond-defibrillation could consistently be achieved, with a defibrillation threshold of 2.3-2.4 kV (over 3 cm), and consistent success at 3 kV (11/11 successful attempts). Shocks uniformly depolarized the tissue, and the threshold energy needed for nanosecond defibrillation was almost an order of magnitude lower than the energy needed for defibrillation with a monophasic 10 ms shock delivered with the same electrode configuration. For the parameters studied here, nanosecond defibrillation caused no baseline shift of the transmembrane potential (that could be indicative of electroporative damage), no changes in action potential duration, and only a brief change of diastolic interval, for one beat after the shock was delivered. Histological staining with tetrazolium chloride and propidium iodide showed that effective defibrillation was not associated with tissue death or with detectable electroporation anywhere in the heart (six hearts). Conclusion Nanosecond-defibrillation is a promising technology that may allow clinical defibrillation with profoundly reduced energies.

Electric field pulses of nano- and picosecond duration are a novel modality for neurostimulation, activation of Ca2+ signaling, and tissue ablation. However it is not known how such brief pulses activate voltage-gated ion channels. We studied excitation and electroporation of hippocampal neurons by 200-ns pulsed electric field (nsPEF), by means of time-lapse imaging of the optical membrane potential (OMP) with FluoVolt dye. Electroporation abruptly shifted OMP to a more depolarized level, which was reached within 10 s), so cells remained above the resting OMP level for at least 20–30 s. Activation of voltage-gated sodium channels (VGSC) enhanced the depolarizing effect of electroporation, resulting in an additional tetrodotoxin-sensitive OMP peak in 4–5 ms after nsPEF. Omitting Ca2+ in the extracellular solution did not reduce the depolarization, suggesting no contribution of voltage-gated calcium channels (VGCC). In 40% of neurons, nsPEF triggered a single action potential (AP), with the median threshold of 3 kV/cm (range: 1.9–4 kV/cm); no APs could be evoked by stimuli below the electroporation threshold (1.5–1.9 kV/cm). VGSC opening could already be detected in 0.5 ms after nsPEF, which is too fast to be mediated by the depolarizing effect of electroporation. The overlap of electroporation and AP thresholds does not necessarily reflect the causal relation, but suggests a low potency of nsPEF, as compared to conventional electrostimulation, for VGSC activation and AP induction.

Nanosecond pulsed electric fields are emerging as a new modality for tissue and tumor ablation. We previously reported that cells exposed to pulsed electric fields develop hypersensitivity to subsequent pulsed electric field applications. This phenomenon, named electrosensitization, is evoked by splitting the pulsed electric field treatment in fractions (split-dose treatments) and causes in vitro a 2- to 3-fold increase in cytotoxicity. The aim of this study was to show the benefit of split-dose treatments for in vivo tumor ablation by nanosecond pulsed electric field. KLN 205 squamous carcinoma cells were embedded in an agarose gel or grown subcutaneously as tumors in mice. Nanosecond pulsed electric field ablations were produced using a 2-needle probe with a 6.5-mm interelectrode distance. In agarose gel, splitting a pulsed electric field dose of 300, 300-ns pulses (20 Hz, 4.4-6.4 kV) in 2 equal fractions increased cell death up to 3-fold compared to single-train treatments. We then compared the antitumor effectiveness of these treatments in vivo. At 24 hours after treatment, sensitizing tumors by a split-dose pulsed electric field exposure (150 + 150, 300-ns pulses, 20 Hz, 6.4 kV) caused a 4- and 2-fold tumor volume reduction as compared to sham and single-train treatments, respectively. Tumor volume reduction that exceeds 75% was 43% for split-dose-treated animals compared to only 12% for single-dose treatments. The difference between the 2 experimental groups remained statistically significant for at least 1 week after the treatment. The results show that electrosensitization occurs in vivo and can be exploited to assist in vivo cancer ablation.

Tumor ablation by nanosecond pulsed electric fields (nsPEF) is an emerging therapeutic modality. We compared nsPEF cytotoxicity for human cell lines of cancerous (IMR-32, Hep G2, HT-1080, and HPAF-II) and non-cancerous origin (BJ and MRC-5) under strictly controlled and identical conditions. Adherent cells were uniformly treated by 300-ns PEF (0–2000 pulses, 1.8 kV/cm, 50 Hz) on indium tin oxide-covered glass coverslips, using the same media and serum. Cell survival plotted against the number of pulses displayed three distinct regions (initial resistivity, logarithmic survival decline, and residual resistivity) for all tested cell types, but with differences in LD(50) spanning as much as nearly 80-fold. The non-cancerous cells were less sensitive than IMR-32 neuroblastoma cells but more vulnerable than the other cancers tested. The cytotoxic efficiency showed no apparent correlation with cell or nuclear size, cell morphology, metabolism level, or the extent of membrane disruption by nsPEF. Increasing pulse duration to 9 μs (0.75 kV/cm, 5 Hz) produced a different selectivity pattern, suggesting that manipulation of PEF parameters can, at least for certain cancers, overcome their resistance to nsPEF ablation. Identifying mechanisms and cell markers of differential nsPEF susceptibility will critically contribute to the proper choice and outcome of nsPEF ablation therapies.

A unique aspect of electrostimulation (ES) with nanosecond electric pulses (nsEP) is the inhibition of effects when the polarity is reversed. This bipolar cancellation feature makes bipolar nsEP less efficient at biostimulation than unipolar nsEP. We propose to minimize stimulation near pulse-delivering electrodes by applying bipolar nsEP, whereas the superposition of two phase-shifted bipolar nsEP from two independent sources yields a biologically-effective unipolar pulse remotely. This is accomplished by electrical compensation of all nsEP phases except the first one, resulting in the restoration of stimulation efficiency due to cancellation of bipolar cancellation (CANCAN-ES). We experimentally proved the CANCAN-ES paradigm by measuring YO-PRO-1 dye uptake in CHO-K1 cells which were permeabilized by multiphasic nsEP (600 ns per phase) from two generators; these nsEP were synchronized either to overlap into a unipolar pulse remotely from electrodes (CANCAN), or not to overlap (control). Enhancement of YO-PRO-1 entry due to CANCAN was observed in all sets of experiments and reached ~3-fold in the center of the gap between electrodes, exactly where the unipolar pulse was formed, and equaled the degree of bipolar cancellation. CANCAN-ES is promising for non-invasive deep tissue stimulation, either alone or combined with other remote stimulation techniques to improve targeting.

Intense nanosecond pulsed electric field (nsPEF) is a novel modality for cell activation and nanoelectroporation. Applications of nsPEF in research and therapy are hindered by a high electric field requirement, typically from 1 to over 50 kV/cm to elicit any bioeffects. We show how this requirement can be overcome by engaging temporal summation when pulses are compressed into high-rate bursts (up to several MHz). This approach was tested for excitation of ventricular cardiomyocytes and peripheral nerve fibers; for membrane electroporation of cardiomyocytes, CHO, and HEK cells; and for killing EL-4 cells. MHz compression of nsPEF bursts (100–1000 pulses) enables excitation at only 0.01–0.15 kV/cm and electroporation already at 0.4–0.6 kV/cm. Clear separation of excitation and electroporation thresholds allows for multiple excitation cycles without membrane disruption. The efficiency of nsPEF bursts increases with the duty cycle (by increasing either pulse duration or repetition rate) and with increasing the total time “on” (by increasing either pulse duration or number). For some endpoints, the efficiency of nsPEF bursts matches a single “long” pulse whose amplitude and duration equal the time-average amplitude and duration of the bursts. For other endpoints this rule is not valid, presumably because of nsPEF-specific bioeffects and/or possible modification of targets already during the burst. MHz compression of nsPEF bursts is a universal and efficient way to lower excitation thresholds and facilitate electroporation.

Accumulating data indicates that some cancer treatments can restore anticancer immunosurveillance through the induction of tumor immunogenic cell death (ICD). Nanosecond pulsed electric fields (nsPEF) have been shown to efficiently ablate melanoma tumors. In this study we investigated the mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors. Our data show that in vitro nsPEF (20–200, 200-ns pulses, 7 kV/cm, 2 Hz) caused a rapid dose-dependent cell death which was not accompanied by caspase activation or PARP cleavage. The lack of nsPEF-induced apoptosis was confirmed in vivo in B16F10 tumors. NsPEF also failed to trigger ICD-linked responses such as necroptosis and autophagy. Our results point at necrosis as the primary mechanism of cell death induced by nsPEF in B16F10 cells. We finally compared the antitumor immunity in animals treated with nsPEF (750, 200-ns, 25 kV/cm, 2 Hz) with animals were tumors were surgically removed. Compared to the naïve group where all animals developed tumors, nsPEF and surgery protected 33% (6/18) and 28.6% (4/14) of the animals, respectively. Our data suggest that, under our experimental conditions, the local ablation by nsPEF restored but did not boost the natural antitumor immunity which stays dormant in the tumor-bearing host.

INTRODUCTION Opening of voltage-gated sodium channels takes tens to hundreds of microseconds, and mechanisms of their opening by nanosecond pulsed electric field (nsPEF) stimuli remain elusive. This study was aimed at uncovering the mechanisms of how nsPEF elicits action potentials (APs) in cardiomyocytes. METHODS AND RESULTS Fluorescent imaging of optical APs (FluoVolt) and Ca2+ -transients (Fluo-4) was performed in enzymatically isolated murine ventricular cardiomyocytes stimulated by 200-nanosecond trapezoidal pulses. nsPEF stimulation evoked tetrodotoxin-sensitive APs accompanied or preceded by slow sustained depolarization (SSD) and, in most cells, by transient afterdepolarization waves. SSD threshold was lower than the AP threshold (1.26 ± 0.03 vs 1.34 ± 0.03 kV/cm, respectively, P

The initiation of action potentials (APs) by membrane depolarization occurs after a brief vulnerability period, during which excitation can be abolished by the reversal of the stimulus polarity. This vulnerability period is determined by the time needed for gating of voltage-gated sodium channels (VGSC). We compared nerve excitation by ultra-short uni- and bipolar stimuli to define the time frame of bipolar cancellation and of AP initiation. Propagating APs in isolated frog sciatic nerve were elicited by cathodic pulses (200 ns–300 µs), followed by an anodic (canceling) pulse of the same duration after a 0–200-µs delay. We found that the earliest and the latest boundaries for opening the critical number of VGSC needed to initiate AP are, respectively, between 11 and 20 µs and between 100 and 200 µs after the onset of depolarization. Stronger depolarization accelerated AP initiation, apparently due to faster VGSC opening, but not beyond the 11-µs limit. Bipolar cancellation was augmented by reducing pulse duration, shortening the delay between pulses, decreasing the amplitude of the cathodic pulse, and increasing the amplitude of the anodic one. Some of these characteristics contrasted the bipolar cancellation of cell membrane electroporation (Pakhomov et al. in Bioelectrochemistry 122:123–133, 2018; Gianulis et al. in Bioelectrochemistry 119:10–19, 2017), suggesting different mechanisms. The ratio of nerve excitation thresholds for a unipolar cathodic pulse and a symmetrical bipolar pulse increased as a power function as the pulse duration decreased, in remarkable agreement with the predictions of SENN model of nerve excitation (Reilly and Diamant in Health Phys 83(3):356–365, 2002).

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD.

Conventional electric stimuli of micro- and millisecond duration excite or activate cells at voltages 10–100 times below the electroporation threshold. This ratio is remarkably different for nanosecond electric pulses (nsEP), which caused excitation and activation only at or above the electroporation threshold in diverse cell lines, primary cardiomyocytes, neurons, and chromaffin cells. Depolarization to the excitation threshold often results from (or is assisted by) the loss of the resting membrane potential due to ion leaks across the membrane permeabilized by nsEP. Slow membrane resealing and the build-up of electroporation damages prevent repetitive excitation by nsEP. However, peripheral nerves and muscles are exempt from this rule and withstand multiple cycles of excitation by nsEP without the loss of function or signs of electroporation. We show that the damage-free excitation by nsEP may be enabled by the membrane charging time constant sufficiently large to (1) cap the peak transmembrane voltage during nsEP below the electroporation threshold, and (2) extend the post-nsEP depolarization long enough to activate voltage-gated ion channels. The low excitatory efficacy of nsEP compared to longer pulses makes them advantageous for medical applications where the neuromuscular excitation is an unwanted side effect, such as electroporation-based cancer and tissue ablation.

Electropermeabilization of cell membranes by micro- and nanosecond-duration stimuli has been studied extensively, whereas effects of picosecond electric pulses (psEP) remain essentially unexplored. We utilized whole-cell patch clamp and Di-8-ANEPPS voltage-sensitive dye measurements to characterize plasma membrane effects of 500 ps stimuli in rat hippocampal neurons (RHN), NG108, and CHO cells. Even a single 500-ps pulse at 190 kV/cm increased membrane conductance and depolarized cells. These effects were augmented by applying brief psEP bursts (5–125 pulses), whereas the rate of pulse delivery (8 Hz–1 kHz) played little role. psEP-treated cells displayed large inward current at negative membrane potentials but modest or no conductance changes at positive potentials. A 1-kHz burst of 25 pulses increased the whole-cell conductance in the range (−100)–(−60) mV to 22–26 nS in RHN and NG108 cells (from 3 and 0.7 nS, respectively), but only to 5 nS in CHO (from 0.3 nS). The conductance increase was reversible within about 2 min. Such pattern of cell permeabilization, with characteristic inward rectification and slow recovery, was similar to earlier reported effects of 60- and 600-ns pulses, pointing to the similarity of structural membrane rearrangements in spite of a different membrane charging mechanism., Highlights • 500-ps pulses at 190 kV/cm electroporate mammalian cells. • Cells lose membrane potential and increase conductance at negative potentials. • Permeabilization is enhanced by increasing pulse number but not the rate. • Membrane resealing takes minutes.

Ca2 + activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca2 + transients was expectedly higher for nsEP (36 kV/cm) than for msEP (0.09 kV/cm) but the respective doses were similar (190 and 460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca2 + level by > 10 nM in cells with blocked voltage-gated Ca2 + channels and depleted Ca2 + depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These electric field values were at 150–230% of stimulation thresholds for both msEP and nsEP, notwithstanding a 400,000-fold difference in pulse duration. For comparable levels of electroporative Ca2 + uptake, msEP caused at least 10-fold greater uptake of propidium than nsEP, suggesting increased yield of larger pores. Electroporation by msEP started Ca2 + entry abruptly and locally at the electrode-facing poles of cell, followed by a slow diffusion to the center. In a stark contrast, nsEP evoked a “supra-electroporation” pattern of slower but spatially uniform Ca2 + entry. Thus nsEP and msEP had comparable dose efficiency, but differed profoundly in the size and localization of electropores.

We tested if picosecond electric pulses (psEP; 190 kV/cm, 500 ps at 50% height), which are much shorter than channel activation time, can activate voltage-gated (VG) channels. Cytosolic Ca 2 + was monitored by Fura-2 ratiometric imaging in GH3 and NG108 cells (which express multiple types of VG calcium channels, VGCC), and in CHO cells (which express no VGCC). Trains of up to 100 psEP at 1 kHz elicited no response in CHO cells. However, even a single psEP significantly increased Ca 2 + in both GH3 (by 114 ± 48 nM) and NG108 cells (by 6 ± 1.1 nM). Trains of 100 psEP amplified the response to 379 ± 33 nM and 719 ± 315 nM, respectively. Ca 2 + responses peaked within 2–15 s and recovered for over 100 s; they were 80–100% inhibited by verapamil and ω-conotoxin, but not by the substitution of Na + with N-methyl-D-glucamine. There was no response to psEP in Ca 2 + -free medium, but adding external Ca 2 + even 10 s later evoked Ca 2 + response. We conclude that electrical stimuli as short as 500 ps can cause long-lasting opening of VGCC by a mechanism which does not involve conventional electroporation, heating (which was under 0.06 K per psEP), or membrane depolarization by opening of VG Na + channels.

The permeabilized condition of the cell membrane after electroporation can last minutes but the underlying mechanisms remain elusive. Previous studies suggest that lipid peroxidation could be responsible for the lasting leaky state of the membrane. The present study aims to link oxidation within the plasma membrane of live cells to permeabilization by electric pulses. We have introduced a method for the detection of oxidation by ratiometric fluorescence measurements of BODIPY-C11 dye using total internal reflection fluorescence (TIRF) microscopy, limiting the signal to the cell membrane. CHO-K1 cells were cultured on glass coverslips coated with an electroconductive indium tin oxide (ITO) layer, which enabled electroporation with micro- and submicrosecond pulses. No oxidation was observed with the electric field directed towards the ITO (cathode), even at field strengths much higher than that needed for permeabilization. Oxidation was readily detectable with the opposite polarity of pulses, but with the threshold higher than the permeabilization threshold. Moreover, a decrease in the medium conductance had opposite effects on permeabilization and lipid oxidation (it enhanced the former and suppressed the latter). We conclude that lipid oxidation can indeed occur at the plasma membrane after electric pulses, but it is not the cause of lasting membrane permeabilization.

We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca2+. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca2+ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca2+ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells.