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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.

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.).

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.

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.

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1–TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor–receptor contact.

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.

Electric fields can be a powerful tool to interact with enzymes or proteins, with an intriguing perspective to allow protein manipulation. Recently, researchers have focused the interest on intracellular enzyme modifications triggered by the application of nanosecond pulsed electric fields. These findings were also supported by theoretical predictions from molecular dynamics simulations focussing on significant variations in protein secondary structures. In this work, a theoretical study utilizing molecular dynamics simulations is proposed to explore effects of electric fields of high intensity and very short nanosecond duration applied to the superoxide dismutase (Cu/Zn-SOD or SOD-1), an important enzyme involved in the cellular antioxidant defence mechanism. The effects of 100-nanosecond pulsed electric fields, with intensities ranging from 108 to 7x108 V/m, on a single SOD1 enzyme are presented. We demonstrated that the lowest intensity of 108 V/m, although not inducing structural changes, can produce electrostatic modifications on the reaction centre of the enzyme, as apparent from the dipolar response and the electric field distribution of the protein active site. Electric pulses above 5x108 V/m produced a fast transition between the folded and a partially denatured state, as inferred by the secondary structures analysis. Finally, for the highest field intensity used (7x108 V/m), a not reversible transition toward an unfolded state was observed.

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.

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.

Cytosolic DNA sensors are a subgroup of pattern recognition receptors (PRRs) and are activated by the abnormal presence of the DNA in the cytosol. Their activation leads to the upregulation of pro-inflammatory cytokines and chemokines and can also induce cell death. The presence of cytosolic DNA sensors and inflammatory cytokines in TS/A murine mammary adenocarcinoma and WEHI 164 fibrosarcoma cells was demonstrated using real time reverse transcription polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbent assay (ELISA). After electrotransfer of plasmid DNA (pDNA) using two pulse protocols, the upregulation of DNA-depended activator of interferon regulatory factor or Z-DNA binding protein 1 (DAI/ZBP1), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60) and interferon-inducible protein 204 (p204) mRNAs was observed in both tumor cell lines, but their expression was pulse protocol dependent. A decrease in cell survival was also observed; it was cell type, DNA concentration and pulse protocol dependent. Furthermore, the different protocols of electrotransfer led to different cell death outcomes, necrosis and apoptosis, as indicated by an annexin V and 7AAD assays. The obtained data provide new insights on the presence of cytosolic DNA sensors in tumor cells and the activation of different types of cells death after electrotransfer of pDNA. These observations have important implications on the planning of gene therapy or DNA vaccination protocols.

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.

Experiments with CHO cells exposed to 60 and 300 ns pulsed electric fields with amplitudes in the range from several kV/cm to tens of kV/cm, showed a decrease of the uptake of calcium ions by more than an order of magnitude when, immediately after a first pulse, a second one of opposite polarity was applied. This effect is assumed to be due to the reversal of the electrophoretic transport of ions through the electroporated membrane during the second phase of the bipolar pulse. This assumption, however, is only valid if electrophoresis is the dominant transport mechanism, rather than diffusion. Comparison of calculated calcium ion currents with experimental results showed that for nanosecond pulses, electrophoresis is at least as important as diffusion. By delaying the second pulse with respect to the first one, the effect of reverse electrophoresis is reduced. Consequently, separating nanosecond pulses of opposite polarity by up to approximately hundred microseconds allows us to vary the uptake of ions from very small values to that obtained with two pulses of the same polarity. The measured calcium ion uptake obtained with bipolar pulses also allowed us to determine the membrane pore recovery time. The calculated recovery time constants are on the order of ten microseconds.

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.

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.

We demonstrate that conditioning of mammalian cells by electroporation with nanosecond pulsed electric field (nsPEF) facilitates their response to the next nsPEF treatment. The experiments were designed to unambiguously separate the electroporation-induced sensitization and desensitization effects. Electroporation was achieved by bursts of 300-ns, 9 kV/cm pulses (50 Hz, n = 3–100) and quantified by propidium dye uptake within 11 min after the nsPEF exposure. We observed either sensitization to nsPEF or no change (when the conditioning was either too weak or too intense, or when the wait time after conditioning was too short). Within studied limits, conditioning never caused desensitization. With settings optimal for sensitization, the second nsPEF treatment became 2.5 times (25 °C) or even 6 times (37 °C) more effective than the same nsPEF treatment delivered without conditioning. The minimum wait time required for sensitization development was 30 s, with still longer delays increasing the effect. We show that the delayed hypersensitivity was not mediated by either cell swelling or oxidative effect of the conditioning treatment; biological mechanisms underlying the delayed electrosensitization remain to be elucidated. Optimizing nsPEF delivery protocols to induce sensitization can reduce the dose and adverse side effects of diverse medical treatments which require multiple pulse applications.

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca(2+) after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca(2+) level from the nominal 2-5μM to 2mM for the first 60-90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca(2+), the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca(2+) in the medium. With the nominal Ca(2+), most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca(2+), the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca(2+)-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.

Summary Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591-amino-acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modelling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca2+ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca2+ imaging with Fura-2. In a Ca(2+)-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2+ transients by mobilization of Ca2+ from the endoplasmic reticulum. With 2 mM external Ca2+, the transients included both extra- and intracellular components. The recruitment of intracellular Ca2+ increased as the stimulus duration decreased. At the threshold of 200-300 nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca2+ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca2+ can be controlled by the duration of the stimulus.

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-μs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-μs EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization.

Electroporation by nanosecond electric pulses (nsEP) is an emerging modality for tumor ablation. Here we show the efficient induction of apoptosis even by a non-toxic nsEP exposure when it is followed by a 30-min chilling on ice. This chilling itself had no impact on the survival of U-937 or HPAF-II cells, but caused more than 75% lethality in nsEP-treated cells (300 ns, 1.8-7 kV/cm, 50-700 pulses). The cell death was largely delayed by 5-23 hr and was accompanied by a 5-fold activation of caspase 3/7 (compared to nsEP without chilling) and more than 60% cleavage of poly-ADP ribose polymerase (compared to less than 5% in controls or after nsEP or chilling applied separately). When nsEP caused a transient permeabilization of 83% of cells to propidium iodide, cells placed at 37 °C resealed in 10 min, whereas 60% of cells placed on ice remained propidium-permeable even in 30 min. The delayed membrane resealing caused cell swelling, which could be blocked by an isosmotic addition of a pore-impermeable solute (sucrose). However, the block of swelling did not prevent the delayed cell death by apoptosis. The potent enhancement of nsEP cytotoxicity by subsequent non-damaging chilling may find applications in tumor ablation therapies.

Previous studies reported a delayed increase of sensitivity to electroporation (termed “electrosensitization”) in mammalian cells that had been subjected to electroporation. Electrosensitization facilitated membrane permeabilization and reduced survival in cell suspensions when the electric pulse treatments were split in fractions. The present study was aimed to visualize the effect of sensitization and establish its utility for cell ablation. We used KLN 205 squamous carcinoma cells embedded in an agarose gel and cell spheroids in Matrigel. A local ablation was created by a train of 200 to 600 of 300-ns pulses (50 Hz, 300–600 V) delivered by a two-needle probe with 1-mm inter-electrode distance. In order to facilitate ablation by engaging electrosensitization, the train was split in two identical fractions applied with a 2- to 480-s interval. At 400–600 V (2.9–4.3 kV/cm), the split-dose treatments increased the ablation volume and cell death up to 2–3-fold compared to single-train treatments. Under the conditions tested, the maximum enhancement of ablation was achieved when two fractions were separated by 100 s. The results suggest that engaging electrosensitization may assist in vivo cancer ablation by reducing the voltage or number of pulses required, or by enabling larger inter-electrode distances without losing the ablation efficiency.

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1-13 kV/cm) was assessed by oxidation of 2',7'-dichlorodihydrofluoresein (H(2)DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H(2)DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2',7'-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H(2)DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H(2)O(2) in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H(2)O(2) and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.10-ns EP caused apoptotic or necrotic death within 2-20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S=alphaD((-K)), where coefficients K and alpha determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas alpha was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms.Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

The exclusion of polar dyes by healthy cells is widely employed as a simple and reliable test for cell membrane integrity. However, commonly used dyes (propidium, Yo-Pro-1, trypan blue) cannot detect membrane defects which are smaller than the dye molecule itself, such as nanopores that form by exposure to ultrashort electric pulses (USEPs). Instead, here we demonstrate that opening of nanopores can be efficiently detected and studied by fluorescent measurement of Tl(+) uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl(+)-sensitive fluorophore FluxOR and subjected to USEPs in a Tl(+)-containing bath buffer, displayed an immediate (within100 ms), dose-dependent surge of fluorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl(+) by 600-ns USEP was at 1-2 kV/cm, and the rate of Tl(+) uptake increased linearly with increasing the electric field. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 1-1.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca(2+) did not block the USEP effect. Addition of a Tl(+)-containing buffer within less than 10 min after USEP also caused a fluorescence surge, which confirms the minutes-long lifetime of nanopores. Overall, the technique of fluorescent detection of Tl(+) uptake proved highly effective, noninvasive and sensitive for visualization and analysis of membrane defects which are too small for conventional dye uptake detection methods.

Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltage-sensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is approximately 1nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated.

Significance The airway pathogen Mycoplasma pneumoniae ( Mp ) produces a virulence factor with ADP-ribosyltransferase and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Mammalian cells exposed to recombinant CARDS TX exhibit distinct patterns of ADP-ribosylated proteins and unusual vacuolation phenotypes with disruption of monolayer integrity, leading to cell death. Recombinant CARDS TX administered to naive mice induces an allergic-type inflammatory response and airway hyperreactivity, suggesting that an analogous response in humans may play a causal role in Mp -associated asthma. Here we present the structure of CARDS TX, the newest member of the well-recognized group of ADP-ribosylating toxins. The structure enhances understanding of Mp pathogenicity and provides a platform for the development of therapies to treat Mp -associated acute and chronic airway diseases.

Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591-amino-acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modelling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.

Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca(2+) mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and amplitudes (15-60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-µs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.

Community-acquired respiratory distress syndrome toxin (CARDS TX) is a 591-amino-acid protein with ADP-ribosyltransferase and vacuolating activities that damages the cells lining the respiratory tracts of patients infected with the bacterial pathogen Mycoplasma pneumoniae. Crystals of CARDS TX were grown in space group C2, with unit-cell parameters a = 191.4, b = 107.4, c = 222.1 A, beta = 90.6 degrees. A complete 2.2 A data set was obtained from a single CARDS TX crystal.

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.

Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms.

In this study, we determined the LD(50) (50% lethal dose) for cell death, and the ED(50) (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD(50) varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD(50) lower than the ED(50) for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

Chemical and physical insults such as electroporation may compromise the membrane integrity and allow leak ion currents through de-novo formed lipid pores. These pores are thought to short-circuit the membrane and hinder ion channels' physiological function rather than to complement it. However, here we report that brief electric stimuli can trigger formation of membrane pores with specific behaviors that are traditionally considered to be unique for protein ion channels; still other behaviors of these pores distinguish them from both “conventional” electropores and any known ion channels. We found that a single electric shock (600-ns duration, 1 to 5 kV/cm) causes minutes-long increase of membrane electrical conductance due to formation of long-lived, voltage- and current-sensitive, rectifying, cation-selective, asymmetrical pores of nanometer diameter (“nanopores”). Once induced, nanopores oscillate between open and quasi-open (electrically silent) states, followed by either gradual resealing or abrupt breakdown into larger pores, with immediate loss of nanopore-specific behaviors. The formation and extended lifetime of nanopores were verified by non-electrophysiological methods, namely by fluorescent detection of Tl+ uptake and of phosphatidylserine externalization. Apparently, nanopores are not unique to cell stimulation with nanosecond electric pulses, but may form under various physiological and pathological conditions. Nanopores appear adequately equipped for certain functions that are traditionally ascribed to ion channels. Clear distinction between nanopores- and ion channels-mediated currents may be critical for understanding how these currents are controlled.Supported by NIH (NCI) R01CA125482.

The signal recognition particle (SRP) RNA helix 6 of archaea and eukaryotes is essential for the binding of protein SRP19 and the assembly of a functional complex. The conserved adenosine at the third position of the tetraloop of helix 6 (A149) is crucial for the binding of protein SRP19 in the mammalian SRP. Here we investigated the significance of the equivalent adenosine residue at position 159 (A159) of Archaeoglobus fulgidus SRP RNA. The A159 of A. fulgidus and A149 of human SRP RNA were changed to C, G or U, and fragments containing helix 6 or helices 6 and 8 were synthesized by run-off transcription with T7 RNA polymerase. The ability of recombinant A. fulgidus and human SRP19 to form ribonucleoprotein complexes was measured in vitro. The simultaneous presence of A149 and helix 8 is required for the high-affinity binding of SRP19 to the human SRP RNA. In contrast, A. fulgidus SRP19 binds to the SRP RNA fragments with high affinity irrespective of the nature of the nucleotide, demonstrating that A159 does not directly participate in protein binding. Instead, as indicated by the resistance of the wild-type A. fulgidus RNA towards digestion by RNase A, this residue allows the formation of a tightly folded RNA molecule. The high affinity between A. fulgidus SRP19 and RNA molecules that contain both helices 6 and 8 suggests that A159 is likely to initiate archaeal SRP assembly by forming a conserved tertiary RNA– RNA interaction.

Background Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. Methodology/Principal Findings We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1–500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1–5 min delay enhanced the effects severalfold. Conclusions/Significance For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency.

Olga N. Pakhomovaa, Yumin Cuia, Christian Zwiebb & Andrew P. Hincka,∗ aDepartment of Biochemistry, Center for Biomolecular Structure Analysis, Allied Health Building/Biochemistry 5.206, University of Texas Health Science Center at San Antonio, MC 7760, 7730 Floyd Curl Drive, San Antonio, TX 78229, U.S.A. bDepartment of Molecular Biology and Biochemistry, The University of Texas Health Science Center at Tyler, Tyler, TX 75710, U.S.A.