Silencing of ATP1A1 attenuates cell membrane disruption by nanosecond electric pulses.

This study explored the role of the Na/K-ATPase (NKA) in membrane permeabilization induced by nanosecond electric pulses. Using CRISPR/Cas9 and shRNA, we silenced the ATP1A1 gene, which encodes α1 NKA subunit in U937 human monocytes. Silencing reduced the rate and the cumulative uptake of YoPro-1 dye after electroporation by 300-ns, 7-10 kV/cm pulses, while ouabain, a specific NKA inhibitor, enhanced YoPro-1 entry. We conclude that the α1 subunit supports the electropermeabilized membrane state, by forming or stabilizing electropores or by hindering repair mechanisms, and this role is independent of NKA's ion pump function.

Urine protects urothelial cells against killing with nanosecond pulsed electric fields.

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.

Ca2+ dependence and kinetics of cell membrane repair after electropermeabilization.

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.

How to alleviate cardiac injury from electric shocks at the cellular level.

Electric shocks, the only effective therapy for ventricular fibrillation, also electroporate cardiac cells and contribute to the high-mortality post-cardiac arrest syndrome. Copolymers such as Poloxamer 188 (P188) are known to preserve the membrane integrity and viability of electroporated cells, but their utility against cardiac injury from cardiopulmonary resuscitation (CPR) remains to be established. We studied the time course of cell killing, mechanisms of cell death, and protection with P188 in AC16 human cardiomyocytes exposed to micro- or nanosecond pulsed electric field (µsPEF and nsPEF) shocks. A 3D printer was customized with an electrode holder to precisely position electrodes orthogonal to a cell monolayer in a nanofiber multiwell plate. Trains of nsPEF shocks (200, 300-ns pulses at 1.74 kV) or µsPEF shocks (20, 100-µs pulses at 300 V) produced a non-uniform electric field enabling efficient measurements of the lethal effect in a wide range of the electric field strength. Cell viability and caspase 3/7 expression were measured by fluorescent microscopy 2-24 h after the treatment. nsPEF shocks caused little or no caspase 3/7 activation; most of the lethally injured cells were permeable to propidium dye already at 2 h after the exposure. In contrast, µsPEF shocks caused strong activation of caspase 3/7 at 2 h and the number of dead cells grew up to 24 h, indicating the prevalence of the apoptotic death pathway. P188 at 0.2-1% reduced cell death, suggesting its potential utility in vivo to alleviate electric injury from defibrillation.

The role of ESCRT-III and Annexin V in the repair of cell membrane permeabilization by the nanosecond pulsed electric field.

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 interplay of excitation and electroporation in nanosecond pulse stimulation.

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.

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions.

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 <1 ms. The threshold for one 300-ns pulse was at 1.5-2 kV/cm, much lower than >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.

Electropermeabilization does not correlate with plasma membrane lipid oxidation.

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.

Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer.

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.

Excitation and electroporation by MHz bursts of nanosecond stimuli.

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.

Mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors.

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.

Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field.

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.

Excitation and injury of adult ventricular cardiomyocytes by nano- to millisecond electric shocks.

Intense electric shocks of nanosecond (ns) duration can become a new modality for more efficient but safer defibrillation. We extended strength-duration curves for excitation of cardiomyocytes down to 200 ns, and compared electroporative damage by proportionally more intense shocks of different duration. Enzymatically isolated murine, rabbit, and swine adult ventricular cardiomyocytes (VCM) were loaded with a Ca2+ indicator Fluo-4 or Fluo-5N and subjected to shocks of increasing amplitude until a Ca2+ transient was optically detected. Then, the voltage was increased 5-fold, and the electric cell injury was quantified by the uptake of a membrane permeability marker dye, propidium iodide. We established that: (1) Stimuli down to 200-ns duration can elicit Ca2+ transients, although repeated ns shocks often evoke abnormal responses, (2) Stimulation thresholds expectedly increase as the shock duration decreases, similarly for VCMs from different species, (3) Stimulation threshold energy is minimal for the shortest shocks, (4) VCM orientation with respect to the electric field does not affect the threshold for ns shocks, and (5) The shortest shocks cause the least electroporation injury. These findings support further exploration of ns defibrillation, although abnormal response patterns to repetitive ns stimuli are of a concern and require mechanistic analysis.

Electropermeabilization by uni- or bipolar nanosecond electric pulses: The impact of extracellular conductivity.

Cellular effects caused by nanosecond electric pulses (nsEP) can be reduced by an electric field reversal, a phenomenon known as bipolar cancellation. The reason for this cancellation effect remains unknown. We hypothesized that assisted membrane discharge is the mechanism for bipolar cancellation. CHO-K1 cells bathed in high (16.1mS/cm; HCS) or low (1.8mS/cm; LCS) conductivity solutions were exposed to either one unipolar (300-ns) or two opposite polarity (300+300-ns; bipolar) nsEP (4-40kV/cm) with increasing interpulse intervals (0.1-50µs). Time-lapse YO-PRO-1 (YP) uptake revealed enhanced membrane permeabilization in LCS compared to HCS at all tested voltages. The time-dependence of bipolar cancellation was similar in both solutions, using either identical (22kV/cm) or isoeffective nsEP treatments (12 and 32kV/cm for LCS and HCS, respectively). However, cancellation was significantly stronger in LCS when the bipolar nsEP had no, or very short (<1µs), interpulse intervals. Finally, bipolar cancellation was still present with interpulse intervals as long as 50µs, beyond the time expected for membrane discharge. Our findings do not support assisted membrane discharge as the mechanism for bipolar cancellation. Instead they exemplify the sustained action of nsEP that can be reversed long after the initial stimulus.

Activation of the phospholipid scramblase TMEM16F by nanosecond pulsed electric fields (nsPEF) facilitates its diverse cytophysiological effects.

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.

Delayed hypersensitivity to nanosecond pulsed electric field in electroporated cells.

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.

Effect of Cooling On Cell Volume and Viability After Nanoelectroporation.

Electric pulses of nanosecond duration (nsEP) are emerging as a new modality for tissue ablation. Plasma membrane permeabilization by nsEP may cause osmotic imbalance, water uptake, cell swelling, and eventual membrane rupture. The present study was aimed to increase the cytotoxicity of nsEP by fostering water uptake and cell swelling. This aim was accomplished by lowering temperature after nsEP application, which delayed the membrane resealing and/or suppressed the cell volume mechanisms. The cell diameter in U-937 monocytes exposed to a train of 50, 300-ns pulses (100 Hz, 7 kV/cm) at room temperature and then incubated on ice for 30 min increased by 5.6 +/- 0.7 µm (40-50%), which contrasted little or no changes (1 +/- 0.3 µm, <10%) if the incubation was at 37 °C. Neither this nsEP dose nor the 30-min cooling caused cell death when applied separately; however, their combination reduced cell survival to about 60% in 1.5-3 h. Isosmotic addition of a pore-impermeable solute (sucrose) to the extracellular medium blocked cell swelling and rescued the cells, thereby pointing to swelling as a primary cause of membrane rupture and cell death. Cooling after nsEP exposure can potentially be employed in medical practice to assist tissue and tumor ablation.

Selective susceptibility to nanosecond pulsed electric field (nsPEF) across different human cell types.

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 LD50 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.

The cytotoxic synergy of nanosecond electric pulses and low temperature leads to apoptosis.

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.

Cell Electrosensitization Exists Only in Certain Electroporation Buffers.

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.