Inflammasome Activation and IL-1ß Release Triggered by Nanosecond Pulsed Electric Fields in Murine Innate Immune Cells and Skin.

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

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.

Modulation of biological responses to 2 ns electrical stimuli by field reversal.

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

Transport of charged small molecules after electropermeabilization - drift and diffusion.

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

Asymmetric Patterns of Small Molecule Transport After Nanosecond and Microsecond Electropermeabilization.

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

Quantitative Limits on Small Molecule Transport via the Electropermeome - Measuring and Modeling Single Nanosecond Perturbations.

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

Nanometer-Scale Permeabilization and Osmotic Swelling Induced by 5-ns Pulsed Electric Fields.

High-intensity nanosecond pulsed electric fields (nsPEFs) permeabilize cell membranes. Although progress has been made toward an understanding of the mechanism of nsPEF-induced membrane poration, the dependence of pore size and distribution on pulse duration, strength, number, and repetition rate remains poorly defined experimentally. In this paper, we characterize the size of nsPEF-induced pores in living cell membranes by isosmotically replacing the solutes in pulsing media with polyethylene glycols and sugars before exposing Jurkat T lymphoblasts to 5 ns, 10 MV/m electric pulses. Pore size was evaluated by analyzing cell volume changes resulting from the permeation of osmolytes through the plasma membrane. We find that pores created by 5 ns pulses have a diameter between 0.7 and 0.9 nm at pulse counts up to 100 with a repetition rate of 1 kHz. For larger number of pulses, either the pore diameter or the number of pores created, or both, increase with increasing pulse counts. But the prevention of cell swelling by PEG 1000 even after 2000 pulses suggests that 5 ns, 10 MV/m pulses cannot produce pores with a diameter larger than 1.9 nm.

Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength.

BACKGROUND: Electroporation, a method for increasing the permeability of membranes to ions and small molecules, is used in the clinic with chemotherapeutic drugs for cancer treatment (electrochemotherapy). Electroporation with calcium causes ATP (adenosine triphosphate) depletion and cancer cell death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. METHODS: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability was determined after treatment with 1, 3, or 5 mM calcium and eight 99 µs pulses with 0.8, 1.0, 1.2, 1.4 or 1.6 kV/cm. Fitting analysis was applied to quantify the cell-killing efficacy in presence of calcium. Post-treatment intracellular ATP was measured in H69 and SW780 cells. Post-treatment intracellular ATP was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. RESULTS: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p<0.05) and reduced viability. The 50% effective cell kill was found at 3.71 kV/cm (H69) and 3.28 kV/cm (SW780), reduced to 1.40 and 1.15 kV/cm (respectively) with 1 mM calcium (lower EC50 for higher calcium concentrations). Quinacrine fluorescence intensity of calcium-electroporated U937 cells was one third lower than in controls (p<0.0001). CONCLUSIONS: Calcium electroporation dose-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field. GENERAL SIGNIFICANCE: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials.