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Although transport of molecules into cells via electroporation is a common biomedical procedure, its protocols are often based on trial and error. Despite a long history of theoretical effort, the underlying mechanisms of cell membrane electroporation are not sufficiently elucidated, in part, because of the number of independent fitting parameters needed to link theory to experiment. Here, we ask if the electroporation behavior of a reduced cell membrane is consistent with time-resolved, atomistic, molecular dynamics (MD) simulations of phospholipid bilayers responding to electric fields. To avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 10–50-ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes.

Molecular dynamics (MD) simulations of electrophoretic transport of monovalent ions through field-stabilized electropores in POPC lipid bilayers permit systematic characterization of the conductive properties of lipid nanopores. The radius of the electropore can be controlled by the magnitude of the applied sustaining external electric field, which also drives the transport of ions through the pore. We examined pore conductances for two monovalent salts, NaCl and KCl, at physiological concentrations. Na+conductance is significantly less than K+and Cl–conductance and is a nonlinear function of pore radius over the range of pore radii investigated. The single pore electrical conductance of KCl obtained from MD simulation is comparable to experimental values measured by chronopotentiometry.

In unexcitable, noncardiac cells, ultrashort (nanosecond) high-voltage (megavolt-per-meter) pulsed electrical fields (nsPEF) can mobilize intracellular Ca2+ and create transient nanopores in the plasmalemma. We studied Ca2+ responses to nsPEF in cardiac cells. Fluorescent Ca2+ or voltage signals were recorded from isolated adult rat ventricular myocytes deposited in an electrode microchamber and stimulated with conventional pulses (CPs; 0.5–2.4 kV/cm, 1 ms) or nsPEF (10–80 kV/cm, 4 ns). nsPEF induced Ca2+ transients in 68/104 cells. Repeating nsPEF increased the likelihood of Ca2+ transient induction (61.8% for <10 nsPEF vs. 80.6% for ≥10 nsPEF). Repetitive Ca2+ waves arising at the anodal side and Ca2+ destabilization occurred after repeated nsPEF (12/29) or during steady-state single nsPEF delivery at 2 Hz. Removing extracellular Ca2+ abolished responses to nsPEF. Verapamil did not affect nsPEF-induced Ca2+ transients, but decreased responses to CP. Tetrodotoxin and KB-R7943 increased the repetition threshold in response to nsPEF: 1–20 nsPEF caused local anodal Ca2+ waves without Ca2+ transients, and ≥20 nsPEF caused normal transients. Ryanodine-thapsigargin and caffeine protected against nsPEF-induced Ca2+ waves and showed less recovery of diastolic Ca2+ levels than CP. Voltage recordings demonstrated action potentials triggered by nsPEF, even in the presence of tetrodotoxin. nsPEF can mobilize intracellular Ca2+ in cardiac myocytes by inducing action potentials. Anodal Ca2+ waves and resistance to Na+ and Ca2+ channel blockade suggest nonselective ion channel transport via sarcolemmal nanopores as a triggering mechanism.

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+levels due to Ca2+influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

Reversible electroporation is used to increase the uptake of chemotherapeutic drugs in local tumor treatment (electrochemotherapy) by applying the pulsing protocol (8 rectangular pulses, 1000 V/cm, 100 µs) standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy multicenter trial. Currently, new electrochemotherapy strategies are under development to extend its applicability to tumors with different histology. Electrical parameters and drug type are critical factors. A possible approach is to test pulse parameters different from European Standard Operating Procedure on Electrochemotherapy but with comparable electroporation yield (European Standard Operating Procedure on Electrochemotherapy-equivalent protocols). Moreover, the use of non-toxic drugs combined with electroporation represents the new frontier for electrochemotherapy applications; calcium electroporation has been recently proposed as a simple tool for anticancer therapy. In vitroinvestigations facilitate the optimization of electrical parameters and drugs for in vivoand clinical testing. In this optimization study, new pulsing protocols have been tested by increasing the pulse number and reducing the electric field with respect to the standard. European Standard Operating Procedure on Electrochemotherapy-equivalent protocols have been identified in HL-60 and A431 cancer cell models, and a higher sensitivity in terms of electroporation yield has been recorded in HL-60 cells. Moreover, cell killing efficacy of European Standard Operating Procedure on Electrochemotherapy-equivalent protocols has been demonstrated in the presence of increasing calcium concentrations on both cell lines. Equivalent European Standard Operating Procedure on Electrochemotherapy protocols can be used to optimize the therapeutic effects in the clinic, where different regions of the same cancer tissue, with different electrical properties, might result in a differential electroporation yield of the standard protocol over the same tissue, or, eventually, in an override of the operational limits of the instrument. Moreover, using calcium can help overcome the drawbacks of standard drugs (side effects, high costs, difficult handling, preparation, and storage procedures). These results support the possibility of new treatment options in both standard electrochemotherapy and calcium electroporation, with clear advantages in the clinic.

Nanosecond, megavolt-per-meter electric pulses cause permeabilization of cells to small molecules, programmed cell death (apoptosis) in tumor cells, and are under evaluation as a treatment for skin cancer. We use nanoelectroporation and fluorescence imaging to construct two-dimensional maps of the electric field associated with delivery of 15 ns, 10 kV pulses to monolayers of the human prostate cancer cell line PC3 from three different electrode configurations: single-needle, five-needle, and flat-cut coaxial cable. Influx of the normally impermeant fluorescent dye YO-PRO-1 serves as a sensitive indicator of membrane permeabilization. The level of fluorescence emission after pulse exposure is proportional to the applied electric field strength. Spatial electric field distributions were compared in a plane normal to the center axis and 15-20 μm from the tip of the center electrode. Measurement results agree well with models for the three electrode arrangements evaluated in this study. This live-cell method for measuring a nanosecond pulsed electric field distribution provides an operationally meaningful calibration of electrode designs for biological applications and permits visualization of the relative sensitivities of different cell types to nanoelectropulse stimulation. PACS Codes: 87.85.M-

We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation—the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.