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

Oxidative Effects of Nanosecond Pulsed Electric Field Exposure in Cells and Cell-Free Media

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 (H2DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H2DCF-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 H2DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H2O2 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 H2O2 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

Electroporation-Induced Electrosensitization

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

Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media

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 20,70-dichlorodihydrofluoresein (H2DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H2DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 20,70-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 H2DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H2O2 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 H2O2 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 damageBiologijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Enhancement of the EP cytotoxic effect by exposure fractionation.

<p>U937 cells were exposed 0.3-µs EPs; the pulse number, amplitude, and delivery mode, are indicated in the figure. Cell survival was measured as a percentage of propidium-excluding cells at 4 hr post exposure (mean +/− s.e., n = 3–7). Survival in sham-exposed samples was over 95% (data not shown). <b><i>A</i></b>: Exposure to 150 pulses was significantly more effective at 1 Hz than at 1,000 Hz. However, splitting the 1,000-Hz train in two fractions of 75 pulses each, with a 150-s interval, made it as efficient as the 1-Hz treatment. <b><i>B</i></b>: Splitting of a single high-rate train in two same size fractions with 150-s interval enhanced the effect EP of 4.5 and 3 kV/cm EPs, but not at the lower EP amplitude of 1.8 kV/cm.</p

The role of fraction size in the enhancement of the EP effect by fractionation.

<p>Survival of U937 cells was determined by propidium exclusion at 4 hr following exposure to 0.3 µs EPs at either 4.5 kV/cm (top graph) or 9 kV/cm. The total number of 600 pulses was split into two fractions which were delivered with a 6-min interval. The number of pulses in the first train varied form 1% to 100% of the total. The latter value corresponded to delivering all pulses in a single train, and the respective survival levels are shown by shaded areas. Mean+/− s.e., n = 4–6. Solid lines are best fit approximations using second degree polynomial function. Dashed lines delimit the borders of 95% confidence intervals for the best fit.</p

Effective reduction of the lethal dose (LD) by exposure fractionation.

<p>U937 cells were exposed to 100 (left) or 200 (right) of 0.3-µs duration EPs. The exposure was delivered either as a single train or as two equal fractions (50+50 and 100+100) with a 5-min interval. EPs were applied at different E-fields amplitudes (values are given above the abscissa), resulting in different absorbed doses. The graphs show cell survival (mean+/− s.e., n = 3–6) versus the dose for different EP treatments. Dashed lines are the best fit data approximations using exponential function; shaded areas denote 95% confidence intervals. Cell survival was measured by propidium exclusion at 4 hr post exposure. Legends show LD values for elimination of 50% and 90% of cells (LD₅₀ and LD₉₀) by the tested exposure protocols.</p

The effect of the pulse repetition rate (left column) and of the total duration of the treatment (right column) on cell survival.

<p>Each plot (except those at the bottom) represents a separate series of experiments where cells were exposed to a fixed number of pulses of a given amplitude and duration (see legends within the figure; e.g., for the top plot the legend means “500 pulses of 0.3 µs duration at 4.5 kV/cm). The only variable in each series was the pulse repetition rate and, consequentially, the total duration of the treatment. Other data in the legends are the cell type and the timepoint after exposure when the cell survival was measured. At 4 hr, cell survival was measured using dye exclusion/quenching method; at 24 hr, it was measured using MTT assay (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017100#s4" target="_blank">Methods</a>” for detail). Each datapoint is the mean +/− s.e for 3–12 independent experiments. For the bottom plots, the curves from all series of experiments were collapsed together; shown are only the connecting lines; the mean value symbols and error bars have been omitted for clarity. See text for more detail.</p