Electroporation-induced electrosensitization

Abstract

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.

Figure 1. The effect of the pulse repetition rate (left column) and of the total duration of the treatment (right column) on cell survival.

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 “Methods” 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.

Figure 2. Enhancement of the EP cytotoxic effect by exposure fractionation.

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). A: 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: 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.

Figure 3. The role of fraction size in the enhancement of the EP effect by fractionation.

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.

Figure 4. Two distinct modes of propidium uptake in EP-exposed cells.

A group of three CHO cells attached to a coverslip was exposed to an EP train at 50 s into the experiment. Exposure parameters are indicated in the figure. Insets show differential-interference contrast and fluorescent images of cells at selected timepoints. Scale bars: 10 µm. The images were taken every 10 s throughout the experiment. The graph shows the time dynamics of Pr uptake by cells 1, 2 and 3. Note the immediate increase in Pr fluorescence in all three cells, caused by electropore opening and transient Pr uptake. Following the transient uptake, the intensity of fluorescence displayed little changes until membrane rupture and massive Pr entry in cells 1 and 2, but not in cell 3.

Figure 5. The effect of PRF (A, B) and exposure fractionation (B) on the incidence of delayed membrane rupture in EP-exposed CHO cells.

A and B are two separate and independent series of experiments; within each series, different exposure regimens were alternated in random order. Membrane integrity was probed by Pr uptake; each curve corresponds to Pr uptake in an individual exposed cell, as measured by cell imaging every 10 s throughout the experiment. EP exposure caused transient Pr uptake due to electroporation in all cells and delayed/accelerated Pr uptake due to secondary membrane rupture in some cells (see Fig. 4 and text for more detail). For clarity, for each type of treatment, cells that showed only transient Pr uptake (blue curves, bottom graphs) are separated from cells that showed both transient and delayed Pr uptake (red curves, top graphs). The number of cells that fell into each of the two categories is shown to the right of the graphs. In all groups, cells were exposed to 100, 60-ns pulses at 13.3 kV/cm, whereas the PRF and pulse delivery protocols varied (see legends in the figure). The legends also give the percent of ruptured cells in each group. Note the increase of EP exposure efficiency at lower PRF (A and B) and also when the 20-Hz train was split in two fractions (B). Single-train 20- and 0.2-Hz exposures in A and B are identical treatments, although in B both showed somewhat higher efficiency.

Figure 6. Effective reduction of the lethal dose (LD) by exposure fractionation.

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.