Delayed hypersensitivity to nanosecond pulsed electric field in electroporated cells

Abstract

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.

Figure 1

Different effects of conditioning in case of transient effects (A–D) and long-lasting cumulative effects (E–H). The top row cartoons (A and E) show the hypothetical effects of a stimulus applied without conditioning. In other cartoons, the test stimulus (TS) is preceded by a conditioning stimulus (CS). The CS is shown smaller than TS, but it could likewise be made larger or the same as TS. The measured endpoint is the amplitude of the response to TS when it is applied alone or preceded by CS. For easier comparison, the response to TS alone is copied to other panels and shown by a dotted line. Dashed line in panels (F–H) shows the expected development of the CS effect when no TS were applied. In these examples, conditioning had no effect (B,F); caused desensitization (C,G); or caused sensitization (D,H).

Figure 2

The experiment design and timeline (A) and measured endpoints (B). A: After aliquoting cell suspension into four electroporation cuvettes (C1-C4) they were placed in a waterbath kept at either 25 or 37 °C. In 4 min, C2 and C3 were subjected to conditioning stimulation (CS) and returned to the waterbath. After a wait time, which was varied from 0 to 8 min, C3 and C4 were subjected to test stimulation (TS) and returned to the waterbath. In 11 min, all suspensions were diluted and processed for measuring propidium (PR) emission in 15 min. (B) The expected time course of PR uptake in C1-C4. PR uptake in conditioned and naïve samples (PR_TS and PR_TS0, respectively) was measured as the emission difference between C3 and C2, and between C4 and C1.

Figure 3

The effect of pulse number in the conditioning stimulation train (A) or in the test stimulation train (B) on the conditioning index (CI, %). The interval between CS and TS was 2 min and the temperature was 25 °C. 300-ns wide, 9 kV/cm pulses were delivered at 50 Hz. The test train was always 12 pulses (A), or the conditioning train was always 12 pulses (B). Mean ± s.e.m., n = 4. CI = 100% corresponds to no effect of conditioning and CI > 100% signifies sensitization (*p < 0.05, **p < 0.01, one-sample t-test for the difference from 100%). ^#p < 0.05, ^##p < 0.01 for the difference between the groups using two-tailed t-test.

Figure 4

The effect of the pulse number per train on the conditioning index (CI, %). The number of pulses was varied concurrently in both the conditioning and the test trains. The wait interval between the trains was 2 min; the incubation temperature was set to either 25 °C or 37 °C (see legends). Mean ± s.e.m., n = 3. *p < 0.05, one-sample t-test for the difference from 100%. See text and Fig. 3 for more details.

Figure 5

Split dose nsPEF delivery is more efficient for membrane permeabilization than a single dose. Propidium (PR) emission, as measured in 17 min after the first nsPEF treatment, is plotted against the total number of pulses. For the split dose treatment, half of the pulses were delivered by each train; the interval between the trains was 2 min. The incubation temperature was 37 °C. Mean ± s.e.m., n = 3. ^#p < 0.05, ^##p < 0.01 for the difference between the two conditions using two-tailed t-test. Error bars may be not visible when they are smaller than the central symbol. See text and Figs 3 and 4 for more details.

Figure 6

Increasing the wait time after the conditioning nsPEF exposures increases sensitization to the second nsPEF exposure. The conditioning index (CI, %) was plotted against the wait time interval, s. Both the conditioning and test trains consisted of 12 pulses (300-ns wide, 9 kV/cm, 50 Hz). The incubation temperature was 25 °C. Mean ± s.e.m., n = 3. *p < 0.05, **p < 0.01, one-sample t-test for the difference from 100%.

Figure 7

Electrosensitization is not mediated by cell swelling. (A) Histograms of cell diameter distribution at 2 min after a sham exposure (solid gray bars) or permeabilization with 12 pulses (300-ns, 9 kV/cm, 50 Hz; semi-transparent colored bars). Dashed and solid lines are the normal (Gaussian) distribution best fits for sham and nsPEF exposure data, respectively. Note diameter increase in RPMI-0 medium (left panel) and its blockage in the presence of sucrose (right). (B) Cells in both media displayed strong sensitization, as manifested by the CI of about 200%; mean +/− s.e., n = 4, *p < 0.05, one-sample t-test for the difference from 100%. The interval between conditioning and test treatments (each of 12 nsPEF) was 2 min, at 25 °C. (C) Split-dose nsPEF delivery protocol (12 pulses + 12 pulses in 2 min) was more efficient than a single dose of 24 pulses in both types of media. Mean ± s.e.m., n = 5. ^##p < 0.01 with a two-tailed t-test.

Figure 8

Electrosensitization is not prevented by Trolox. (A) 100 µM of Trolox was most efficient to prevent cell loss from incubation with 100 µM cumene hydroperoxide for 2 hours. Cell survival was measured 24 hr later and expressed in % to vehicle-treated parallel controls. Mean +/− s.e., n = 4. See text for more details. (B) Trolox did not prevent sensitization, as manifested by the CI of over 200%; mean +/− s.e., n = 3, **p < 0.01, one-sample t-test for the difference from 100%. The interval between conditioning and test treatments (each of 12 nsPEF, 300-ns, 9 kV/cm, 50 Hz) was 2 min, at 25 °C. (C) Trolox did not change the higher efficiency of a split-dose nsPEF delivery (12 pulses + 12 pulses in 2 min) compared to a single dose of 24 pulses. Mean ± s.e.m., n = 4, ^##p < 0.01 with a two-tailed t-test.