Ultra-Low Intensity Post-Pulse Affects Cellular Responses Caused by Nanosecond Pulsed Electric Fields

Abstract

High-intensity nanosecond pulse electric fields (nsPEF) can preferentially induce various effects, most notably regulated cell death and tumor elimination. These effects have almost exclusively been shown to be associated with nsPEF waveforms defined by pulse duration, rise time, amplitude (electric field), and pulse number. Other factors, such as low-intensity post-pulse waveform, have been completely overlooked. In this study, we show that post-pulse waveforms can alter the cell responses produced by the primary pulse waveform and can even elicit unique cellular responses, despite the primary pulse waveform being nearly identical. We employed two commonly used pulse generator designs, namely the Blumlein line (BL) and the pulse forming line (PFL), both featuring nearly identical 100 ns pulse durations, to investigate various cellular effects. Although the primary pulse waveforms were nearly identical in electric field and frequency distribution, the post-pulses differed between the two designs. The BL's post-pulse was relatively long-lasting (~50 µs) and had an opposite polarity to the main pulse, whereas the PFL's post-pulse was much shorter (~2 µs) and had the same polarity as the main pulse. Both post-pulse amplitudes were less than 5% of the main pulse, but the different post-pulses caused distinctly different cellular responses. The thresholds for dissipation of the mitochondrial membrane potential, loss of viability, and increase in plasma membrane PI permeability all occurred at lower pulsing numbers for the PFL than the BL, while mitochondrial reactive oxygen species generation occurred at similar pulsing numbers for both pulser designs. The PFL decreased spare respiratory capacity (SRC), whereas the BL increased SRC. Only the PFL caused a biphasic effect on trans-plasma membrane electron transport (tPMET). These studies demonstrate, for the first time, that conditions resulting from low post-pulse intensity charging have a significant impact on cell responses and should be considered when comparing the results from similar pulse waveforms.

Keywords: charging current; intracellular effects; nanosecond pulse; post-pulse; spare respiratory capacity.

Figure 1

The waveforms were generated using two pulse generators: the Blumlein line (BL) and the pulse forming line (PFL). (a): The BL exhibits an opposite polarity post-pulse compared to the main pulse, whereas the PFL has a post-pulse with the same polarity. In the figure, the BL pulse is intentionally inverted to match the text, although it should be positive due to the negative charging power supply concerning the ground. The grey arrow: is pulse current; The orange arrow: post pulse current. (b): The waveforms display a voltage increase from −1.5 kV to −5 kV. Each waveform represents the average of 30 consecutive waveforms. (c): The charges delivered to the load are calculated by integrating the current over time. In this case, the load was a cuvette. (d): The energy deposited into the load.

Figure 2

The pulse waveforms were analyzed in both the time and frequency domains. (a) The intervals of interest in the waveform, including the prepulse (−450 ns to −100 ns), main pulse (−100 ns to 500 ns), and post-pulse (500 ns to 1600 ns); the spectrum of the pulses was calculated for each interval using FFT: (b) the prepulse; (c) the main pulse; and (d) the post-pulse; (e,f) zoomed-in views of the post-pulses for both PFL and BL on a smaller voltage and longer time scale.

Figure 3

The spectrums of the pulses over time were calculated for the PFL and BL waveforms using STFT performed on the data shown in Figure 1b. Top row: the PFL voltages (−1.5 kV to −5 kV); bottom row: the BL voltages (−1.5 kV to −5 kV). The color bars show the magnitude of the spectrum.

Figure 4

The potential drops were simulated using a linear equivalent cell model by applying a clean pulse (CP), a PFL pulse, and a BL pulse at 1 μs. The potential between the outer membrane (OMP) and the potential between an intracellular organelle (e.g., mitochondrion) (IMP) are shown in (a) on a larger scale (both in voltage and time) and (b) on a smaller scale. (c) The equivalent cell model in Pspice (Version 9.1) along with the parameters (R_ext= 1 kΩ, C_om = 100 pF, R_cyt2 = 100 Ω, C_im = 10 pF, R_cyt = 10 kΩ) [33].

Figure 5

nsPEF effects of BL and PFL pulsers on tPMET and PI uptake. The tPMET rates defined as the rate of increase in WST-8 absorbance per min of reaction (left axis, solid lines), and PI fluorescence (Right axis, doted lines) were determined by plate reader (10–35 min) and flow cytometry (5 min) respectively in a different assay. B16F10 cells were exposed to different pulsing numbers with BL or PFL (green and blue color code respectively) with a fixed electric field of 40 kV/cm. BL pulser showed the inhibitory effect on tPMET (significant decrease start at 20 pulses compared to control) while the PFL showed the biphasic effect on tPMET with a significant increase at fewer pulsing numbers (5 pulses, showed by red **) and then decrease for high pulsing number (significant decrease at 10 pulses). Significant differences were observed between these two pulsers in regard to an increase in PI uptake (at 10, 15, 20, and 30 Pulses), indicated by the (****). (n = 3) ** p < 0.05 and **** p < 0.0001.

Figure 6

nsPEF effects of BL and PFL pulsers cell viability. Cell viability of B16F10 cells was determined using a plate reader after 24 h for (a) various pulsing numbers with a fixed electric field of 40 kV/cm, or (b) different electric fields (0, 30, 40, and 50 kV/cm) of 10 pulses, with BL (green) or PFL (blue) pulsers. In (a), significant differences were observed between these two pulsers, particularly at 5 and 10 pulses. In (b), the viability did not show a significant decrease compared to the control at 30 and 40 kV/cm with BL pulsing, whereas with PFL pulsing, a significant decrease in viability was observed (**** p < 0.0001).

Figure 7

nsPEF effects of BL and PFL pulsers on the reactive oxygen species and mitochondria membrane potential at 20 min after pulsing. B16F10 cells were exposed to different pulsing numbers with BL or PFL (green and blue color code respectively) with a fixed electric field of 40 kV/cm. Dotted lines represent the TMRE and solid lines represent the MSOX. The IC-50 is mentioned at the top. Significant differences were observed between these two pulsers in regard to a decrease in mitochondrial membrane potential (at 10, 15, and 20 pulses), indicated by the (**** with p < 0.0001).

Figure 8

nsPEF effects of BL and PFL pulsers on mitochondrial oxidative metabolism in B16F10 melanoma cell lines. The oxygen consumption rate (OCR) of cells was measured 15 h after pulsing with 5 pulses. The x-axis represents time (up to 75 min), which aligns with the recommended test profile in the Seahorse assay for measuring mitochondrial respiration. The electric field was maintained at 40 kV/cm for both pulsers. The cells were maintained at 37 °C during the 15 h while they adhered. The different states of mitochondrial respiration are indicated: basal respiration (Basal), proton leak (respiration after oligomycin exposure), maximal respiratory capacity (respiration after FCCP, MRC), and non-mitochondrial respiration (after rotenone and antimycin A) (NM). * p < 0.05 compared to control. Cells treated with PFL pulses showed a lower SRC compared to the control group (** p < 0.002).

Figure 9

The timeline of the cell responses to PFL and BL pulses at different time intervals: 10–50 min, 15 h, and 24 h after pulsing. The magnitude of cell responses is represented by the extension of azimuthal angels (larger angle meaning larger response). Created with BioRender.com.