Ablation therapies aim at eradication of tumors with minimal impact on surrounding healthy tissues. Conventional pulsed electric field (PEF) treatments cause pain and muscle contractions far beyond the ablation area. The ongoing quest is to identify PEF parameters efficient at ablation but not at stimulation. We measured electroporation and cell killing thresholds for 150 ns–1 ms PEF, uni- and bipolar, delivered in 10- to 300-pulse trains at up to 1 MHz rates. Monolayers of murine colon carcinoma cells exposed to PEF were stained with YO-PRO-1 dye to detect electroporation. In 2–4 h, dead cells were labeled with propidium. Electroporation and cell death thresholds determined by matching the stained areas to the electric field intensity were compared to nerve excitation thresholds (Kim et al. in Int J Mol Sci 22(13):7051, 2021). The minimum fourfold ratio of cell killing and stimulation thresholds was achieved with bipolar nanosecond PEF (nsPEF), a sheer benefit over a 500-fold ratio for conventional 100-µs PEF. Increasing the bipolar nsPEF frequency up to 100 kHz within 10-pulse bursts increased ablation thresholds by < 20%. Restricting such bursts to the refractory period after nerve excitation will minimize the number of neuromuscular reactions while maintaining the ablation efficiency and avoiding heating.

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Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength–duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.

Electric shocks, the only effective therapy for ventricular fibrillation, also electroporate cardiac cells and contribute to the high-mortality post-cardiac arrest syndrome. Copolymers such as Poloxamer 188 (P188) are known to preserve the membrane integrity and viability of electroporated cells, but their utility against cardiac injury from cardiopulmonary resuscitation (CPR) remains to be established. We studied the time course of cell killing, mechanisms of cell death, and protection with P188 in AC16 human cardiomyocytes exposed to micro- or nanosecond pulsed electric field (μsPEF and nsPEF) shocks. A 3D printer was customized with an electrode holder to precisely position electrodes orthogonal to a cell monolayer in a nanofiber multiwell plate. Trains of nsPEF shocks (200, 300-ns pulses at 1.74 kV) or μsPEF shocks (20, 100-μs pulses at 300 V) produced a non-uniform electric field enabling efficient measurements of the lethal effect in a wide range of the electric field strength. Cell viability and caspase 3/7 expression were measured by fluorescent microscopy 2–24 h after the treatment. nsPEF shocks caused little or no caspase 3/7 activation; most of the lethally injured cells were permeable to propidium dye already at 2 h after the exposure. In contrast, μsPEF shocks caused strong activation of caspase 3/7 at 2 h and the number of dead cells grew up to 24 h, indicating the prevalence of the apoptotic death pathway. P188 at 0.2–1% reduced cell death, suggesting its potential utility in vivo to alleviate electric injury from defibrillation.

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