Subnanosecond electric pulses (200 ps) at electric field intensities on the order of 20 kV/cm cause the death of B16.F10 murine melanoma cells when applied for minutes with a pulse repetition rate of 10 kHz. The lethal effect of the ultrashort pulses is found to be caused by a combination of thermal effects and electrical effects. Studies on the cellular level show increased transport across the membrane at much lower exposure times or number of pulses. Exposed to 2000 pulses, NG108 cells exhibit an increase in membrane conductance, but only allow transmembrane currents to flow, if the medium is positively biased with respect to the cell interior. This means that the cell membrane behaves like a rectifying diode. This increase in membrane conductance is a nonthermal process, since the temperature rise due to the pulsing is negligible.

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.

Nanosecond electric pulses are an effective power source in plasma medicine and biological stimulation, in which biophysical responses are governed by peak power and not energy. While uniphasic nanosecond pulse generators are widely available, the recent discovery that biological effects can be uniquely modulated by reversing the polarity of nanosecond duration pulses calls for the development of a multimodal pulse generator. This paper describes a method to generate nanosecond multiphasic pulses for biomedical use, and specifically demonstrates its ability to cancel or enhance cell swelling and blebbing. The generator consists of a series of the fundamental module, which includes a capacitor and a MOSFET switch. A positive or a negative phase pulse module can be produced based on how the switch is connected. Stacking the modules in series can increase the voltage up to 5 kV. Multiple stacks in parallel can create multiphase outputs. As each stack is independently controlled and charged, multiphasic pulses can be created to produce flexible and versatile pulse waveforms. The circuit topology can be used for high-frequency uniphasic or biphasic nanosecond burst pulse production, creating numerous opportunities for the generator in electroporation applications, tissue ablation, wound healing, and nonthermal plasma generation.

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca²⁺, Cd²⁺, Zn²⁺, and Ba²⁺ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca²⁺ and Ba²⁺ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within 7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba²⁺ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca²⁺ entry could elicit calcium-induced calcium release (CICR) followed by Ca²⁺ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca²⁺ and Ba²⁺ proved as sensitive nanoporation markers, with Ba²⁺ being more reliable for monitoring membrane damage and resealing.