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High-intensity nanosecond pulsed electric fields (nsPEFs) permeabilize cell membranes. Although progress has been made toward an understanding of the mechanism of nsPEF-induced membrane poration, the dependence of pore size and distribution on pulse duration, strength, number, and repetition rate remains poorly defined experimentally. In this paper, we characterize the size of nsPEF-induced pores in living cell membranes by isosmotically replacing the solutes in pulsing media with polyethylene glycols and sugars before exposing Jurkat T lymphoblasts to 5 ns, 10 MV/m electric pulses. Pore size was evaluated by analyzing cell volume changes resulting from the permeation of osmolytes through the plasma membrane. We find that pores created by 5 ns pulses have a diameter between 0.7 and 0.9 nm at pulse counts up to 100 with a repetition rate of 1 kHz. For larger number of pulses, either the pore diameter or the number of pores created, or both, increase with increasing pulse counts. But the prevention of cell swelling by PEG 1000 even after 2000 pulses suggests that 5 ns, 10 MV/m pulses cannot produce pores with a diameter larger than 1.9 nm. [ABSTRACT FROM AUTHOR]

Intense nanosecond pulsed electric fields (nsPEFs) have been shown to induce, on intracellular structures, interesting effects dependent on electrical exposure conditions (pulse length and amplitude, repetition frequency and number of pulses), which are known in the literature as “bioelectrical effects” (Schoenbach et al., IEEE Trans Plasma Sci 30:293–300, ). In particular, pulses with a shorter width than the plasma membrane charging time constant (about 100 ns for mammalian cells) can penetrate the cell and trigger effects such as permeabilization of intracellular membranes, release of Ca2+ and apoptosis induction. Moreover, the observed effects have led to exploration of medical applications, like the treatment of melanoma tumors (Nuccitelli et al., Biochem Biophys Res Commun 343:351–360, ). Pulsed electric fields allowing such effects usually range from several tens to a few hundred nanoseconds in duration and from a few to several tens of megavolts per meter in amplitude (Schoenbach et al., IEEE Trans Diel Elec Insul 14:1088–1109, ); however, the biological effects of subnanosecond pulses have been also investigated (Schoenbach et al., IEEE Trans Plasma Sci 36:414–422, ). The use of such a large variety of pulse parameters suggests that highly flexible pulse-generating systems, able to deliver wide ranges of pulse durations and amplitudes, are strongly required in order to explore effects and applications related to different exposure conditions. The Blumlein pulse-forming network is an often-employed circuit topology for the generation of high-voltage electric pulses with fixed pulse duration. An innovative modification to the Blumlein circuit has been recently devised which allows generation of pulses with variable amplitude, duration and polarity. Two different modified Blumlein pulse-generating systems are presented in this article, the first based on a coaxial cable configuration, matching microscopic slides as a pulse-delivery system, and the other based on microstrip transmission lines and designed to match cuvettes for the exposure of cell suspensions. [ABSTRACT FROM AUTHOR]

The design and realization of a nanosecond, high-voltage electric pulse generator for bioelectrical applications is reported in this paper. A Blumlein type architecture was adopted, with some modifications, and realized in a microstrip line configuration with meander-shaped conducting strips, and with ultra-fast, high voltage solid state switches. Three microstrip-lines have been realized in such a way that, being interchangeable within the structure, they allow to match different load impedances. A fiber optic-based system was also realized to separate the high voltage side of the system from the switch-control circuit. The system is suitable for applying high voltage nanosecond electric pulses, with variable pulse duration, amplitude, repetition rate and polarity, to liquid media with different electromagnetic characteristics, hosted in electroporation cuvettes with different gap dimensions. [ABSTRACT FROM AUTHOR]

A numerical model, usually employed to analyze the electroporation phenomenon in eukaryotic cells exposed to high voltage electric pulses with duration in the ms to ?s time scale, is extended to investigate the electroporation onset and dynamics induced by nanosecond pulsed electric fields (nsPEFs). The model allows to track the primary events and dynamics of the electroporation process on a time scale where available experimental methods do not provide reliable information. In particular the model, solved in the time domain, provides the spatial and temporal distribution of the transmembrane voltage and pore density of fixed radii around the plasma membrane. Moreover, it allows to optimize the experimental design of biological investigations in the framework of the mechanistic understanding of nsPEF. The obtained simulation results are correlated to biological observations of plasma membrane permeabilization in human lymphoblastoid Jurkat T-cells exposed to nsPEFs with variable pulse amplitude, as assessed by YO-PRO1 dye uptake. [ABSTRACT FROM AUTHOR]