Here, the sizes of the pores created by square-wave electric pulses with the duration of 100 μs and 2 ms are compared for pulses with the amplitudes close to the threshold of electroporation. Experiments were carried out with three types of cells: mouse hepatoma MH-22A cells, Chinese hamster ovary (CHO) cells, and human erythrocytes. In the case of a short pulse (square-wave with the duration of 100 μs or exponential with the time constant of 22 μs), in the large portion (30-60%) of electroporated (permeable to potassium ions) cells, an electric pulse created only the pores, which were smaller than the molecule of bleomycin (molecular mass of 1450 Da, r≈0.8 nm) or sucrose (molecular mass of 342.3 Da, radius-0.44-0.52 nm). In the case of a long 2-ms duration pulse, in almost all cells, which were electroporated, there were the pores larger than the molecules of bleomycin and/or sucrose. Kinetics of pore resealing depended on the pulse duration and was faster after the shorter pulse. After a short 100-μs duration pulse, the disappearance of the pores permeable to bleomycin was completed after 6-7 min at 24-26°C, while after a long 2-ms duration pulse, this process was slower and lasted 15-20 min. Thus, it can be concluded that a short 100-μs duration pulse created smaller pores than the longer 2-ms duration pulse. This could be attributed to the time inadequacy for pores to grow and expand during the pulse, in the case of short pulses.

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The electroporation threshold was compared at various electric pulse durations for three cell lines: two non-tumor cell lines (human erythrocytes and Chinese hamster ovary cells) and one tumor cell line (rat glioma C6 cells). First, the dependences of the fraction of electroporated cells on the pulse intensity were obtained for the cells exposed to single square-wave electric pulses with the durations of 0.02-2 ms. Then, the average cell radii were measured for each cell line and the transmembrane potential induced by the external electric field was calculated. The obtained values of the transmembrane potential were in the range of 480-930 mV and decreased with increasing pulse duration. The obtained dependences of the transmembrane potential required to electroporate 50% of cells on the pulse duration were close to each other for all cell lines studied.

Here, theoretical relationships between the parameters of the electric pulse, which is necessary to porate the cell by electric pulse of various shapes, have been obtained. The theoretical curves were compared with the experimental relationships. Experiments were carried out with human erythrocytes, Chinese hamster ovary and mouse hepatoma MH-22A cells. The fraction of electroporated MH-22A cells was determined from the extent of the release of intracellular potassium ions and erythrocytesfrom the extent of their hemolysis after long (20-24 h) incubation in 0.63% NaCl solution at 4°C. The dependence of the fraction of electroporated cells on the amplitude of the electric field pulse was determined for pulses with the duration from 95 ns to 2 ms. The shapes of theoretical dependencies are in agreement with experimental ones. The cell poration time depended on the intensity of the pulse: the shorter the pulse duration, the higher the electric field strength has to be. This dependence is much more pronounced for pulses <1 µs. For example, if the pulse amplitude required to electroporate 50% of human erythrocytes increased from 1.0 to 1.76 kV/cm, when the duration of a squarewave pulse was reduced from 2 ms to 20 µs, it increased from 3 to 12 kV/cm, when the pulse duration was reduced from 950 to 95 ns. The relationships between the electric field strength required for electroporation and the frequency of the applied ac field were calculated for different pulselengths. It has been obtained that although the electric field strength is constant for frequencies <10 kHz but its value depends on the pulselength decreasing with increasing pulse duration. At higher frequencies, electric field strength is dependent on the frequency of the ac field.

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