Effects of extracellular medium conductivity on cell response in the context of sub-microsecond range calcium electroporation

Abstract

In the present study, we report the effects of extracellular medium conductivity on cell response in the context of sub-microsecond range (100 ns-900 ns) electroporation, calcium electroporation and cell size. The effects of 25 ns and microsecond range (100 μs) pulses were also covered. As a model, three different cancer cell lines of various size (C32, MCF-7/DX and MC38/0) were used and the results indicated different size-dependent susceptibility patterns to the treatment. The applied pulsed electric field (PEF) protocols revealed a significant decrease of cell viability when calcium electroporation was used. The dependence of calcium ion transport and finally the anticancer effect on the external medium conductivity was determined. It was shown that small differences in conductivity do not alter viability significantly, however, mostly affect the permeabilization. At the same, MC38/0 cell line was the least susceptible to calcium electroporation, while the C32 line the most. In all cases calcium electroporation was mostly dependent on the sensitivity of cells to electroporation and could not be effectively improved by the increase of CaCl 2 concentration from 2 mM to 5 mM. Lastly, sub-microsecond PEF stimulated aquaporin-4 and VDAC1/Porin immunoreactions in all treated cells lines, which indicated that cell water balance is affected, ions exchange is increased and release of mitochondrial products is occurrent.Electroporation is a phenomenon of pulsed electric field (PEF) initiated permeabilization of biological cell plasma membrane, which serves as a basepoint of many successful biomedical and biotechnological methodologies 1,2 . The mechanism of electroporation is dependent on cell polarization in PEF and transient charge accumulation on the cell membrane (known as transmembrane potential, TMP) 3,4 . When a threshold TMP is reached, the permeability of cell membrane is increased due to occurrence of transient hydrophilic pores 5 . It is believed that oxidative effects can be a separate mechanism responsible for the formation of pores during electroporation 6,7 , nevertheless, polarization of the cell is a primary trigger.Polarization of the cell depends on the parameters of PEF, however, also on the permittivity and conductivity of both the cells and the medium 3,8 . Therefore, the effects of extracellular medium conductivity on electroporation efficiency have been focused for decades 9-12 . Absolute majority of the scientific works focus the micro-millisecond range of pulses, while a new modality of shorter (nanosecond range) pulses was introduced and is gaining popularity 13,14 . Currently, there are two contributions 10,11 experimentally focusing the 12-102 ns range of pulses in the context of extracellular medium conductivity, while the sub-microsecond range is not covered in literature. Both papers are published by the same group Silve et al., however the upmost interest lies in the peculiar cell response to 12 ns pulses. According to these studies, the same DC3-F cell line indicated an opposite respons...

The Impact of Extracellular Ca2+ and Nanosecond Electric Pulses on Sensitive and Drug-Resistant Human Breast and Colon Cancer Cells

“…However, the electrophoretic component of longer (microsecond) pulses is much higher compared to ultra-short pulses; therefore, a similar treatment outcome indicates that passive diffusion of calcium was the dominant mechanism of delivery through a permeabilized membrane. Indeed, calcium is a small ion, and thus passive diffusion is sufficient to ensure significant electrotransfer, even in the nanosecond range when the electrophoresis influence is negligible [34]. We have shown that during supra-electroporation, when the pulse duration is significantly shorter than the polarization time, electric field strength should be increased several-fold to trigger the same permeabilization rates as in the case of longer pulses.…”

“…Cell suspension was kept on ice before electroporation and then pulses were delivered in a cuvette (Bio-Rad) between 1 mm gap parallel plate aluminum electrodes. Electroporation protocols were also combined with CaCl 2 (Sigma-Aldrich) in a 2 mM concentration, based on our previous study [34]. Calcium chloride solution was prepared in the same buffer as for electroporation alone.…”

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“…The percentage of stained cells was shown in the table, after which the intensity of the staining was estimated. The intensity of the immunohistochemical staining was assessed as (-) negative (no reaction), (+) weak, (++) moderate and (+++) strong [ 34 ].…”