Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltagesensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is ~1 nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.
The exclusion of polar dyes by healthy cells is widely employed as a simple and reliable test for cell membrane integrity. However, commonly used dyes (propidium, Yo-Pro-1, trypan blue) cannot detect membrane defects which are smaller than the dye molecule itself, such as nanopores that form by exposure to ultrashort electric pulses (USEPs). Instead, here we demonstrate that opening of nanopores can be efficiently detected and studied by fluorescent measurement of Tl+ uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl+-sensitive fluorophore FluxOR™ and subjected to USEPs in a Tl+-containing bath buffer, displayed an immediate (within<100 ms), dose-dependent surge of fluorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl+ by 600-ns USEP was at 1–2 kV/cm, and the rate of Tl+ uptake increased linearly with increasing the electric field. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 1–1.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca2+ did not block the USEP effect. Addition of a Tl+-containing buffer within less than 10 min after USEP also caused a fluorescence surge, which confirms the minutes-long lifetime of nanopores. Overall, the technique of fluorescent detection of Tl+ uptake proved highly effective, noninvasive and sensitive for visualization and analysis of membrane defects which are too small for conventional dye uptake detection methods.
In recent years, research into biological and medical effects of millimeter waves (MMW) has expanded greatly. This paper analyzes general trends in the area and briefly reviews the most significant publications, proceeding from cell-free systems, dosimetry, and spectroscopy issues through cultured cells and isolated organs to animals and humans. The studies reviewed demonstrate effects of lowintensity MMW (10 mW/cm 2 and less) on cell growth and proliferation, activity of enzymes, state of cell genetic apparatus, function of excitable membranes, peripheral receptors, and other biological systems. In animals and humans, local MMW exposure stimulated tissue repair and regeneration, alleviated stress reactions, and facilitated recovery in a wide range of diseases (MMW therapy). Many reported MMW effects could not be readily explained by temperature changes during irradiation. The paper outlines some problems and uncertainties in the MMW research area, identifies tasks for future studies, and discusses possible implications for development of exposure safety criteria and guidelines.
Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in cell plasma membrane, followed by cell volume changesdue to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by 50, 60-ns EP (10 Hz, ~13 kV/cm) and 5, 600-ns EP (1 Hz, ~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with (polyethylene glycols and sugars. Such replacement reduced cell swelling and/or turned it into a transient or sustained shrinking, depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as indicated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.
Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca2+ mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1–60), and amplitudes (15–60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-μs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.
High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.
Background-Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8-and 9-μs EP.Methods-Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.Results-10-ns EP caused apoptotic or necrotic death within 2-20 hrs. Survival (S, %) followed the absorbed dose (D, J/g) as: S=αD (−K) , where coefficients K and α determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas α was cell type-and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.Conclusions-1.8-and 9-μs EP cause cell death efficiently and indiscriminately (LD 50 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD 50 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms.General significance-Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.
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