First-principles molecular dynamics simulations have been performed on the solvation of Na+ in water. Consistent with the available experimental data, we find that the first solvation shell of Na+ contains on average 5.2 water molecules. A significant number of water exchanges between the first and second solvation shells are observed. However, the simulations are not long enough to reliably measure the rate of water exchange. Contrary to several previous studies, we do not find any effect of Na+ on the orientation of water molecules outside of the first solvation shell. Furthermore, the complete set of structural properties determined by first-principles molecular dynamics is not predicted by any of the known classical simulations.

Electrical models for biological cells predict that reducing the duration of applied electrical pulses to values below the charging time of the outer cell membrane (which is on the order of 100 ns for mammalian cells) causes a strong increase in the probability of electric field interactions with intracellular structures due to displacement currents. For electric field amplitudes exceeding MV/m, such pulses are also expected to allow access to the cell interior through conduction currents flowing through the permeabilized plasma membrane. In both cases, limiting the duration of the electrical pulses to nanoseconds ensures only nonthermal interactions of the electric field with subcellular structures. This intracellular access allows the manipulation of cell functions. Experimental studies, in which human cells were exposed to pulsed electric fields of up to 30 MV/m amplitude with durations as short as 3 ns, have confirmed this hypothesis and have shown that it is possible to selectively alter the behavior and/or survival of cells. Observed nanosecond pulsed effects at moderate electric fields include intracellular release of calcium and enhanced gene expression, which could have long term implications on cell behavior and function. At increased electric fields, the application of nanosecond pulses induces a type of programmed cell death, apoptosis, in biological cells. Cell survival studies with 10 ns pulses have shown that the viability of the cells scales inversely with the electrical energy density, which is similar to the "dose" effect caused by ionizing radiation. On the other hand, there is experimental evidence that, for pulses of varying durations, the onset of a range of observed biological effects is determined by the electrical charge that is transferred to the cell membrane during pulsing. This leads to a similarity law for nanosecond pulse effects, with the product of electric field intensity, pulse duration, and the square root of the number of pulses as the similarity parameter. The similarity law allows one not only to predict cell viability based on pulse parameters, but has also been shown to be applicable for inducing platelet aggregation, an effect which is triggered by internal calcium release. Applications for nanosecond pulse effects cover a wide range: from a rather simple use as preventing biofouling in cooling water systems, to advanced medical applications, such as gene therapy and tumor treatment. Results of this continuing research are leading to the development of wound healing and skin cancer treatments, which are discussed in some detail.

The barrier function of plasma membrane in nsPEF-exposed mammalian cells was examined using whole-cell patch-clamp techniques. A specialized setup for nsPEF exposure of individual cells in culture was developed and characterized for artifact-free compatibility with the patch-clamp method. For the first time, our study provides experimental evidence that even a single 60-ns pulse at 12 kV/cm can cause a profound and long-lasting (minutes) reduction of the cell membrane resistance (R(m)), accompanied by the loss of the membrane potential. R(m) measured in GH3, PC-12, and Jurkat cells (but not in HeLa cells) in 80-120 s after nsPEF exposure was decreased about threefold, and its gradual recovery could take 15 min. Multiple pulses enhanced permeabilization, for example, R(m) in GH3 cells fell about 10-fold after a train of five pulses. Within studied limits, permeabilization did not depend on the presence of Ca(2+), Mg(2+), K(+), Cs(+), Cd(2+), EGTA, tetraethylammonium, or 4-aminopyridine in the pipette or bath solutions. Our results supported theoretical model predictions of plasma membrane poration by nsPEF. However, the extended decrease in R(m), assumed to be related to the life span of the pores, and different nsPEF sensitivity of individual cell lines have yet to be explained. The phenomenon of long-lived membrane permeabilization provides new insights on the nature of nsPEF-opened conductance pores and on molecular mechanisms that underlie nsPEF bioeffects.

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200 -700 nM, respectively, above basal levels (ϳ100 nM), while the uptake of propidium iodide was absent. This suggests that increases in intracellular calcium were not because of plasma membrane electroporation. nsPEF and the purinergic agonist UTP induced calcium mobilization in the presence and absence of extracellular calcium with similar kinetics and appeared to target the same inositol 1,4,5-trisphosphate-and thapsigargin-sensitive calcium pools in the endoplasmic reticulum. For cells exposed to either nsPEF or UTP in the absence of extracellular calcium, there was an electric field-dependent or UTP dose-dependent increase in capacitative calcium entry when calcium was added to the extracellular media. These findings suggest that nsPEFs, like ligand-mediated responses, release calcium from similar internal calcium pools and thus activate plasma membrane calcium influx channels or capacitative calcium entry.