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.

Human dermal fibroblasts exhibit comparable cellular and molecular effects when exposed to THz radiation and hyperthermic stress. These findings suggest that radiation at 2.52 THz generates primarily thermal effects in mammalian cells. Therefore, we conclude that THz-induced bioeffects may be accurately predicted with conventional thermal damage models.

In this study, we determined the LD50 (50% lethal dose) for cell death, and the ED50 (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD50 varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD50 lower than the ED50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

Terahertz spectrometers and imaging systems are currently being evaluated as biomedical tools for skin burn assessment. These systems show promise, but due to their size and weight, they have restricted portability, and are impractical for military and battlefield settings where space is limited. In this study, we developed and tested the performance of a compact, light, and portable THz time-domain spectroscopy (THz-TDS) device. Optical properties were collected with this system from 0.1 to 1.6 THz for water, ethanol, and several ex vivo porcine tissues (muscle, adipose, skin). For all samples tested, we found that the index of refraction (n) decreases with frequency, while the absorption coefficient (μ(a)) increases with frequency. Muscle, adipose, and frozen/thawed skin samples exhibited comparable n values ranging between 2.5 and 2.0, whereas the n values for freshly harvested skin were roughly 40% lower. Additionally, we found that the freshly harvested samples exhibited higher μ(a) values than the frozen/thawed skin samples. Overall, for all liquids and tissues tested, we found that our system measured optical property values that were consistent with those reported in the literature. These results suggest that our compact THz spectrometer performed comparable to its larger counterparts, and therefore may be a useful and practical tool for skin health assessment.

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, Propidium Iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (15 minutes) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.

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A bipolar (BP) nanosecond electric pulse (nsEP) exposure generates reduced calcium influx compared to a unipolar (UP) nsEP. This attenuated physiological response from a BP nsEP exposure is termed “bipolar cancellation” (BPC). The predominant BP nsEP parameters that induce BPC consist of a positive polarity (↑) front pulse followed by the delivery of a negative polarity (↓) back pulse of equal voltage and width; thereby the duration is twice a UP nsEP exposure. We tested these BPC parameters, and discovered that a BP nsEP with symmetrical pulse widths is not required to generate BPC. For example, our data revealed the physiological response initiated by a ↑900 nsEP exposure can be cancelled by a second pulse that is a third of its duration.  However, we observed a complete loss of BPC from a ↑300 nsEP followed by a ↓900 nsEP exposure. Spatiotemporal analysis revealed these asymmetrical BP nsEP exposures generate distinct local YO-PRO®-1 uptake patterns across the plasma membrane. From these findings, we generated a conceptual model that suggests BPC is a phenomenon balanced by localized charging and discharging events across the membrane.

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10 ns duration and 150 kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10 min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24 h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects.