Search

A unique aspect of electrostimulation (ES) with nanosecond electric pulses (nsEP) is the inhibition of effects when the polarity is reversed. This bipolar cancellation feature makes bipolar nsEP less efficient at biostimulation than unipolar nsEP. We propose to minimize stimulation near pulse-delivering electrodes by applying bipolar nsEP, whereas the superposition of two phase-shifted bipolar nsEP from two independent sources yields a biologically-effective unipolar pulse remotely. This is accomplished by electrical compensation of all nsEP phases except the first one, resulting in the restoration of stimulation efficiency due to cancellation of bipolar cancellation (CANCAN-ES). We experimentally proved the CANCAN-ES paradigm by measuring YO-PRO-1 dye uptake in CHO-K1 cells which were permeabilized by multiphasic nsEP (600 ns per phase) from two generators; these nsEP were synchronized either to overlap into a unipolar pulse remotely from electrodes (CANCAN), or not to overlap (control). Enhancement of YO-PRO-1 entry due to CANCAN was observed in all sets of experiments and reached ~3-fold in the center of the gap between electrodes, exactly where the unipolar pulse was formed, and equaled the degree of bipolar cancellation. CANCAN-ES is promising for non-invasive deep tissue stimulation, either alone or combined with other remote stimulation techniques to improve targeting.

Accumulating data indicates that some cancer treatments can restore anticancer immunosurveillance through the induction of tumor immunogenic cell death (ICD). Nanosecond pulsed electric fields (nsPEF) have been shown to efficiently ablate melanoma tumors. In this study we investigated the mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors. Our data show that in vitro nsPEF (20–200, 200-ns pulses, 7 kV/cm, 2 Hz) caused a rapid dose-dependent cell death which was not accompanied by caspase activation or PARP cleavage. The lack of nsPEF-induced apoptosis was confirmed in vivo in B16F10 tumors. NsPEF also failed to trigger ICD-linked responses such as necroptosis and autophagy. Our results point at necrosis as the primary mechanism of cell death induced by nsPEF in B16F10 cells. We finally compared the antitumor immunity in animals treated with nsPEF (750, 200-ns, 25 kV/cm, 2 Hz) with animals were tumors were surgically removed. Compared to the naïve group where all animals developed tumors, nsPEF and surgery protected 33% (6/18) and 28.6% (4/14) of the animals, respectively. Our data suggest that, under our experimental conditions, the local ablation by nsPEF restored but did not boost the natural antitumor immunity which stays dormant in the tumor-bearing host.

Delivery of large and structurally complex target molecules into cells is vital to the emerging areas of cellular modification and molecular therapy. Inadequacy of prevailing in vivo (viral) and in vitro (liposomal) gene transfer methods for delivery of proteins and a growing diversity of synthetic nanomaterials has encouraged development of alternative physical approaches. Efficacy of injury/diffusion-based delivery via shear mechanoporation is largely insensitive to cell type and target molecule; however, enhanced flexibility is typically accompanied by reduced gene transfer effectiveness. We detail a method to improve transfection efficiency through coordinated mechanical disruption of the cell membrane and electrophoretic insertion of DNA to the cell interior. An array of micromachined nozzles focuses ultrasonic pressure waves, creating a high-shear environment that promotes transient pore formation in membranes of transmitted cells. Acoustic Shear Poration (ASP) allows passive cytoplasmic delivery of small to large nongene macromolecules into established and primary cells at greater than 75% efficiency. Addition of an electrophoretic action enables active transport of target DNA molecules to substantially augment transfection efficiency of passive mechanoporation/diffusive delivery without affecting viability. This two-stage poration/insertion method preserves the compelling flexibility of shear-based delivery, yet substantially enhances capabilities for active transport and transfection of plasmid DNA.

We demonstrate that conditioning of mammalian cells by electroporation with nanosecond pulsed electric field (nsPEF) facilitates their response to the next nsPEF treatment. The experiments were designed to unambiguously separate the electroporation-induced sensitization and desensitization effects. Electroporation was achieved by bursts of 300-ns, 9 kV/cm pulses (50 Hz, n = 3–100) and quantified by propidium dye uptake within 11 min after the nsPEF exposure. We observed either sensitization to nsPEF or no change (when the conditioning was either too weak or too intense, or when the wait time after conditioning was too short). Within studied limits, conditioning never caused desensitization. With settings optimal for sensitization, the second nsPEF treatment became 2.5 times (25 °C) or even 6 times (37 °C) more effective than the same nsPEF treatment delivered without conditioning. The minimum wait time required for sensitization development was 30 s, with still longer delays increasing the effect. We show that the delayed hypersensitivity was not mediated by either cell swelling or oxidative effect of the conditioning treatment; biological mechanisms underlying the delayed electrosensitization remain to be elucidated. Optimizing nsPEF delivery protocols to induce sensitization can reduce the dose and adverse side effects of diverse medical treatments which require multiple pulse applications.

Modern technologies enable deep tissue focusing of nanosecond pulsed electric field (nsPEF) for non-invasive nerve and muscle stimulation. However, it is not known if PEF orders of magnitude shorter than the activation time of voltage-gated sodium channels (VGSC) would evoke action potentials (APs). One plausible scenario requires the loss of membrane integrity (electroporation) and resulting depolarization as an intermediate step. We report, for the first time, that the excitation of a peripheral nerve can be accomplished by 12-ns PEF without electroporation. 12-ns stimuli at 4.1–11 kV (3.3–8.8 kV/cm) evoked APs similarly to conventional stimuli (100–250 μs, 1–5 V, 103–515 V/m), except for having higher selectivity for the faster nerve fibers. Nerves sustained repeated tetanic stimulations (50 Hz or 100 Hz for 1 min) alternately by 12-ns PEF and by conventional pulses. Such tetani caused a modest AP decrease, to a similar extent for both types of stimuli. Nerve refractory properties were not affected. The lack of cumulative damages even from tens of thousands of 12-ns stimuli and the similarities with the conventional stimulation prove VGSC activation by nsPEF without nerve membrane damage.

Electroporation by nanosecond electric pulses (nsEP) is an emerging modality for tumor ablation. Here we show the efficient induction of apoptosis even by a non-toxic nsEP exposure when it is followed by a 30-min chilling on ice. This chilling itself had no impact on the survival of U-937 or HPAF-II cells, but caused more than 75% lethality in nsEP-treated cells (300 ns, 1.8-7 kV/cm, 50-700 pulses). The cell death was largely delayed by 5-23 hr and was accompanied by a 5-fold activation of caspase 3/7 (compared to nsEP without chilling) and more than 60% cleavage of poly-ADP ribose polymerase (compared to less than 5% in controls or after nsEP or chilling applied separately). When nsEP caused a transient permeabilization of 83% of cells to propidium iodide, cells placed at 37 °C resealed in 10 min, whereas 60% of cells placed on ice remained propidium-permeable even in 30 min. The delayed membrane resealing caused cell swelling, which could be blocked by an isosmotic addition of a pore-impermeable solute (sucrose). However, the block of swelling did not prevent the delayed cell death by apoptosis. The potent enhancement of nsEP cytotoxicity by subsequent non-damaging chilling may find applications in tumor ablation therapies.

Previous studies reported a delayed increase of sensitivity to electroporation (termed “electrosensitization”) in mammalian cells that had been subjected to electroporation. Electrosensitization facilitated membrane permeabilization and reduced survival in cell suspensions when the electric pulse treatments were split in fractions. The present study was aimed to visualize the effect of sensitization and establish its utility for cell ablation. We used KLN 205 squamous carcinoma cells embedded in an agarose gel and cell spheroids in Matrigel. A local ablation was created by a train of 200 to 600 of 300-ns pulses (50 Hz, 300–600 V) delivered by a two-needle probe with 1-mm inter-electrode distance. In order to facilitate ablation by engaging electrosensitization, the train was split in two identical fractions applied with a 2- to 480-s interval. At 400–600 V (2.9–4.3 kV/cm), the split-dose treatments increased the ablation volume and cell death up to 2–3-fold compared to single-train treatments. Under the conditions tested, the maximum enhancement of ablation was achieved when two fractions were separated by 100 s. The results suggest that engaging electrosensitization may assist in vivo cancer ablation by reducing the voltage or number of pulses required, or by enabling larger inter-electrode distances without losing the ablation efficiency.

The present study compared electroporation efficiency of bipolar and unipolar nanosecond electric field oscillations (NEFO). Bipolar NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2–4 decreased to 35%, 12% and 7% of the first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4. Membrane permeabilization was quantified in CHO and GH3 cells by uptake of a membrane integrity marker dye YO-PRO-1 (YP) and by the membrane conductance increase measured by patch clamp. For treatments with 1–20 unipolar NEFO, at 9.6–24 kV/cm, 10 Hz, the rate and amount of YP uptake were consistently 2-3-fold higher than after bipolar NEFO treatments, despite delivering less energy. However, the threshold amplitude was about 7 kV/cm for both NEFO waveforms. A single 14.4 kV/cm unipolar NEFO caused a 1.5–2 times greater increase in membrane conductance (p 2+-free conditions and thus cannot be explained by the reversal of electrophoretic flows of Ca2+. Instead, the data indicate that the electric field polarity reversals reduced the pore yield.