Pulsed Power for Wound Healing

Nanosecond and subnanosecond high-voltage pulses have used in new biological applications, such as cancer treatment using an ultrashort pulsed high electric field. For this treatment, the use of high-power pulsed electromagnetic wave has been proposed to obtain the required high electric field. This paper focuses on the design of a compact high-power pulsed electromagnetic wave generator using a nanosecond pulsed power generator for cancer treatment. We developed a pulsed power generator that continuously outputs multiple pulses via a nonlinear transmission line (NLTL) using magnetic switches. The NLTL comprises a ladder of capacitors and saturable inductors. The NLTL generates a pulse train by delaying the propagation of the pulse using the magnetic switch of each ladder. The frequency of repetition of the burst pulse is 13.8 MHz. The peak output voltage is 7 kV at the maximum input voltage of 28 kV. The number of pulses in the pulse train can be varied by the number of units of the magnetic switch.

This paper describes three pulse generators: a spark gap switched coaxial cable, a spark gap switched Blumlein, and a solid state modulator, developed for applying ultrashort electrical pulses to biological materials in culture. Research has shown that ultrashort pulsed electric fields can induce apoptosis in biological cells, and that pulses as short as 10 ns with field amplitude greater than 1 W/m cause membrane phospholipid rearrangement and activation of the effector enzymes of apoptosis. Pulses of very short duration use only tens of mJ per mL per pulse to induce apoptosis and other intracellular effects without causing thermal trauma. The pulse generators discussed here, each of a different topology, deliver ns pulsed electric fields (nsPEF) to cells in liquid suspension, and can be modified to drive electrodes for external, surgical, or endoscopic treatment of tissues in situ.

Due to their ability to accumulate considerable amounts of triglycerides, microalgae are considered as the most promising future biomass feedstock for oil production. Nanosecond pulsed electric field (PEF) can induce growth stimulation on plants and fungi, as we have shown in previous work. In the frame of this study, we transposed such growth stimulation process on microalgae in suspension to increase the cultivation yields in order to reduce biomass production costs. Optical density measurement of cell culture growth revealed to be a very sensitive diagnostic tool for determination of the algae growth and algae lethality exposed to PEF treatments. At first approach, the lethal pulse parameters of PEF treatment of Chlamydomonas rheinhardtii and Chlorella vulgaris algae suspension were determined by monitoring the optical density over the entire cultivation time. As a first important result we found that a 30 min long PEF treatment at field strength of 40 kV/cm and pulse duration of 25 ns doesn't affect algae growth. In this case the specific energy dose was 1.4 kJ/kg. PEF treatments at specific treatment energies below this limit were considered to be non-lethal. We found that the average value of the growth rate (0.08±0,03 h-1) of nsPEF treated C. rheinhardtii suspension doesn't deviate significantly from the average growth rate of the sham cultivation (0.07±0,02 h-1) indicating no stimulation effect. In case of C. vulgaris the 30 min PEF treatment with a non-lethal dose during exponential growth phase led to an increase of biomass yield in the range of 10-20%. This increase was significant towards the end of cultivation after 5 days.

The increasing interest towards biocompatible nanotechnologies in medicine, combined with electric fields stimulation, is leading to the development of electro-sensitive smart systems for drug delivery applications. Common examples of electro-sensitive materials include phospholipids that can be used to design nano-sized vesicles suitable for external electric actuation. To this regard, recently the use of pulsed electric fields to trigger release across phospholipid membranes has been numerically studied, for a deeper understanding of the phenomena at the molecular scale. Aim of this work is to give an experimental validation of the feasibility of controlling drug release from liposomes mediated by nanosecond pulsed electric fields.

EDITORIAL article Front. Oncol., 22 March 2023Sec. Cancer Molecular Targets and Therapeutics Volume 13 - 2023 | https://doi.org/10.3389/fonc.2023.1183900

An electrode microchamber has been constructed for applying nanosecond pulsed strong electric fields to living cells, and fluorescence lifetime microscopy (FLIM) has been used to investigate the effects of external electric fields on dynamics and function of HeLa cells expressing enhanced green fluorescent protein (EGFP). Both morphological change in cells and reduction of the fluorescence lifetime of EGFP have been observed after application of electric fields having a pulsed width of 50 ns and a strength of 4 MV m(-1), indicating that apoptosis, which is a programmed cell death, was induced by nanosecond pulsed electric fields and that fluorescence lifetime of EGFP decreased along with the induction of apoptosis. The reduction of the fluorescence lifetime occurred before the morphological change, indicating that FLIM provides a sensitive and noninvasive detection of the progress of apoptosis induced by application of nanosecond pulsed electric fields.

Platelet-rich plasma: ready or not?

Evaluation of Platelet Gel Characteristics Using Thrombin Produced by the Thrombin Processing Device: A Comparative Study

Recently, pulsed power technologies, including pulsed electric fields (PEFs) and time-modulated plasmas, are starting to be applied actively in agriculture and food processing. In the applications, compact pulsed power generators with moderate peak power and repetitive operation are developed for controlling discharge plasmas and electric field distribution. These applications are mainly based on the biological effects of a spatially distributed electric field and the chemically active species in the plasma. The PEFs are caused by applying pulse voltage between the electrodes and contribute to form pores on the cell membrane or to change conformation of protein. When the applied voltage exceeds the discharge onset criterion, plasmas are generated through the avalanche process of electron accelerated with intense electric field in a gas or liquids medium. The plasmas produce chemically active species, UV radiation, an intense electric field in the vicinity of discharge channel and shock waves, which also have different biological effects. The agricultural applications of pulsed power can be categorized as two phases: pre-harvest and post-harvest phases. The pre-harvest phase consists of seed germination, seedling growth, plant growth and growth mode change from vegetative to reproductive. Pulsed power technologies are used to promotion of seed germination, plant growth enhancement through direct stimulation or indirect effect such as inactivation of bacteria in soil and liquid hydroponic media, and promotion of mushroom fruit body formation. The post-harvest phase consists of harvesting the agricultural produce, storing the products, transporting the products to consumers and food processing such as drying, pasteurization (sterilization of bacteria), permeabilization and fermentation. The pulsed power technologies are also used to keep freshness of agricultural produce through decontaminating airborne, inactivating bacteria and decomposition of plant hormone in the storage containers. The poration of cell membrane by PEF contributes improvement of extraction of juice, nutritional agents, and antioxidant metabolites such as polyphenols from agricultural products. In this review, at first, a basis of pulsed power system for agricultural applications and bio-effect by high-electric field exposure is outlined. After that, pre-harvest and post-harvest agricultural applications are described. The utilization of pulsed power technologies to contribute efficient food processing and improve food safety and quality is also described.

A series of reports in 2007 established that single nucleotide polymorphisms (SNPs) in the 9p21.3 locus are associated with coronary heart disease (CHD), both in patients with acute myocardial infarction and chronic atherosclerosis.1,–,4 Subsequently, many additional studies used a candidate locus approach to replicate the 9p21.3-CHD association. Intriguingly, other phenotypes have also been found to be associated with this genomic region, including type II diabetes, stroke, malignant melanoma, aortic aneurysms, cerebral aneurysms, and periodontitis. Unfortunately, neither the causative variants nor genes have been established. Although genetic associations may permit better disease risk stratification, without the knowledge of the genes and variants we lose opportunities to improve our basic understanding of the disease process and hence the potential for targeted therapies. Article see p 445 Many cellular pathways in multiple tissues contribute to the pathogenic processes resulting in CHD. There is value in using intermediate phenotypes as outcomes in genetic association studies because there is enhanced power to detect gene associations when the number of genes potentially responsible for the phenotype is reduced, thereby increasing the fraction of the variance explained by any single factor or gene. In addition, intermediate traits are usually easier to define (have less heterogeneity) than clinical disease. Somewhat surprisingly, 9p21.3 has not been associated with measures believed to represent the chronic process of atherosclerosis or endothelial cell reactivity,5 suggesting that this locus may contribute to CHD by an alternate pathophysiologic mechanism. There is abundant pathological and clinical evidence demonstrating the vital role played by blood platelets in acute coronary syndromes, which result from the formation of occlusive platelet thrombi in coronary arteries at the sites of ruptured atherosclerotic plaques,6,7 and antiplatelet agents have become a mainstay of therapy in patients with acute coronary syndromes. In addition to …

Different electrical characteristics of cells not only determine their initial electrical responses to pulsed electric field exposure, but also allow devising exposure conditions for pulsed electric field treatments that can preferentially target specific cells, such as cancer cells. We have investigated the dielectric properties of Jurkat cells, a malignant human T-cell line, before and after application of microsecond and nanosecond pulsed electric fields by means of time domain reflectometry dielectric spectroscopy. Jurkat cells in suspension with a 10% volume fraction were exposed with a repetition rate of about 1Hz to either 8 consecutive 1-kV/cm electroporation pulses of 100 μs, or 8 consecutive 18-kV/cm pulses of 300 ns. Electrode polarization has a significant effect on the low frequency measurements (<1 MHz) and needs to be corrected. We modeled the effect with a constant-phase-angle element in series with the impedance of the cell suspension. A Cole-Cole relaxation function was employed to describe the dielectric dispersion of cell suspension. Preliminary data analysis shows that conductivities of cell suspensions increased dramatically following microsecond or nanosecond exposure, indicating that membrane poration had occurred. Further analysis based on combination of a Maxwell-Wagner mixture model and a single shell cell model suggests an increase in plasma membrane conductivity. The changes of low frequency conductivity of the cell suspensions were different for both regimens, indicating different membrane charging and pore forming mechanisms.

Mid-myocardial ventricular arrhythmias are challenging to treat. Cardiac electroporation via pulsed electric fields (PEFs) offers significant promise. We therefore tested PEF delivery using screw-in pacemaker leads as proof-of-concept. In 5 canine models, we applied nanosecond PEF (pulse width 300ns) across the right ventricular (RV) septum using a single lead bipolar configuration (n = 2) and between two leads (n = 3). We recorded electrograms (EGMs) prior to, immediately post, and 5min after PEF. Cardiac magnetic resonance imaging (cMRI) and histopathology were performed at 2weeks and 1month. Nanosecond PEF induced minimal extracardiac stimulation and frequent ventricular ectopy that terminated post-treatment; no canines died with PEF delivery. With 1 lead, energy delivery ranged from 0.64 to 7.28J. Transient ST elevations were seen post-PEF. No myocardial delayed enhancement (MDE) was seen on cMRI. No lesions were noted on the RV septum at autopsy. With 2 leads, energy delivery ranged from 56.3 to 144.9J. Persistent ST elevations and marked EGM amplitude decreases developed post-PEF. MDE was seen along the septum 2weeks and 1month post-PEF. There were discrete fibrotic lesions along the septum; pathology revealed dense connective tissue with < 5% residual cardiomyocytes. Ventricular electroporation is feasible and safe with an active fixation device. Reversible changes were seen with lower energy PEF delivery, whereas durable lesions were created at higher energies. Central illustration: pulsed electric field delivery into ventricular myocardium with active fixation leads.

Nanosecond pulsed electric fields (nsPEFs) could be used to ablate local tumor masses through minimally invasive or open surgery delivery, but further pre-clinical studies are needed. NsPEFs not only induces permanent permeabilization of cells, but also impacts intracellular organelles and result in apoptosis and other forms of cell death in vitro and in vivo. Hepatocellular carcinoma (HCC) is the third most common cancer cause of mortality worldwide. A diagnosis of HCC carries a guarded prognosis, especially when metastasis is documented. Presently, reliable, curative treatments without recurrence for very early, early, and intermediate HCC stages are still in urgent need of attention. In these studies, we specifically test the efficacy of nsPEFs in an orthotopic HCC rat tumor model. Orthotropic insertion of stably transfected (mcherry and/or luciferase) N1-S1 cells in Sprague Dawley rat livers was used as a model for treating HCC. Cells were treated with pulses with 100 ns duration and electric field strengths of 50 kV/cm. Different pulse numbers were applied. Recent results suggest that treatments of 300 or 500 pulses were more effective when separated by a 6 minute interval than when applied as a continuous, uninterrupted single treatment. However over time and treatment of more than 20 animals, 1000 nanosecond pulses treatment consistently showed successful ablation by effectively inducing tumor cell death. A single treatment of 1000 nsPEFs successfully induces cell apoptosis by activating caspase-9, caspase-3, but not caspase-8 in some cells, indicating induction of intrinsic apoptosis mechanisms as well as caspase-independent cell death, like that observed in vitro with human Jurkat cells. More importantly, rats successfully treated with 1000 pulses showed protective responses to a second N1-S1 intra-hepatic challenge injection of N1-S1 cells, suggesting a host immune response, which is under exploration. As an ideal potential therapy for HCC, nsPEFs treatment did not show any damaging impact on liver blood flow. These studies demonstrate for the first time, successful intra-hepatic treatment of HCC with a potential induction of immunogenic cell death and a host immune response after nsPEF ablation. Citation Format: Ru Chen, Nova Sain, Tye Harlow, Peter Shires, Gary Long, Ricahrd Heller, Stephen Beebe. Ablation of N1-S1 hepatocellular carcinoma in rat livers with nanosecond pulsed electric fields and induction of protective immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4753. doi:10.1158/1538-7445.AM2013-4753

Since the beginning of 2020, worldwide attention has been focused on SARS-CoV-2, the second strain of the severe acute respiratory syndrome virus. Although advances in vaccine technology have been made, particularly considering the advent of mRNA vaccines, up to date, no single antigen design can ensure optimal immune response. Therefore, new technologies must be tested as to their ability to further improve vaccines. Nanosecond Pulsed Electric Field (nsPEF) is one such method showing great promise in different biomedical and industrial fields, including the fight against COVID-19. Of note, available research shows that nsPEF directly damages the cell’s DNA, so it is critical to determine if this technology could be able to fragment either viral DNA or RNA so as to be used as a novel technology to produce inactivated pathogenic agents that may, in turn, be used for the production of vaccines. Considering the available evidence, we propose that nsPEF may be used to produce inactivated SARS-CoV-2 viruses that may in turn be used to produce novel vaccines, as another tool to address 20 the current COVID-19 pandemic. Key Messages Viral inactivation by using pulsed electric fields in the nanosecond frequency. DNA fragmentation by a Nanosecond Pulsed Electric Field (nsPEF). Opportunity to apply new technologies in vaccine development.

High-intensity nanosecond pulse electric fields (nsPEF) can preferentially induce various effects, most notably regulated cell death and tumor elimination. These effects have almost exclusively been shown to be associated with nsPEF waveforms defined by pulse duration, rise time, amplitude (electric field), and pulse number. Other factors, such as low-intensity post-pulse waveform, have been completely overlooked. In this study, we show that post-pulse waveforms can alter the cell responses produced by the primary pulse waveform and can even elicit unique cellular responses, despite the primary pulse waveform being nearly identical. We employed two commonly used pulse generator designs, namely the Blumlein line (BL) and the pulse forming line (PFL), both featuring nearly identical 100 ns pulse durations, to investigate various cellular effects. Although the primary pulse waveforms were nearly identical in electric field and frequency distribution, the post-pulses differed between the two designs. The BL's post-pulse was relatively long-lasting (~50 µs) and had an opposite polarity to the main pulse, whereas the PFL's post-pulse was much shorter (~2 µs) and had the same polarity as the main pulse. Both post-pulse amplitudes were less than 5% of the main pulse, but the different post-pulses caused distinctly different cellular responses. The thresholds for dissipation of the mitochondrial membrane potential, loss of viability, and increase in plasma membrane PI permeability all occurred at lower pulsing numbers for the PFL than the BL, while mitochondrial reactive oxygen species generation occurred at similar pulsing numbers for both pulser designs. The PFL decreased spare respiratory capacity (SRC), whereas the BL increased SRC. Only the PFL caused a biphasic effect on trans-plasma membrane electron transport (tPMET). These studies demonstrate, for the first time, that conditions resulting from low post-pulse intensity charging have a significant impact on cell responses and should be considered when comparing the results from similar pulse waveforms.