Nanosecond Pulsed Electric Fields Research Articles

Nanosecond pulse electric field treatment initiates mitochondrial apoptosis pathway by inducing mitochondrial morphological changes in myocardial cells.

As an emerging myocardial ablation technique, the mechanism of nanosecond pulse electric field (nsPEF) ablation is currently less studied. Mitochondria are one of the important membrane structure organelles in cells, participating in numerous life activities within the cell. This study aimed to explore the morphological changes of mitochondria in living cells following nsPEF treatment. Myocardial cells were treated with a self-made solid-state LTD high-voltage nanosecond pulse generator with a pulse width of 100ns for 80 times. The changes in mitochondrial membrane potential and cell apoptosis in rat myocardial cells after nsPEFs were investigated using JC-1 assay kit, apoptosis double staining assay kit, and mitochondrial fluorescence probe. The results showed that after nsPEF treatment, the mitochondrial membrane potential decreased, apoptosis increased, and the average mitochondrial area decreased from 0.48 µm2 in live myocardial cells to 0.16 µm2. The average circumference ranges from 3.17µm dropped to 1.60µm. The shape factor decreased from 1.92 to 1.41. The aspect ratio has decreased from 2.16 to 1.59. nsPEF treatment induces changes in the morphology of myocardial cell mitochondria. Based on the results of mitochondrial membrane potential and apoptosis, it can be inferred that under this equipment and parameter conditions, nsPEF treatment first causes changes in mitochondrial morphology, and then initiates the mitochondrial apoptosis pathway, which may provide experimental basis for investigating the potential mechanism of nsPEF ablation of myocardial cells.

Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Enhanced Cellular Doxorubicin Uptake via Delayed Exposure Following Nanosecond Pulsed Electric Field Treatment: An In Vitro Study.

The combination of nanosecond Pulsed Electric Field (nsPEF) with pharmaceuticals is a pioneering therapeutic method capable of enhancing drug uptake efficacy in cells. Utilizing nsPEFs configured at 400 pulses, an electric field strength of 15 kV/cm, a pulse duration of 100 ns, and a repetition rate of 10 pulses per second (PPS), we combined the nsPEF with a low dose of doxorubicin (DOX) at 0.5 μM. Upon verifying that cells could continuously internalize DOX from the surrounding medium within 1 h post nsPEF exposure, we set the DOX exposure period to 10 min and contrasted the outcomes of varying sequences of DOX and nsPEF administration: pulsing followed by DOX, DOX followed by pulsing, and DOX applied 40 min after pulsing. Flow cytometry, CCK-8 assays, and transmission electron microscopy (TEM) were employed to examine intracellular DOX accumulation, cell viability, apoptosis, cell cycle, and ultrastructural transformations. Our findings demonstrate that exposing cells to DOX 40 min subsequent to nsPEF treatment can effectively elevate intracellular DOX levels, decrease cell viability, and inhibit the cell cycle. This research work presents a novel approach to enhance DOX uptake efficiency with moderate conditions of both DOX and nsPEF.

Efficacy of a surgical cardiac ablation clamp using nanosecond pulsed electric fields: An acute porcine model

ObjectiveTo examine the effectiveness of a recently developed nonthermal technology, nanosecond pulse-field ablation (nsPFA), for surgical ablation of the atria in a beating heart porcine model. MethodsSix pigs underwent sternotomy and ablation using an nsPFA parallel clamp. The ablation electrodes (53 mm long) were embedded in the jaws of the clamp. Nine lesions per pig were created in locations chosen to be representative of the Cox-maze procedure. Four lesions were intended to electrically isolate parts of the atrium: the right atrial appendage, left atrial appendage, right pulmonary veins, and left pulmonary veins. For these lesions, exit block testing was performed both after ablation and before euthanasia; the time between the 2 tests was 3.3 ± 0.5 hours (range, 2-4 hours). Using purse string sutures, 5 more lesions were created up to the superior vena cava, down to the inferior vena cava, across the right atrial free wall, and at 2 distinct locations on the left atrial free wall. The clamp delivered a train of nanosecond duration pulses, with a total duration of 2.5 seconds, independent of tissue thickness. The heart tissue was stained with 1% triphenyltetrazolium chloride after a dwelling period of 2 hours. Subsequently, each lesion was cross sectioned at 5-mm intervals to assess the ablation depth and transmurality. In some sections, transmurality could not be established on the basis of triphenyltetrazolium chloride staining alone; for these lesions, Gomori-trichrome stains were used, and the histologic sections were evaluated for transmurality. ResultsThe ablation time was 2.5 seconds per lesion, for a total of only 22.5 seconds ablation time to create 9 lesions. A total of 53 lesions were created, resulting in 388 separate histologic sections. Transmurality was established in 386 sections (99.5%). Mean tissue thickness was 3.1 ± 1.5 mm (range, 0.2-8.6 mm). Exit block was confirmed in 23 of the 24 lesions (96%) postablation and 23 of 24 (96%) before the animals were humanely killed. Over the course of the procedure, neither pulse-induced arrhythmias nor any other complications were noted. ConclusionsThe novel nsPFA clamp device was effective in creating acute conduction block and transmural lesions in both the right and left atria in an acute porcine model. This nonthermal energy source has great potential to both shorten procedural time and enable effective ablation in the beating heart.

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Ablating Myocardium Using Nanosecond Pulsed Electric Fields: Preclinical Assessment of Feasibility, Safety, and Durability.

Unlike conventional microsecond pulsed electrical fields that primarily target the cell membranes, nanosecond pulses are thought to primarily electroporate intracellular organelles. We conducted a comprehensive preclinical assessment of catheter-based endocardial nanosecond pulsed field ablation in swine. A novel endocardial nanosecond pulsed field ablation system was evaluated in a total of 25 swine. Using either a low-dose (5-second duration) or high-dose (15-second duration) strategy, thoracic veins and discrete atrial and ventricular sites were ablated. Predetermined survival periods were <1 (n=1), ≈2 (n=7), ≈7 (n=6), 14 (n=2), or ≈28 (n=9) days, and venous isolation was assessed before euthanasia. Safety assessments included evaluation of esophageal effects, phrenic nerve function, and changes in venous caliber. All tissues were subject to careful gross pathological and histopathologic examination. All (100%) veins (13 low-dose, 34 high-dose) were acutely isolated, and all reassessed veins (6 low-dose, 15 high-dose) were durably isolated. All examined vein lesions (10 low-dose, 22 high-dose) were transmural. Vein diameters (n=15) were not significantly changed. Of the animals assessed for phrenic palsy (n=9), 3 (33%) demonstrated only transient palsy. There were no differences between dosing strategies. Thirteen mitral isthmus lesions were analyzed, and all 13 (100%) were transmural (depth, 6.4±0.4 mm). Ventricular lesions were 14.7±4.5 mm wide and 7.1±1.3 mm deep, with high-dose lesions deeper than low-dose (7.9±1.2 versus 6.2±0.8 mm; P=0.007). The esophagus revealed nontransmural adventitial surface lesions in 5 of 5 (100%) animals euthanized early (2 days) post-ablation. In the 10 animals euthanized later (14-28 days), all animals demonstrated significant esophageal healing-8 with complete resolution, and 2 with only trace fibrosis. A novel, endocardial nanosecond pulsed field ablation system provides acute and durable venous isolation and linear lesions. Transient phrenic injury and nontransmural esophageal lesions can occur with worst-case assessments suggesting limits to pulsed field ablation tissue selectivity and the need for dedicated assessments during clinical studies.

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Integrated Time Nanosecond Pulse Irreversible Electroporation (INSPIRE): Assessment of Dose, Temperature, and Voltage on Experimental and Clinical Treatment Outcomes.

This study sought to investigate a novel strategy using temperature-controlled delivery of nanosecond pulsed electric fields as an alternative to the 50-100 microsecond pulses used for irreversible electroporation. INSPIRE treatments were carried out at two temperatures in 3D tumor models using doses between 0.001 s and 0.1 s. The resulting treatment zones were quantified using viability staining and lethal electric field intensities were determined numerically. Computational modeling was then used to determine parameters necessary for INSPIRE treatments to achieve equivalent treatment zones to clinical electroporation treatments and evaluate the potential for these treatments to induce deleterious thermal damage. Lethal thresholds between 1109 and 709 V/cm were found for nominal 0.01 s treatments with pulses between 350 ns and 2000 ns at physiological temperatures. Further increases in dose resulted in significant decreases in lethal thresholds. Given these experimental results, treatment zones comparable to clinical electroporation are possible by increasing the dose and voltage used with nanosecond duration pulses. Temperature-controlled simulations indicate minimal thermal cell death while achieving equivalent treatment volumes to clinical electroporation. Nanosecond electrical pulses can achieve comparable outcomes to traditional electroporation provided sufficient electrical doses or voltages are applied. The use of temperature-controlled delivery may minimize thermal damage during treatment. Intense muscle stimulation and the need for cardiac gating have limited irreversible electroporation. Nanosecond pulses can alleviate these challenges, but traditionally have produced significantly smaller treatment zones. This study suggests that larger ablation volumes may be possible with the INSPIRE approach and that future in vivo studies are warranted.

Electric field dependence of the electron drift velocity in n-type InxGa1-xAs1-yBiy epilayer

The effect of Bi incorporation into InxGa1-xAs lattice on the nanosecond pulsed electric field dependence of the drift velocity of electrons in n-type InxGa1-xAs1-yBiy alloys with various doping densities is investigated at room temperature. The electronic band structure of the alloys is calculated by Finite Element Methods (FEM). The electron temperature is determined from analytical modeling of the power dissipation mechanism. The drift velocity of the electrons (vdrift) does not saturate up to 5.4 kV/cm electric field for the lowest doping density sample. The sample is burned out at a higher electric field due to the impact ionization mechanism. However, the vdrift saturates at a high electric field region in the samples with higher doping densities, and the saturation of electric field values of vdrift depends on the doping density for these samples. The highest electron drift mobility (μdrift) is obtained as 6236 cm2/Vs for the sample with the lowest electron density. The heating of the only electrons through the applied electric field is not enough to initiate inter-valley transfer because of scatterings during transport. Hence, the electric field dependence on electron transport occurs only in the central valley. The effect of the scatterings on the electron transport is identified by considering ionized impurity and phonon scatterings.

Effects of Pulse Width and Electrical Energy of Low‐Voltage Nanosecond Pulsed Electric Fields on Mitochondria in Cancer Cells

Nanosecond pulsed electric fields (nsPEFs) have recently shown promise as a cancer therapy. Although nsPEF‐induced apoptotic responses have been observed, the stress on individual cell components has not been quantified. Therefore, the authors built a cell equivalent circuit, including mitochondria, to quantitatively calculate the voltage, electric field, and electrical energy applied to the cell components in the frequency domain. Additionally, pulse width and electrical energy changed in mitochondrial membrane potential over time. The results indicated that the change of pulsed voltage switched the cell stimulation pathway, resulting in different mitochondrial membrane potentials over time. The frequency response analysis confirmed that the potential on mitochondrial membranes increased under shorter pulse width conditions. This suggested that the frequency analysis provided in this paper is useful for relating the site of cellular stimulation to induce physiological effects. © 2024 Institute of Electrical Engineers of Japan and Wiley Periodicals LLC.

Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing.

Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application. To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs. The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification. In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation. Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.

An integrated nanofiller spray and nanosecond pulse electrically-assisted method for synergistically interlaminar toughening and in-situ damage monitoring of CFRP composites

Nanomaterials have already been explored their potential for interlaminar toughening and crack-monitoring capabilities within the field of composite materials. However, efficient enhancement of interlaminar fracture toughness remains an unresolved challenge. Herein, a novel integrated filler spray and nanosecond pulsed electric field (nsPEF)-assisted curing technique is proposed for the fabrication of high-performance composite material system for the first time. The spraying technique proves to be an effective approach for depositing fillers, including carbon nanotubes (CNTs), graphene nanoplatelets (GNPs), and carbon nanofibers (CNFs), onto carbon fiber prepreg. Meanwhile, the nsPEF-assisted technique is utilized to enhance the wettability of resin to fiber, thus reducing void defects. The efficacy of this integrated technique is evidenced by the particularly low content of fillers (0.047 wt%). The experimental results show a substantial reduction of porosity in the composite by 49.36% (from 1.56% to 0.79%). Meanwhile, the mode I initiation and steady-state toughness of samples with CNTs and GNPs are remarkably improved by up to 47% and 82%, respectively, compared to the baseline. This represents the most significant improvement reported to date for non-functionalized CNTs and GNPs at such low concentrations. Moreover, the relative electrical resistance variation (ΔR/R0%) of CNT/GNP sensors used for in-situ damage sensing increased by ∼ 14%. This work pioneers the investigation of the synergistic effects of two promising techniques for preparing composite materials, offering advantages in terms of simplicity, multifunctionality, and the potential for industrial scalability.

Synergistic effects of nanosecond pulsed plasma and electric field on inactivation of pancreatic cancer cells in vitro

Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on the metabolic activities of cells and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for > 2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, > 20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-fivefold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.

Macrophages enhanced with nanosecond pulsed electric fields and TGF-β1 strengthened the recovery of spinal cord injury.

Macrophages enhanced with nanosecond pulsed electric fields and TGF-β1 strengthened the recovery of spinal cord injury.

Investigation of lethal thresholds of nanosecond pulsed electric field in rabbit VX2 hepatic tumors through finite element analysis and verification with a single-needle bipolar electrode: A prospective strategy employing three-dimensional comparisons

Pulsed electric field has emerged as a promising modality for the solid tumor ablation with the advantage in treatment planning, however, the accurate prediction of the lesion margin requires the determination of the lethal electric field (E) thresholds. Herein we employ the highly repetitive nanosecond pulsed electric field (RnsPEF) to ablate the normal and VX2 tumor-bearing livers of rabbits. The ultrasound-guided surgery is operated using the conventional double- and newly devised single-needle bipolar electrodes. Finite element analysis is also introduced to simulate the E distribution in the practical treatments. Two- and three-dimensional investigations are performed on the image measurements and reconstructed calcification models on micro-CT, respectively. Specially, an algorithm considering the model surface, volume and shape is employed to compare the similarities between the simulative and experimental models. Blood vessel injury, temperature and synergistic efficacy with doxorubicin (DOX) are also investigated. According to the three-dimensional calculation, the overall E threshold is 4536.4 ± 618.2 V/cm and the single-needle bipolar electrode is verified to be effective in tissue ablation. Vessels are well preserved and the increment of temperature is limited. Synergy of RnsPEF and DOX shows increased apoptosis and improved long-term tumor survival. Our study presents a prospective strategy for the evaluation of the lethal E threshold, which can be considered to guide the future clinical treatment planning for RnsPEF.

Inflammasome Activation and IL-1β Release Triggered by Nanosecond Pulsed Electric Fields in Murine Innate Immune Cells and Skin.

Although electric field-induced cell membrane permeabilization (electroporation) is used in a wide range of clinical applications from cancer therapy to cardiac ablation, the cellular- and molecular-level details of the processes that determine the success or failure of these treatments are poorly understood. Nanosecond pulsed electric field (nsPEF)-based tumor therapies are known to have an immune component, but whether and how immune cells sense the electroporative damage and respond to it have not been demonstrated. Damage- and pathogen-associated stresses drive inflammation via activation of cytosolic multiprotein platforms known as inflammasomes. The assembly of inflammasome complexes triggers caspase-1-dependent secretion of IL-1β and in many settings a form of cell death called pyroptosis. In this study we tested the hypothesis that the nsPEF damage is sensed intracellularly by the NLRP3 inflammasome. We found that 200-ns PEFs induced aggregation of the inflammasome adaptor protein ASC, activation of caspase-1, and triggered IL-1β release in multiple innate immune cell types (J774A.1 macrophages, bone marrow-derived macrophages, and dendritic cells) and invivo in mouse skin. Efflux of potassium from the permeabilized cell plasma membrane was partially responsible for nsPEF-induced inflammasome activation. Based on results from experiments using both the NRLP3-specific inhibitor MCC950 and NLRP3 knockout cells, we propose that the damage created by nsPEFs generates a set of stimuli for the inflammasome and that more than one sensor can drive IL-1β release in response to electrical pulse stimulation. This study shows, to our knowledge, for the first time, that PEFs activate the inflammasome, suggesting that this pathway alarms the immune system after treatment.

Nanosecond pulsed electric field stimulates CD103+ DC accumulation in tumor microenvironment via NK-CD103+ DC crosstalk

CD103+ DC is crucial for antitumor immune response. As a promising local therapy on cancers, nanosecond pulsed electric field (nsPEF) has been widely reported to stimulate anti-tumor immune response, but the underlying relationship between intratumoral CD103+ DC and nsPEF treatment remains enigmatic. Here, we focused on the behavior of CD103+ DC in response to nsPEF treatment and explored the underlying mechanism. We found that the nsPEF treatment led to the activation and accumulation of CD103+ DC in tumor. Depletion of CD103+ DC via Batf3−/− mice demonstrated CD103+ DC was necessary for intratumoral CD8+ T cell infiltration and activation in response to nsPEF treatment. Notably, NK cells recruited CD103+ DC into nsPEF-treated tumor through CCL5. Inflammatory array revealed CD103+ DC-derived IL-12 mediated the CCL5 secretion in NK cells. In addition, the boosted activation and infiltration of intratumoral CD103+ DC were abolished by cGAS-STING pathway inhibition, following IL-12 and CCL5 decreasing. Furthermore, nsPEF treatment promoting CD103+ DC-mediated antitumor response enhanced the effects of CD47 blockade strategy. Together, this study uncovers an unprecedented role for CD103+ DC in nsPEF treatment-elicited antitumor immune response and elucidates the underlying mechanisms.

Low-Intensity Nanosecond Pulsed Electric Field Accelerates Osteogenic Transformation of Human Dermal Fibroblasts by Enhancing Cell Pluripotency.

Autologous human fibroblasts have the potential to differentiate into the osteogenic lineage under specific conditions and can be utilized for bone regeneration. However, their efficiency is currently unsatisfactory. Recently, low-intensity nanosecond pulsed electric field (nsPEF) stimulation has been demonstrated to enhance cell pluripotency by activating epigenetic regulatory pathways. In this study, human dermal fibroblasts were exposed to different intensities of nsPEF to assess whether these exposures resulted in changes in proliferation rate, calcium salt deposition, and expression of differentiation-related markers in different experimental groups. The results showed a significant increase in cell proliferation, pluripotency, bone marker expression, and osteogenic differentiation efficiency when stimulating cells with 5 kV/cm of nsPEF. However, cell proliferation and differentiation significantly decreased at 25 kV/cm. Additionally, the proliferation and efficiency of osteogenic differentiation were reduced when the nsPEF intensity was increased to 50 kV/cm. Treatment with a 5 kV/cm of nsPEF led to increased and concentrated expression of Yes-Associated Protein (YAP) in the nucleus. These observations suggest that human dermal fibroblasts possess a heightened potential to differentiate into osteogenic cells when activated with nsPEF at 5 kV/cm. Consequently, the nsPEF strengthening strategy shows promise for fibroblast-based tissue-engineered bone repair research.

Dispersive Modeling of Normal and Cancerous Cervical Cell Responses to Nanosecond Electric Fields in Reversible Electroporation Using a Drift-Step Rectifier Diode Generator.

This paper creates an approximate three-dimensional model for normal and cancerous cervical cells using image processing and computer-aided design (CAD) tools. The model is then exposed to low-frequency electric pulses to verify the work with experimental data. The transmembrane potential, pore density, and pore radius evolution are analyzed. This work adds a study of the electrodeformation of cells under an electric field to investigate cytoskeleton integrity. The Maxwell stress tensor is calculated for the dispersive bi-lipid layer plasma membrane. The solid displacement is calculated under electric stress to observe cytoskeleton integrity. After verifying the results with previous experiments, the cells are exposed to a nanosecond pulsed electric field. The nanosecond pulse is applied using a drift-step rectifier diode (DSRD)-based generator circuit. The cells' transmembrane voltage (TMV), pore density, pore radius evolution, displacement of the membrane under electric stress, and strain energy are calculated. A thermal analysis of the cells under a nanosecond pulse is also carried out to prove that it constitutes a non-thermal process. The results showed differences in normal and cancerous cell responses to electric pulses due to changes in morphology and differences in the cells' electrical and mechanical properties. This work is a model-driven microdosimetry method that could be used for diagnostic and therapeutic purposes.

Negative effects of cancellation during nanosecond range High-Frequency calcium based electrochemotherapy in vitro

Drug delivery using nanosecond pulsed electric fields is a new branch of electroporation-based treatments, which potentially can substitute European standard operating procedures for electrochemotherapy. In this work, for the first time, we characterize the effects of ultra-fast repetition frequency (1–2.5 MHz) nanosecond pulses (5–9 kV/cm, 200 and 400 ns) in the context of nano-electrochemotherapy with calcium. Additionally, we investigate the feasibility of bipolar symmetric (↑200 ns + ↓200 ns) and asymmetric (↑200 ns + ↓400 ns) nanosecond protocols for calcium delivery. The effects of bipolar cancellation and the influence of interphase delay (200 ns) are overviewed. Human lung cancer cell lines A549 and H69AR were used as a model. It was shown that unipolar pulses delivered at high frequency are effective for electrochemotherapy with a significant improvement in efficiency when the delay between separate pulses is reduced. Bipolar symmetric pulses trigger the cancellation phenomenon limiting applications for drug delivery and can be compensated by the asymmetry of the pulse (↑200 ns + ↓400 ns or ↑400 ns + ↓200 ns). The results of this study can be successfully used to derive a new generation of nsPEF protocols for successful electrochemotherapy treatments.