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Electroporation technique induced by high-voltage pulses has been successfully used to ablate tumor cells while preserving the function of normal blood vessels. Generally, the tumor blood vessels can provide a pathway to draw nutrients for tumor growth and contribute to invasion and metastasis, which is an obstacle to tumor treatment. The electroporation study of the endothelial cell, which is important in the vasculature microenvironment, is helpful to investigate the influence on both tumor and normal blood vessels. This study built a multicell-layer model of the vascular microenvironment to investigate the discriminative electroporation effect between normal and tumor blood vessels by high-frequency bipolar pulses (HFBPs) and monopolar pulses (MPs). The simulation results showed that both pore number and electroporation region in normal blood vessels are significantly lower than those in tumor blood vessels. The rich vascular smooth muscle cells existed in the normal blood vessels play a protective function for endothelial cells, compared with tumor blood vessels. However, the differences in pore number and electroporation region between normal and tumor blood vessels are gradually smaller with an increased electric field, which demonstrates that the electroporation pulse with higher intensity damages both normal and tumor blood vessels. HFBPs generate a weaker electroporation effect on both normal and tumor blood vessels than traditional MP. However, HFBPs are more suitable to electroporate tumor blood vessels, while preserving the normal blood vessels. Moreover, this study could also provide a multicell-layer model that can be used to analyze the cell electroporation effect in the vascular microenvironment. [ABSTRACT FROM AUTHOR]

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.

Electroporation is a complex, iterative, and nonlinear phenomenon often studied through numerical simulations. In recent years, simulations of tissue electroporation have been conducted with static models. However, the results of a static model simulation are restricted to a fixed protocol signature of the pulsed electric field. In this paper, we describe a novel dynamic model of tissue electroporation that also accounts for tissue dispersion and temperature to allow time-domain simulations. We have implemented the biological dispersion of potato tubers and thermal analysis in a commercial finite-element method software. A cell electroporation model was adapted to account for the increase in tissue conductivity. The model yielded 12 parameters divided into three dynamic states of electroporation. The thermal analysis describes the dependence of tissue conductivity on temperature. The model parameters were evaluated using experiments with vegetal tissue (Solanum tuberosum) under electrochemotherapy protocols. The proposed model can accurately predict the conductivity of tissue under electroporation from 100 to 1000 V/cm. A negligible thermal effect was observed at 1000 V/cm, with a temperature increase of 0.89 ° C. We believe that the proposed model is suitable to describe the electroporation at the tissue level and provides a hint of the effects on the cell membrane. [ABSTRACT FROM AUTHOR]

This document, published in the Journal of Applied Physics, discusses the importance of multiscale modeling and analysis in the field of biophysics. The authors emphasize the need for tools and analyses that integrate across physical scales and time scales in order to advance medical science. The document includes a collection of studies on various topics such as cardiac biomechanics, tissue mechanics, cerebral fluid flow, cell mechanics, protein modeling, and molecular fluorescence microscopy. The authors hope that readers will find these studies informative and inspiring. [Extracted from the article]

From the microscopic electroporation to the irregular distribution of cell populations, the inactivation efficacy of pulsed electric fields (PEFs) from in vitro experiments has lacked a unified physical model due to its cross-scale complexity. Inspired by a coarse-grained approach from the percolation theory, the inactivation process is simulated from a simple yet robust lattice model, where the spatiotemporal heterogeneity of the collective structure and the stochastic PEF strike are portrayed as random matrices, while also accounting for the rules of single-cell electroporation and subsequent death. Beyond successfully simulating the inactivation of monolayer adherent cells and suspended cells, which are in good agreement with in vitro results, our model reveals that (1) macroscopically three-staged inactivation pattern originates from the "accelerate–uniform–decelerate" transition of inactivation velocity, and (2) the inactivation patterns obey a universal scaling law under varied field strength, which is not satisfied under varied pulsed widths. The simulation not only sheds light on the PEF inactivation of the macroscopic cell collectives but also provides a simple and generalized numerical method for predicting PEF efficacy in experiments or engineering. [ABSTRACT FROM AUTHOR]

BACKGROUND: The effect of contact force (CF) on lesion formation is not clear during pulsed field ablation (PFA). The aim of this study was to evaluate the impact of CF, PFA, and their interplay through the PFA index (PF index) formula on the ventricular lesion size in swine. METHODS: PFA was delivered through the CF-sensing OMNYPULSE catheter. Predefined PFA applications (×3, ×6, ×9, and ×12) were delivered maintaining low (5-25 g), high (26-50 g), and very high (51-80 g) CFs. First, PFA lesions were evaluated on necropsy in 11 swine to investigate the impact of CF/PFA-and their integration in the PF index equation-on lesion size (study characterization). Then, 3 different PF index thresholds-300, 450, and 600-were tested in 6 swine to appraise the PF index accuracy to predict the ventricular lesion depth (study validation). RESULTS: In the study characterization data set, 111 PFA lesions were analyzed. CF was 32±17 g. The average lesion depth and width were 3.5±1.2 and 12.0±3.5 mm, respectively. More than CF and PFA dose alone, it was their combined effect to impact lesion depth through an asymptotically increasing relationship. Likewise, not only was the PF index related to lesion depth in the study validation data set (r2=0.66; P

Chimeric antigen receptor T (CAR-T) therapy holds promise for cancer treatment but faces challenges with using fresh patient cells, including manufacturing failures and logistical hurdles. Cryopreserved peripheral blood mononuclear cells (PBMCs) offer a potential solution, and while lentiviral processes have been reported for generating CAR-T from these cells, few studies have demonstrated successful PiggyBac electroporation methods. Therefore, the objectives of our study were twofold: Firstly, to conduct a comparative study on cryopreserved PBMCs, fresh PBMCs, and their respective preparations of CAR-T. Secondly, to establish a PiggyBac electroporation CAR-T preparation process using cryopreserved PBMCs through process optimization. The results revealed that long-term frozen PBMCs viability in a relatively stable manner. CAR-T generated from cryopreserved PBMCs exhibited comparable expansion potential, cell phenotype, differentiation profiles, exhaustion markers, and cytotoxicity against human ovarian cancer cell line (SKOV-3) cells to those derived from fresh PBMCs. Moreover, through process optimization, we further enhanced the proliferation and toxicity of CAR-T. This approach has the potential to revolutionize the CAR-T production model by utilizing healthy donor cells instead of patient cells. This shift could mitigate issues affecting treatment efficacy, such as suboptimal cell condition following illness or delays in cell preparation. [ABSTRACT FROM AUTHOR]

Cold atmospheric plasma (CAP) treatment induces cancer cell death through the generation of reactive oxygen and nitrogen species (RONS). However, the efficacy of RONS delivery into cells remains limited by membrane permeability. Here, we investigated whether combining CAP with pulsed electric fields (PEFs) could enhance cancer cell death through increased intracellular RONS uptake. HeLa cells were treated with argon atmospheric pressure plasma jet (Ar-APPJ), PEF, or their combination. The combined treatment showed significantly enhanced cell death compared to single treatments. While PEF treatment alone induced membrane permeabilization, the combination with Ar-APPJ resulted in more pronounced and sustained membrane disruption, as evidenced by increased calcein leakage. This enhanced effect was attributed to Ar-APPJ-induced lipid peroxidation interfering with membrane resealing after PEF-induced electroporation. We also demonstrated that PEF-induced membrane electroporation facilitates the intracellular uptake of CAP-generated RONS. These findings provide mechanistic insights into the synergistic effects of combined CAP and PEF treatments, suggesting enhanced cell death via multiple pathways. [ABSTRACT FROM AUTHOR]

Electric stimulation (ES) is a versatile technique that uses an electric field to manipulate microorganisms individually. Over the past several decades, the capabilities of ES have expanded from bioremediation to the precise motion control of cells and microorganisms. However, there is limited information on the underlying mechanisms, latest advancement and broader microbial applications of ES in various fields, such as the production of extracellular polymers with upgraded properties. This review article summarizes recent advancements in ES and discusses it as a unique external manipulation technique for microorganisms with wide applications in bioremediation, industry, biofilm deactivation, disinfection, and controlled biosynthesis. One specific application of ES discussed in this review is the extracellular biosynthesis, regulation, and organization of extracellular polymers, such as bacterial cellulose nanofibrils, curdlan, and microbial nanowires. Overall, this review aims to provide a platform for microbial biotechnologists and synthetic biologists to leverage the manipulation of microorganisms using ES for bio-based applications, including the production of extracellular polymers with enhanced properties. Researchers can engineer, manipulate, and control microorganisms for various applications by harnessing the potential of electric fields. [ABSTRACT FROM AUTHOR]

Electroporation and electrofusion are efficient methods, which have been widely used in different areas of biotechnology and medicine. Pulse strength and width, as an external condition, play an important role in the process of these methods. However, comparatively little work has been done to explore the effects of pulsed electric field parameters on electroporation and electrofusion. Herein, influences of pulse strength and width on the electroporation and electrofusion of phospholipid bilayers were systematically investigated by using experiments combined with molecular dynamics simulations. Experimental results and machine learning-based regression analysis showed that the number of pores is mainly determined by pulse strength, while the sizes of pores were enlarged by increasing the pulse widths. In addition, the formation of large-size pores is the most crucial factor that affects the fusion rate of myeloma cells. The same trend has taken place on coarse-grained and all-atom MD simulations. The result suggested that electroporation events occur only in an electric field exceeding the strength of threshold, and the unbalanced degree of electric potential between two membranes leads to pores formation during the process of electroporation. Generally, this work provides a comprehensive understanding of how pulse strength and width govern the poration event of bilayer lipid membranes, as well as guidance on the experimental design of electrofusion. [ABSTRACT FROM AUTHOR]

Background: In the realm of cancer treatment, sono–X-ray–photodynamic therapy (SXPDT) has garnered significant interest as a novel therapeutic approach. The essential part of SXPDT is the sensitizer, which under X-ray photon and ultrasound sono-irradiation may transform sono and photo-energy into cytotoxic molecules. Photon absorption, targeting, penetration, and oxygen dependence remain challenges in sono–X-ray–photosensitizer (SXPs) design. Rapid advancements in material science have prompted the creation of several SXPs that create cytotoxic species with great selectivity, safety, and noninvasiveness for the treatment of tumors. The current study aims to provide an advanced method of activated cancer treatment by using electroporation to assist the delivery of Roussin salts porphyrin-based conjugated carbon nanoparticles (EP@RRBP-CNP) for the sono–X-ray–photodynamic prostate cancer (PCa) in vivo and in vitro treatment. Materials and methods: Human PCa cells (DU-145) were used in the in vitro study, and the in vivo application groups of the study protocol were Swiss albino mice treated with N-methyl-N-nitrosourea (MNU) / testosterone only; they were not given any treatment to induce PCa. The study treatment protocol started only after PCa induction, and involved daily administration of EP@RRBP-CNP as SXPDT sensitizer whether or not to be exposed to photo–(X-ray) or sono–(US) or a combination of them for 3 min for a period of 2 weeks. Results: Indicated that CNP is a useful RRBP delivery mechanism that targets PCa cells directly. Furthermore, EP@RRBP-CNP is a promising SXPS that, when used in conjunction with SXPDT, can be very effective in in vitro treating PCa-DU-145 (in a dose-dependent manner cell viability declined, an increase in the cells population during the G0/G1-phase indicates that the cell cycle was arrested, and an increase in cell population in the Pre-G, autophagic cell death, as well as necrosis and early and late apoptosis, indicate that cell death was induced) and MNU/testosterone-PCa-induced mice in vivo (induced antiproliferative genes, p53, Bax, TNFalpha, caspase 3,9, repressed antiangiogenic and antiapoptotic genes, VEGF and Bcl2, respectively), successfully slowing the growth of tumors and even killing cancer cells, as well as lowering oxidative stress (MDA), improving the functions of the kidneys (urea, creatinine), liver (ALT, AST), and antioxidants (GPx, GPx, GST, CAT, GSH, TAC). SXPDT, the X-ray photo- or sono-chemical RRBP activation mechanism, and the antioxidant capacity of non-activated RRBP can all be linked to this process. Conclusion: On the bases of the findings, EP@RRBP-CNP shows a great promise as a novel, efficient selective delivery system for localized SXPDT-activated prostate cancer treatment. [ABSTRACT FROM AUTHOR]

Gelonin is a ribosome-inactivating protein with extreme intracellular toxicity but poor permeation into cells. Targeted disruption of cell membranes to facilitate gelonin entry is explored for cancer and tissue ablation. We demonstrate a hundreds- to thousands-fold enhancement of gelonin cytotoxicity by pulsed electric fields in the T24, U-87, and CT26 cell lines. The effective gelonin concentration to kill 50% of cells (EC50) after electroporation ranged from <1 nM to about 100 nM. For intact cells, the EC50 was unattainable even at the highest gelonin concentration of 1000 nM, which reduced cell survival by only 5–15%. For isoeffective electroporation treatments using 300 ns, 9 µs, and 100 µs pulses, longer pulses were more efficient at lowering gelonin EC50. Increasing the electric field strength of 8, 100 µs pulses from 0.65 to 1.25 kV/cm reduced gelonin EC50 from 128 nM to 0.72 nM. Conversely, the presence of 100 nM gelonin enabled a more than 20-fold reduction in the number of pulses required for equivalent cell killing. Pulsed electric field-mediated delivery of gelonin shows promise for hyperplasia ablation at concentrations sufficiently low to minimize or avoid systemic toxicity. [ABSTRACT FROM AUTHOR]

This study investigated the effects of pulsed electric field (PEF) treatment on the peeling efficiency and textural properties of whiteleg shrimp (Litopenaeus vannamei). Shrimp samples were treated at field strengths of 0, 1.0, 1.5, and 2.0 kV/cm to assess PEF impact on peeling force, incomplete peeling percentage, and texture profile. The results showed that PEF treatment significantly reduced the peeling force from 50.88 N in controls to 42.99 N at 2.0 kV/cm, while the percentage of incompletely peeled shrimp decreased from 27.5% to 15.9%. Texture profile analysis indicated that PEF treatment had no impact on the key properties of hardness and chewiness (no significant difference), with a reduction in springiness observed at higher field strengths. Improvements in peelability are attributed to electroporation, which disrupts collagen in the connective tissue between the shrimp shell and muscle. These findings indicate that PEF treatment is an efficient, non-thermal method for enhancing shrimp peeling processes while preserving textural integrity. PEF technology offers a promising alternative to traditional mechanical and thermal methods in the seafood processing industry. [ABSTRACT FROM AUTHOR]

This study aims to explore the effect of pulsed electric field (PEF) treatment as a method very likely to result in reversible electroporation of Scutellaria baicalensis Georgi underground organs, resulting in increased mass transfer and secondary metabolites leakage. PEF treatment with previously established empirically tailored parameters [E = 0.3 kV/cm (U = 3 kV, d = 10 cm), t = 50 µs, N = 33 f = 1 Hz] was applied 1–3 times to S. baicalensis roots submerged in four different Natural Deep Eutectic Solvents (NADES) media (1—choline chloride/xylose (1:2) + 30% water, 2—choline chloride/glucose (1:2) + 30% water, 3—choline chloride/ethylene glycol (1:2), and 4—tap water (EC = 0.7 mS/cm). Confocal microscopy was utilized to visualize the impact of PEF treatment on the root cells in situ. As a result of plant cell membrane permeabilization, an extract containing major active metabolites was successfully acquired in most media, achieving the best results using medium 1 and repeating the PEF treatment twice (baicalein

Objective Electroporation (EP) is a method in which the membrane permeability is increased by applying electrical pulses. The determination of modifications that occur in cells subsequent to EP with varying pulse parameters holds significant importance in establishing the foundations of EP theory. Therefore, we sought clarification regarding the phenomenon of pore formation on the membrane of the electroporated human cervical cancer cell line (HeLa) cells. Methods The pores created on the cell membrane due to EP was observed using a scanning electron microscope. The change in the viability and mitochondrial membrane potential (ΔΨm) of cells was determined by WST-8 and JC-1 assays. Results The surface of the electroporated cell membrane exhibited a relatively uniform pore population. The viability of HeLa cells was significantly reduced with increasing electric field intensities. A slight decrease in ΔΨm was observed between the control and the 0.8 and 1.6 kV/cm EP groups, but ΔΨm was higher in the 2.4 and 3.2 kV/cm EP groups compared to the control group. Conclusion In conclusion, our study showed that the application of EP to the cervical cancer cell line resulted in the formation of pores of varying sizes on the membrane. While cell viability decreased with increasing electric field amplitude, no significant change was observed in ΔΨm between EP treatment and control groups. It should be noted that further research is needed to determine the pore distributions in electroporated cells and the resulting changes at different electric field amplitudes. Keywords:Electroporation, cervical cancer, pore size, cell viability, mitochondrial membrane potential: [ABSTRACT FROM AUTHOR]

The primary method of treatment for patients suffering from drug-resistant focal-onset epilepsy is resective surgery, which adversely impacts neurocognitive function. Radio frequency (RF) ablation and laser ablation are the methods with the most promise, achieving seizure-free rates similar to resection but with less negative impact on neurocognitive function. However, there remains a number of concerns and open technical questions about these two methods of thermal ablation, with the primary ones: (1) heating; (2) hemorrhage and bleeding; and (3) poor directionality. Irreversible electroporation (IRE) is a proven method of focal ablation, which circumvents all three of the primary concerns regarding focal RF and laser ablation. Here, we demonstrate the in vivo application of a flexible implant with organic electrodes for focal ablation of epilepsy foci using high-frequency IRE (H-FIRE) in mice. Our results show that local, targeted ablation is possible in the close neighborhood of the electrode, paving the way for the clinical application in the treatment of focal epilepsy. [ABSTRACT FROM AUTHOR]

Purpose: Pulsed electrical field (PEF) ablation is an energy-based technique used to treat a range of cancers by irreversible electroporation (IRE). Our objective was to use computational and plant-based models to characterize the electric field distribution and ablation zones induced with a commercial 8-needle array-based applicator intended for treatment of skin cancer when high-frequency IRE (H-FIRE) pulses are applied. Electric field characterisation of this device was not previously assessed. Methods: Vegetable experimental were conducted using parallel plate electrodes to obtain the lethal threshold for H-FIRE pulses. Then a 3D computational model of the applicator was built mimicking the experimental conditions. The computational ablation zones were validated with the experiments for different voltage levels ranging from 220 to 525 V. Results: A threshold of 453 V/cm was estimated for H-FIRE pulses, which was used to predict computationally the ablation zones. It was found that the model showed a low prediction error, ranging from 2% for the minor diameter to 4.5% for the depth compared with experiments. Voltages higher than 370 V created an ablation volume with a rectangular prism shape determined by the positions of the needles, whereas lower voltages provoked the appearance of untreated areas (gaps). Conclusions: Our computer model predicts reasonably well the ablation zone induced by H-FIRE pulses, suggesting that a sufficiently large voltage must be applied to avoid the appearance of gaps. The validated model with vegetable experiments could serve as the basis for future computer studies to predict the behaviour of this device on heterogeneous tissues. [ABSTRACT FROM AUTHOR]

Transdermal drug delivery (TDD) represents a transformative paradigm in drug administration, offering advantages such as controlled drug release, enhanced patient adherence, and circumvention of hepatic first-pass metabolism. Despite these benefits, the inherent barrier function of the skin, primarily attributed to the stratum corneum, remains a significant impediment to the efficient permeation of therapeutic agents. Recent advancements have focused on macromolecular-assisted permeation enhancers, including carbohydrates, lipids, amino acids, nucleic acids, and cell-penetrating peptides, which modulate skin permeability by transiently altering its structural integrity. Concurrently, innovative methodologies such as iontophoresis, electroporation, microneedles, ultrasound, and sonophoresis have emerged as potent tools to enhance drug transport by creating transient microchannels or altering the skin's microenvironment. Among the novel approaches, the development of nanocarriers such as Liposome, niosomes, and transethosomes etc. has garnered substantial attention. These elastic vesicular systems, comprising lipids and edge activators, exhibit superior skin penetration owing to their deformability and enhanced payload delivery capabilities. Furthermore, the integration of nanocarriers with physical enhancement techniques demonstrates a synergistic potential, effectively addressing the limitations of conventional TDD systems. This comprehensive convergence of macromolecular-assisted enhancers, advanced physical techniques, and next-generation nanocarriers underscores the evolution of TDD, paving the way for optimized therapeutic outcomes. [ABSTRACT FROM AUTHOR]

Background: We have previously developed a candidate therapeutic HPV DNA vaccine (pBI-11) encoding mycobacteria heat shock protein 70 linked to HPV16/18 E6/E7 proteins for the control of advanced HPV-associated oropharyngeal cancer (NCT05799144). While naked DNA vaccines are readily produced, stable, and well tolerated, their potency is limited by the delivery efficiency. Here we compared three different IM delivery strategies, including intramuscular (IM) injection, either with a needle alone or with electroporation at the injection site, and a needle-free injection system (NFIS), for their ability to elicit gene expression and to improve the potency of pBI-11 DNA vaccine. Results: We found that electroporation after IM injection significantly increases gene expression from a luciferase-encoding DNA construct compared to IM injection alone or NFIS. We also showed that single administration of pBI-11 DNA via electroporation-mediated delivery generates the greatest increase in HPV antigen-specific CD8 + T cell-mediated immune responses, resulting in the most potent antitumor effect compared to the other two methods. We further compared the response to three repeat immunizations via each of these different methods. We found that electroporation-mediated delivery of pBI-11 DNA generates the greatest HPV antigen-specific CD8 + T cell immune responses and therapeutic antitumor effects compared to the other two methods. Monitoring of mouse behaviors and body weight, and necropsy indicated that electroporation-mediated delivery of clinical grade pBI-11 DNA vaccine was well-tolerated and presented no evident local or systemic toxicity. Conclusions: These findings provide rationale for clinical testing of pBI-11 DNA vaccine delivered by electroporation for the control of HPV16/18-associated infections and/or cancers. [ABSTRACT FROM AUTHOR]

Wearing face masks is an effective non‐pharmaceutical strategy to inhibit the transmission of airborne diseases. Nonetheless, most commercial face masks lack disinfecting capabilities, posing risks of secondary transmission, while those with antimicrobial coatings are incapable of instant pathogen inactivation. Herein, a proof‐of‐concept instant disinfecting face mask is demonstrated consisting of a Cu(OH)2‐nanowire electroporation filter powered by a respiration‐driven triboelectric nanogenerator (R‐TENG). The R‐TENG comprises electrospun polyvinyl alcohol (PVA) and polyvinylidene fluoride (PVDF) membranes. A dome‐shaped PVA membrane is specifically designed to maximize the effective contact area. Coupling the novel dome‐shape design with maximized β‐phase crystallites of PVDF, the enhanced R‐TENG generates an open‐circuit voltage of 120 V under the normal breathing rate of an adult, resulting in an amplified electric field of 19 MV m−1 at the nanowire tips of the electroporation filter. Under this high localized electric field, effective inactivation of bacteria (>99.9%) is achieved via a synergy of electroporation and physical penetration. Conversely, when the R‐TENG is disconnected, only partial inactivation (90%–99%) via physical penetration is realized. For the first time, it shows that R‐TENG powered electroporation is a viable solution to achieve instant, effective disinfection, paving the way for next‐generation face masks. [ABSTRACT FROM AUTHOR]

Gene electrotransfer (GET) is a physical method of gene delivery to various tissues utilizing pulsed electric fields to transiently permeabilize cell membranes to allow for genetic material transfer and expression. Optimal pulsing parameters dictate gene transfer efficiency and cell survival, which are critical for the wide adaptation of GET as a gene therapy technique. Tissue heterogeneity complicates the delivery process, requiring the extensive optimization of pulsing protocols currently empirically optimized. These experiments are time-consuming and resource-intensive, requiring large numbers of animals for in vivo optimization. Advances in machine learning (ML) and computing power, data analysis, and model generation using ML techniques, such as neural networks, enable predictive modeling for GET. ML models have been used previously to predict ablation performance in irreversible electroporation procedures and single-cell electroporation platforms. In this work, we present ML predictive models that could be used to optimize pulsing parameters based on already completed experiments. The models were trained on 132 data points from 19 papers with the Matlab Statistics and Machine Learning Toolbox. An artificial neural network (ANN) was generated that could predict binary treatment outcomes with an accuracy of 71.8%. Support vector machines (SVMs) using selected features based on χ 2 tests were also explored. All models used a maximum of 24 features as input, spread across target species, needle configuration, pulsing parameters, and plasmid parameters. Pulse voltage and pulse width dominated as the critical parameters, followed by field strength, dose, and electrode with the greatest impact on GET efficiency. This study elucidates areas where predictive ML algorithms may ideally inform GET study design to accelerate optimization and improve efficiencies upon the further training of these models. [ABSTRACT FROM AUTHOR]

Bacillus subtilis has been widely used as a biological control agent in agricultural production. Environmental strains of B. subtilis are an important source of biological control agents. However, due to its low genetic transformation efficiency, the genetic manipulation of the environmental and nondomesticated strains is challenging. In this study, the impact of competent cell preparation, pulse electroporation, and recovery culture on the electroporation efficiency of B. subtilis GLB191 was assessed utilizing response surface methodology. Results indicated that the concentration of glycine, DL-threonine, and Tween 80 used in a cell wall weakening solution during competent cell preparation, and the voltage applied during pulse electroporation were the primary factors affecting electroporation efficiency. Optimization of these factors led to nearly a three-fold increase (reaching 74.00 ± 5.10 CFU/µg DNA) in electroporation efficiency. The elimination of dam and dcm modifications to mitigate the influence of host restriction-modification systems was integrated to further increase the electroporation efficacy. An electroporation efficiency for replicative plasmids of 1.96 ± 0.05 × 106 CFU/µg DNA was achieved using the optimized strategy. Utilizing this improved methodology, the temperature-sensitive plasmid pJOE8899 was efficiently transformed into B. subtilis GLB191, resulting in a markerless knockout of pdeH. The optimized transformation strategy significantly enhances the efficiency of markerless genome editing of nondomesticated B. subtilis, offering the potential for future interpretation of their modes of action, which is critical for the development of the nondomesticated B. subtilis strains. [ABSTRACT FROM AUTHOR]

Creating genetically modified (GM) animals using CRISPR/Cas mediated through the electroporation of two-cell stage embryos, rather than fertilized eggs, holds considerable potential. The full potential of genome editing using two-cell stage embryos is only beginning to be explored. We developed an improved electroporation method to prevent blastomere fusion in two-cell-stage embryos, enabling efficient genome editing. Using this method, we demonstrated that the indel mutation rates and ssODN knock-in (KI) efficiencies in two-cell-stage embryos are comparable to those in fertilized eggs, with a tendency for higher efficiency in long DNA KI. This study highlights the potential value of two-cell-stage embryos and provides enhanced animal model production opportunities. Furthermore, realizing genome editing in two-cell-stage embryos extends the editing timeframe from fertilized egg to two-cell-stage embryo, offering promising avenues for future research in embryo genome editing techniques. [ABSTRACT FROM AUTHOR]

Simple Summary: Although in most cases breast cancer is curable, some specific subtypes and late stages of the disease are difficult to treat, and treatment-related side effects often are severe. Two methods that focus on the local treatment of breast cancer, regional chemotherapy and electroporation, can be combined to increase the treatment efficacy at the tumor site and decrease systemic side effects. This publication explains the technique, systemic impact on circulating tumor cells, and first results of this treatment approach. Background: Relapsed unresectable triple-negative breast cancer is a demanding disease with only a few treatment options. Especially for patients with unresectable tumor masses, a treatment that offers rapid tumor shrinkage is needed. If patients are exhausted from several treatment lines, systemic side effects have to be avoided. Reversible electroporation has shown to be effective for breast cancer if combined with systemic bleomycin and/or cisplatin. To enhance the local effect and reduce the systemic side effects, we combined reversible electroporation with regional chemotherapy. Materials and Methods: Patients with advanced metastasized and relapsed breast cancer received regional chemotherapy via intra-arterial infusion and isolated thoracic perfusion combined with percutanous reversible electroporation. Circulating tumor cells (CETCs/CTCs) were counted before and 24 h after the treatment. Tumor response was evaluated by CT (computer tomography) control. Results: A total of 21 treatments were conducted for 14 patients who had a mean tumor size of 7.6 cm (standard deviation 3.3 cm). Higher local drug levels are present with arterial infusion compared to venous infusion and result in enhanced response rates. Circulating tumor cells decreased or stayed stable for 24 h after the treatment for 11 and 8 cases, respectively. An increase was observed in two cases. A total of 13 patients showed a clinical response with tumor shrinkage that led to resectability. One patient did not respond to the treatment regimen. Conclusions: The combination of reversible electroporation with intra-arterial chemotherapy is feasible and results in a good clinical response with neglectable side effects. The treatment is repeatable and can lead to resectability. [ABSTRACT FROM AUTHOR]

Simple Summary: The DIRECT Registry study is a prospective trial of irreversible electroporation (IRE) using the NanoKnife System for the treatment of patients with Stage 3 pancreatic adenocarcinoma (PDAC). The data from this United States Food and Drug Administration Investigational Device Exemption study aims to assess the safety and effectiveness of IRE in a real-world setting in the treatment of Stage 3 PDAC after induction chemotherapy when combined with standard of care (SOC) compared to SOC alone. Background/Objectives: Overall survival for patients with Stage 3 pancreatic ductal adenocarcinoma (PDAC) remains limited, with a median survival of 12 to 15 months. Irreversible electroporation (IRE) is a local tumor ablation method that induces cancerous cell death by disrupting cell membrane homeostasis. The DIRECT Registry study was designed to assess the effectiveness and safety of IRE when combined with standard of care (SOC) treatment for Stage 3 PDAC versus SOC alone in a real-world setting after at least 3 months of induction chemotherapy; Methods: Patients with Stage 3 PDAC treated with IRE plus SOC or SOC alone were prospectively enrolled in a multicenter registry study. Enrollment required 3 months of active multi-agent chemotherapy with no progression before enrollment. Endpoints were 30- and 90-day mortality and adverse events (AEs). Results: Eighty-seven IRE and 27 SOC subjects were enrolled in the registry. Mean ages were 64.0 ± 8.4 and 66.4 ± 9.9 years, and mean anterior/posterior tumor diameters were 2.2 ± 0.7 cm and 3.2 ± 1.3 for the IRE and SOC groups respectively (p = 0.0066). All IRE procedures were performed using an open approach. The 90-day all-cause mortality was 5/83 (6.0%) and 2/27 (7.4%) for the IRE and SOC groups, respectively. Two subjects in the IRE group died from treatment-related complications, and one patient in the SOC group died due to chemotherapy-related complications. Conclusions: Initial results from the DIRECT registry study indicate the use of IRE for curative intent tumor ablation in combination with induction chemotherapy has equivalent morbidity and mortality rates when compared to standard-of-care chemotherapy alone. [ABSTRACT FROM AUTHOR]

Background and purpose:Intracellular delivery is crucial in biological and medical studies. Although many molecular tools have been created for cell-based gene therapies, it remains challenging to introduce external molecules into cells. As one of the most popular non-viral transfection methods, electroporation induces transient pores in the cell membrane by applying an external electric field. Unsatisfactory transfection efficiency and low cell viability are the major drawbacks of electroporation. To overcome these issues, the current study investigated the effect of urea on electroporation-mediated transfection efficiency. Experimental approach: Three voltages of electroporation, including 100, 120, and 140 V, and 3 concentrations of urea buffer, including 0.25%, 0.5%, and 1% W/V, were considered as variables in this study. The HEK-293 cell line was used for transfection, and green fluorescent protein (GFP) expression was evaluated using flow cytometry and fluorescence microscopy. Findings/Results: The results showed that the combination of electroporation and urea increased electroporation efficacy, but the effect depended on voltage and urea concentration. When different concentrations of urea were added to HEK-293 cells at a voltage of 100 V, the number of cells transfected by pEGFP-N1 increased (from 12.3 ± 0.2% in untreated cells to 17.35 ± 0.55%, 23.3 ± 0.3%, and 14 ± 0.1% at urea concentrations of 0.25%, 0.5%, and 1% W/V, respectively). The electroporation buffer containing 0.5% W/V urea showed the highest EGFP expression (23.3 ± 0.3%) and high cell viability (over 90%). Conclusion and implications: This research offers a new perspective for improving gene transfection efficiency once electroporation is utilized. [ABSTRACT FROM AUTHOR]

Microneedle technology is a pivotal component of third-generation transdermal drug delivery systems featuring tiny needles that create temporary microscopic channels in the stratum corneum which facilitate drug penetration in the dermis. This review offers a detailed examination of the current types of microneedles, including solid, coated, dissolving, hollow, and swelling microneedles, along with their preparation techniques as well as their benefits and challenges. Use of 3D printing technology is especially gaining significant attention due to its ability to achieve the high dimensional accuracy required for precise fabrication. Additionally, its customisability presents significant potential for exploring new designs and creating personalised microneedles products. Furthermore, this review explores next generation microneedles, especially stimuli-responsive microneedle, bioinspired microneedle and microneedles combined with other transdermal technology like sonophoresis, electroporation and iontophoresis. Regulatory aspects, characterisation techniques, safety considerations, and cost factors have also been addressed which are crucial for translation from lab to the market. [ABSTRACT FROM AUTHOR]

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure. [Display omitted] • New gene transfection method for embryos at E7 and E8 before neural tube closure. • Only optical fiber and injector needed. • Gene transfection into progenitors in ecto- and endodermal tissues. • Useful to analyze the role of lethal genes during embryogenesis, such as Ldb1. [ABSTRACT FROM AUTHOR]

Drug delivery through electroporation could be highly beneficial for the treatment of different types of diseased tissues within the human body. In this work, a mathematical model of reversible tissue electroporation is presented for injecting drug into the diseased cells. The model emphasizes the tissue boundary where the drug is injected as a point source. In addition, the effect of drug loss at tissue boundaries through extracellular space is studied elaborately. Multiple pulses are applied to deliver a sufficient amount of drug into the targeted cells. The set of differential equations that model the physical circumstances are solved numerically. This model obtains a mass transfer coefficient (MTC), in terms of pore fraction coefficient and drug permeability that controls the drug transport from extracellular to intracellular space. The drug penetration throughout the tissue is captured for the application of different pulses. The boundary effects on drug concentration are highlighted in this study. The advocated model is able to perform homogeneous drug transport into the cells so that the affected tissue is treated completely. This model can be applied to optimize clinical experiments by avoiding the lengthy and costly in vivo and in vitro experiments. [ABSTRACT FROM AUTHOR]

Cell-based therapies hold great potential for cancer immunotherapy. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasability of combining gene electrotransfer and electrofusion into a single-step technique and evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity for single or combined method of gene delivery or cell fusdion. We determined the percentage of fused cells expressing green fluorescence protein (GFP) in a murine cell model of melanoma B16F1, cell line used in our previous studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single step. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Furthermore, our results suggest that cell membrane fluidity is not related to the efficiency of the gene electrotransfer and electrofusion. The protocol is compatible with microfluidic devices, however further optimization of electric pulse parameters and buffers is still needed. [ABSTRACT FROM AUTHOR]

Background: Tumor-reactive T cells play a crucial role in anti-tumor responses, but T cells induced by DNA vaccination are time-consuming processes and exhibit limited anti-tumor efficacy. Therefore, we evaluated the anti-tumor effectiveness of reactive T cells elicited by electroporation (EP)-mediated DNA vaccine targeting epidermal growth factor receptor variant III (pEGFRvIII plasmid), in conjunction with adoptive cell therapy (ACT), involving the transfer of lymphocytes from a pEGFRvIII EP-vaccinated healthy donor. Methods: The validation of the established pEGFRvIII plasmid and EGFRvIII-positive cell model was confirmed through immunofluorescence and western blot analysis. Flow cytometry and cytotoxicity assays were performed to evaluate the functionality of antigen-specific reactive T cells induced by EP-mediated pEGFRvIII vaccines, ACT, or their combination. The anti-tumor effectiveness of EP-mediated pEGFRvIII vaccines alone or combined with ACT was evaluated in the B16F10-EGFRvIII tumor model. Results: EP-mediated pEGFRvIII vaccines elicited serum antibodies and a robust cellular immune response in both healthy and tumor-bearing mice. However, this response only marginally inhibited early-stage tumor growth in established tumor models. EP-mediated pEGFRvIII vaccination followed by adoptive transfer of lymphocytes from vaccinated healthy donors led to notable anti-tumor efficacy, attributed to the synergistic action of antigen-specific CD4+ Th1 cells supplemented by ACT and antigen-specific CD8+ T cells elicited by the EP-mediated DNA vaccination. Conclusions: Our preclinical studies results demonstrate an enhanced anti-tumor efficacy of EP-mediated DNA vaccination boosted with adoptively transferred, vaccinated healthy donor-derived allogeneic lymphocytes. [ABSTRACT FROM AUTHOR]

Objective: The potential risk of inducing ventricular arrhythmias was explored by employing pulsed field ablation (PFA) through the administration of both monophasic and biphasic waveform deliveries. Methods: PFA was applied to specific locations in the right ventricle (RV, n = 5 sites) in swine (n = 2), utilizing identical settings with consistent amplitude, pulse width, and a number of pulses for both monophasic and biphasic waveforms. PFA deliveries were precisely timed in 10‐ms intervals across the entire T wave. Results: Using monophasic waveforms, ventricular fibrillation (VF) was induced 7/7 times when PFA pulses were delivered during the T wave. Biphasic waveforms resulted in no ventricular arrhythmias when PFA was delivered across the entire duration of T wave. Conclusions: Our findings indicate that VF can be consistently induced with monophasic pulse waveforms, but not with biphasic pulse waveforms in a porcine model. [ABSTRACT FROM AUTHOR]