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The microfluidic channel with a planar inductive microcoil for the cell membrane permeabilization and the integrated planar electrodes for cell dielectrophoretic manipulation is proposed and analyzed in the study. The analyzed setup is based on the dielectrophoretic entrapment of the biological cell followed by membrane permeabilization using high pulsed magnetic field. The finite element method analysis of the DEP force and the generated pulsed magnetic field is performed. Based on finite element method analysis the potential applications of the setup in the fields of drug delivery, biomedicine and biotechnology are discussed.

This study aims to identify changes in the expression profile of antigens defining lymphocyte maturity and function. Also, we proved that application of electric field treatment could modulate the expression profile of lymphocytes while avoiding cell death. Based on our studies, we propose a mechanism in which the cells: (1) permeabilize the cell membrane, (2) increase the expression of lymphocyte-activation antigens, and (3) downregulate the expression of CD7. [Extracted from the article]

Candida lusitaniae is an opportunistic yeast pathogen, which can readily develop resistance to antifungal compounds and result in a complex long-term treatment. The efficient treatment is difficult since structure and metabolic properties of the fungal cells are similar to those of eukaryotic host. One of the potential methods to improve the inhibition rate or the cell permeability to inhibitors is the application of electroporation. In this work we investigated the dynamics of the growth inhibition and membrane permeabilization of C. lusitaniae by utilizing the various pulse shape and duration electric field pulses. Our results indicated that single electroporation procedure using 8 kV/cm electric field may result in up to 51±5% inhibition rate. Also it has been experimentally shown that the electroporation pulse shape may influence the inhibitory effect; however, the amplitude of the electric field and the pulse energy remain the most important parameters for definition of the treatment outcome. The dynamics of the cell membrane permeabilization in the 2–8 kV/cm electric field were overviewed. [ABSTRACT FROM AUTHOR]

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE - European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h  after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca 2+ electrochemotherapy in vitro. We provide experimental proof that 7.5-12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca 2+ electrochemotherapy, ensuring excellent cytotoxic efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background/Objectives: Electrochemotherapy (ECT) is a safe and efficient method of targeted drug delivery using pulsed electric fields (PEF), one that is based on the phenomenon of electroporation. However, the problems of electric field homogeneity within a tumor can cause a diminishing of the treatment efficacy, resulting only in partial response to the procedure. This work used gold nano-particles for electric field amplification, introducing the capability to improve available elec-trochemotherapy methods and solve problems associated with field non-homogeneity. Methods: We characterized the potential use of gold nanoparticles of 13 nm diameter (AuNPs: 13 nm) in combination with microsecond (0.6-1.5 kV/cm × 100 μs × 8 (1 Hz)) and nanosecond (6 kV/cm × 300-700 ns × 100 (1, 10, 100 kHz and 1 MHz)) electric field pulses. Finally, we tested the most prominent protocols (microsecond and nanosecond) in the context of bleomycin-based electrochemotherapy ( 4T1 mammary cancer cell line). Results: In the nano-pulse range, the synergistic effects (improved permeabilization and electrotransfer) were profound, with increased pulse burst frequency. Addi-tionally, AuNPs not only reduced the permeabilization thresholds but also affected pore resealing. It was shown that a saturated cytotoxic response with AuNPs can be triggered at significantly lower electric fields and that the AuNPs themselves are non-toxic for the cells either separately or in combination with bleomycin. Conclusions: The used electric fields are considered sub-threshold and/or not applicable for electrochemotherapy, however, when combined with AuNPs results in successful ECT, indicating the methodology's prospective applicability as an anticancer treatment method.

Crocus sativus L. is a widely cultivated traditional plant for obtaining dried red stigmas known as "saffron," the most expensive spice in the world. The response of C. sativus to pre-sowing processing of corms with cold plasma (CP, 3 and 5 min), vacuum (3 min), and electromagnetic field (EMF, 5 min) was assessed to verify how such treatments affect plant performance and the quality and yield of herbal raw materials. The results show that applied physical stressors did not affect the viability of corms but caused stressor-dependent changes in the kinetics of sprouting, growth parameters, leaf trichome density, and secondary metabolite content in stigmas. The effect of CP treatment on plant growth and metabolite content was negative, but all stressors significantly (by 42-74%) increased the number of leaf trichomes. CP3 treatment significantly decreased the length and dry weight of flowers by 43% and 60%, respectively, while EMF treatment increased the length of flowers by 27%. However, longer CP treatment (5 min) delayed germination. Vacuum treatment improved the uniformity of germination by 28% but caused smaller changes in the content of stigma compounds compared with CP and EMF. Twenty-six compounds were identified in total in Crocus stigma samples by the HPLC-DAD method, including 23 crocins, rutin, picrocrocin, and safranal. Processing of Crocus corms with EMF showed the greatest efficiency in increasing the production of secondary metabolites in saffron. EMF increased the content of marker compounds in stigmas (crocin 4: from 8.95 to 431.17 mg/g; crocin 3: from 6.27 to 164.86 mg/g; picrocrocin: from 0.4 to 1.0 mg/g), although the observed effects on growth were neutral or slightly positive. The obtained findings indicate that treatment of C. sativus corms with EMF has the potential application for increasing the quality of saffron by enhancing the amounts of biologically active compounds.

The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca 2+ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3-7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca 2+ electrochemotherapy ( B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals.

Recently, magnetic fields (MFs) have received major attention due to their potential therapeutic applications and biological effects. This review provides a comprehensive analysis of the cellular and molecular impacts of MFs, with a focus on both in vitro and in vivo studies. We investigate the mechanisms by which MFs influence cell behavior, including modifications in gene expression, protein synthesis, and cellular signaling pathways. The interaction of MFs with cellular components such as ion channels, membranes, and the cytoskeleton is analyzed, along with their effects on cellular processes like proliferation, differentiation, and apoptosis. Molecular insights are offered into how MFs modulate oxidative stress and inflammatory responses, which are pivotal in various pathological conditions. Furthermore, we explore the therapeutic potential of MFs in regenerative medicine, cancer treatment, and neurodegenerative diseases. By synthesizing current findings, this article aims to elucidate the complex bioeffects of MFs, thereby facilitating their optimized application in medical and biotechnological fields.

Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells' plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.

In the context of climate change, methods to improve the resistance of coniferous trees to biotic and abiotic stress are in great demand. The common plant response to exposure to vastly different stressors is the generation of reactive oxygen species (ROS) followed by activation of the defensive antioxidant system. We aimed to evaluate whether seed treatment with physical stressors can activate the activity of antioxidant enzymes and radical scavenging activity in young Picea abies (L.) H. Karst seedlings. For this, we applied seed treatment with cold plasma (CP) and electromagnetic field (EMF) and compared the response in ten different half-sib families of Norway spruce. The impact of the treatments with CP (1 min-CP1; 2 min-CP2) and EMF (2 min) on one-year-old and two-year-old P. abies seedlings was determined by the emergence rate, parameters of growth, and spectrophotometric assessment of antioxidant capacity (enzyme activity; DPPH and ABTS scavenging) in needles. The results indicated that the impact of seed treatment is strongly dependent on the genetic family. In the 577 half-sib family, the activity of antioxidant enzymes catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX), and glutathione reductase (GR) increased after EMF-treatment in one-year-old seedlings, while similar effects in 477 half-sib family were induced by CP2 treatment. In two-year-old seedlings, CP1-treatment increased CAT, APX, POX, GR, SOD, DPPH, and ABTS activity in the 457 half-sib family. However, no significant impact of the treatment with CP1 was determined in one-year-old seedlings in this family. The application of novel technologies and the consideration of the combinatory impact of genetic and physical factors could have the potential to improve the accumulation of compounds that play an essential role in the defense mechanisms of P. abies . Nevertheless, for different resistance and responses to stressors of plants, their genetic properties play an essential role. A comprehensive analysis of interactions among the stress factors (CP and EMF), genetic properties, and changes induced in the antioxidant system can be of importance both for the practical application of seed treatment in forestry and for understanding fundamental adaptation mechanisms in conifers.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Radzevičiūtė-Valčiukė, Gečaitė, Balevičiūtė, Szewczyk, Želvys, Lekešytė, Malyško-Ptašinskė, Mickevičiūtė, Malakauskaitė, Kulbacka and Novickij.)

Short electric field pulses represent a novel potential approach for achieving uniform electroporation within tissue containing elongated cells oriented in various directions, such as electroporation-based cardiac ablation procedures. In this study, we investigated how electroporation with nanosecond pulses with respect to different pulse shapes (unipolar, bipolar, and asymmetric) influences cardiomyocyte permeabilization and gene transfer. For this purpose, rat cardiomyocytes (H9c2) were used. The efficacy of the pulsed electric field protocols was assessed by flow cytometry and electrogene transfer by fluorescent and holotomographic microscopy. The response of the cells was assessed by the metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assay), F-actin distribution in cells by confocal microscopy, and muscle atrophy F-box (MAFbx) marker. We show nano- and microsecond pulse protocols, which are not cytotoxic for cardiac muscle cells and can be efficiently used for gene electrotransfection. Asymmetric nanosecond pulsed electric fields were similarly efficient in plasmid delivery as microsecond and millisecond protocols. However, the millisecond protocol induced a higher MAFbx expression in H9c2 cells., (Copyright 2024, Mary Ann Liebert, Inc., publishers.)

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., (© 2024. The Author(s).)

Head and neck cancer continues to be among the most prevalent types of cancer globally, yet it can be managed with appropriate treatment approaches. Presently, chemotherapy and radiotherapy stand as the primary treatment modalities for various groups and regions affected by head and neck cancer. Nonetheless, these treatments are linked to adverse side effects in patients. Moreover, due to tumor resistance to multiple drugs (both intrinsic and extrinsic) and radiotherapy, along with numerous other factors, recurrences or metastases often occur. Electrochemotherapy (ECT) emerges as a clinically proven alternative that offers high efficacy, localized effect, and diminished negative factors. Electrochemotherapy involves the treatment of solid tumors by combining a non-permeable cytotoxic drug, such as bleomycin, with a locally administered pulsed electric field (PEF). It is crucial to employ this method effectively by utilizing optimal PEF protocols and drugs at concentrations that do not possess inherent cytotoxic properties. This review emphasizes an examination of diverse clinical practices of ECT concerning head and neck cancer. It specifically delves into the treatment procedure, the choice of anti-cancer drugs, pre-treatment planning, PEF protocols, and electroporation electrodes as well as the efficacy of tumor response to the treatment and encountered obstacles. We have also highlighted the significance of assessing the spatial electric field distribution in both tumor and adjacent tissues prior to treatment as it plays a pivotal role in determining treatment success. Finally, we compare the ECT methodology to conventional treatments to highlight the potential for improvement and to facilitate popularization of the technique in the area of head and neck cancers where it is not widespread yet while it is not the case with other cancer types., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Morozas, Malyško-Ptašinskė, Kulbacka, Ivaška, Ivaškienė and Novickij.)

Calcium electroporation is a modality of electrochemotherapy (ECT), which is based on intracellular electric field-mediated delivery of cytotoxic doses of calcium into the cells resulting in rapid cell death. In this work, we have developed a CHO-K1 luminescent cell line, which allowed the estimation of cell membrane permeabilization, ATP depletion and cytotoxicity evaluation without the use of additional markers and methodologies. We have shown the high efficiency of nanosecond pulses compressed into a MHz burst for application in calcium ECT treatments. The 5 kV/cm and 10 kV/cm nanosecond (100 and 600 ns) pulses were delivered in bursts of 10, 50 and 100 pulses (a total of 12 parametric protocols) and then compared to standard microsecond range sequences (100 µs × 8) of 0.4-1.4 kV/cm. The effects of calcium-free, 2 mM and 5 mM calcium electroporation treatments were characterized. It was shown that reversible electroporation is accompanied by ATP depletion associated with membrane damage, while during calcium ECT the ATP depletion is several-fold higher, which results in cell death. Finally, efficacy-wise equivalent pulse parameters from nanosecond and microsecond ranges were established, which can be used for calcium nano-ECT as a better alternative to ESOPE (European Standard Operating Procedures on Electrochemotherapy) protocols., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Improving noninvasive antenatal diagnosis of fetal inflammatory response syndrome (FIRS) can assist in the evaluation of prenatal risk and reduce perinatal outcomes. This study aimed to determine whether soluble urokinase-type plasminogen activator receptor (suPAR) in vaginally collected amniotic fluid is significant in identifying FIRS after preterm premature rupture of membranes before 34 weeks of gestation., Methods: This was a prospective cohort study of 114 pregnant women and their newborns after preterm premature rupture of membranes at 22-34 +6  weeks of gestation. SuPAR was evaluated using an enzyme-linked immunosorbent assay in vaginally collected amniotic fluid. Patients were classified according to the presence or absence of FIRS. FIRS was defined by umbilical cord blood interleukin-6 level > 11 pg/mL or histological funisitis. The data were analyzed using the R package (R-4.0.5)., Results: SuPAR was detected in all amniotic fluid samples with a median of 26.23 ng/mL (interquartile range (IQR), 15.19-51.14). The median level of suPAR was higher in the FIRS group than in the non-FIRS group, 32.36 ng/mL (IQR, 17.27-84.16) vs. 20.46 ng/mL (IQR, 11.49-36.63) (P = 0.01), respectively. The presence of histological chorioamnionitis significantly increased the suPAR concentration in the FIRS group (P < 0.001). The areas under the curve for FIRS and FIRS with histological chorioamnionitis were 0.65 and 0.74, respectively, with an optimum cutoff value of 27.60 ng/mL. Controlling for gestational age, the cutoff of suPAR more than 27.60 ng/mL predicted threefold higher odds for FIRS and sixfold higher odds for FIRS with histologic chorioamnionitis., Conclusion: Soluble urokinase-type plasminogen activator receptor in vaginally obtained amniotic fluid may assist in evaluating prenatal risk of FIRS in patients after preterm premature rupture of membranes before 34 weeks of gestation., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Vitalij Novickij reports financial support was provided by Research Council of Lithuania. Julita Kulbacka reports financial support was provided by National Science Centre Poland.]., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Electrochemotherapy (ECT) involves combining anticancer drugs with electroporation, which is induced by pulsed electric fields (PEFs), while the effects vary in effectiveness based on the specific parameters of the electrical pulses and susceptibility of the cells to a specific drug. In this work, we utilized conventional microsecond electroporation protocols (0.8 - 1.5 kV/cm × 100 μs × 8, 1 Hz) and the new modality of nanosecond pulses (4 and 8 kV/cm × 500 ns × 100, 1 kHz and 1 MHz), which are compressed into a high frequency burst. Sensitive and resistant lung, breast and ovarian human cancer cell lines were used in the study. In order to overcome drug-resistance, we have investigated the feasibility to use anticancer drug cocktails i.e., bleomycin and cisplatin combinations with metformin, vinorelbine and Dp44mT. The different susceptibility of various human cancer cells lines to electric pulses was determined, the efficacy of ECT was characterized and the type of cell death depending on the combinations of drugs was investigated. The results indicate that synergistic effects of PEFs with drug cocktails may be used to overcome drug-resistance in cancer, while the application of nsPEF provides more flexibility in parametric protocols and modulation of cancer cell death., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Julita Kulbacka reports financial support was provided by National Science Centre. Vitalij Novickij reports financial support was provided by Research Council of Lithuania.]., (Copyright © 2023 Elsevier B.V. All rights reserved.)

T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) expression has been a trending topic in recent years due to its differential expression in a wide range of neoplasms. TIM-3 is one of the key immune checkpoint receptors that interact with GAL-9, PtdSer, HMGB1 and CEACAM1. Initially identified on the surface of T helper 1 (Th1) lymphocytes and later on cytotoxic lymphocytes (CTLs), monocytes, macrophages, natural killer cells (NKs), and dendritic cells (DCs), TIM-3 plays a key role in immunoregulation. Recently, a growing body of evidence has shown that its differential expression in various tumor types indicates a specific prognosis for cancer patients. Here, we discuss which types of cancer TIM-3 can serve as a prognostic factor and the influence of coexpressed immune checkpoint inhibitors, such as LAG-3, PD-1, and CTLA-4 on patients' outcomes. Currently, experimental medicine involving TIM-3 has significantly enhanced the anti-tumor effect and improved patient survival. In this work, we summarized clinical trials incorporating TIM-3 targeting monoclonal and bispecific antibodies in monotherapy and combination therapy and highlighted the emerging role of cell-based therapies., (© 2023. The Author(s).)

The application of negative polarity electrical pulse (↓) following positive polarity pulses (↑) may induce bipolar cancellation (BPC), a unique physiological response believed to be specific to nanosecond electroporation (nsEP). The literature lacks analysis of bipolar electroporation (BP EP) involving asymmetrical sequences composed of nanosecond and microsecond pulses. Moreover, the impact of interphase interval on BPC caused by such asymmetrical pulse needs consideration. In this study, the authors utilized the ovarian clear carcinoma cell line (OvBH-1) model to investigate the BPC with asymmetrical sequences. Cells were exposed to pulses delivered in 10-pulse bursts but as uni- or bipolar, symmetrical, or asymmetrical sequences with a duration of 600 ns or 10 µs and electric field strength equal to 7.0 or 1.8 kV/cm, respectively. It was shown that the asymmetry of pulses influences BPC. The obtained results have also been investigated in the context of calcium electrochemotherapy. The reduction of cell membrane poration, and cell survival have been observed following Ca 2+ electrochemotherapy. The effects of interphase delays (1 and 10 µs) on the BPC phenomenon were reported. Our findings show that the BPC phenomenon can be controlled using pulse asymmetry or delay between the positive and negative polarity of the pulse., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Julita Kulbacka reports financial support was provided by National Science Centre Poland. Vitalij Novickij reports financial support was provided by Research Council of Lithuania.’, (Copyright © 2023 Elsevier B.V. All rights reserved.)

Checkpoint molecules such as PD-1, LAG-3, and TIM-3 are currently under extensive investigation for their roles in the attenuation of the immune response in cancer. Various methods have been applied to overcome the challenges in this field. This study investigated the effects of nanosecond pulsed electric field (nsPEF) treatment on the expression of immune checkpoint molecules in A375 and C32 melanoma cells. The researchers found that the nsPEF treatment was able to enhance membrane permeabilization and morphological changes in the cell membrane without being cytotoxic. We found that the effects of nsPEFs on melanoma included (1) the transport of vesicles from the inside to the outside of the cells, (2) cell contraction, and (3) the migration of lipids from inside the cells to their peripheries. The treatment increased the expression of PD-1 checkpoint receptors. Furthermore, we also observed potential co-localization or clustering of MHC class II and PD-1 molecules on the cell surface and the secretion of cytokines such as TNF-α and IL-6. These findings suggest that nsPEF treatment could be a viable approach to enhance the delivery of therapeutic agents to cancer cells and to modulate the tumor microenvironment to promote an antitumor immune response. Further studies are needed to explore the mechanisms underlying these effects and their impacts on the antitumor immune response, and to investigate the potential of nsPEF treatment in combination with immune checkpoint inhibitors to improve clinical outcomes for cancer patients.

High-Intensity Pulsed Electromagnetic Fields (HI-PEMF) treatment is an emerging noninvasive and contactless alternative to conventional electroporation, since the electric field inside the tissue is induced remotely by an externally applied pulsed magnetic field. Recently, HI-PEMF has been successfully used in the transfer of plasmid DNA and siRNA in vivo, with no or minimal infiltration of immune cells. In addition to gene electrotransfer, treatment with HI-PEMF has also shown potential for electrochemotherapy, where activation of the immune response contributes to the treatment outcome. The immune response can be triggered by immunogenic cell death that is characterized by the release of damage-associated molecular patterns (DAMPs) from damaged or/and dying cells. In this study, the release of the best-known DAMP molecules, i.e., adenosine triphosphate (ATP), calreticulin and high mobility group box 1 protein (HMBG1), after HI-PEMF treatment was investigated in vitro on three different cell lines of different tissue origin and compared with conventional electroporation treatment parameters. We have shown that HI-PEMF by itself does not cause the release of HMGB1 or calreticulin, whereas the release of ATP was detected immediately after HI-PEMF treatment. Our results indicate that HI-PEMF treatment causes no to minimal release of DAMP molecules, which results in minimal/limited activation of the immune response.

In the context of climate change, strategies aimed at enhancing trees' resistance to biotic and abiotic stress are particularly relevant. We applied an electromagnetic field (EMF) seed treatment to observe changes in the establishment and content of biochemical compounds in silver birch seedlings induced by a short (1 min) seed exposure to a physical stressor. The impact of EMF treatment was evaluated on seedling emergence and growth of one-year-old and two-year-old seedlings from seven half-sib families of silver birch. The effects on numerous biochemical parameters in seedling leaves, such as total phenolic content (TPC), total flavonoid content (TFC), amounts of photosynthetic pigments, total soluble sugars (TSS), level of lipid peroxidation level, antioxidant activity and activity of antioxidant enzymes, were compared using spectrophotometric methods. The results indicated that, in one-year-old seedlings, two of seven (60th and 73rd) half-sib families exhibited a positive response to seed treatment with EMFs in nearly all analyzed parameters. For example, in the 60th family, seed treatment with EMFs increased the percentage of emergence by 3 times, one-year-old seedling height by 71%, leaf TPC by 47%, antioxidant activity by 2 times and amount of chlorophyll a by 4.6 times. Meanwhile, the other two (86th and 179th) families exhibited a more obvious positive response to EMF in two-year-old seedlings as compared to one-year-old seedling controls. The results revealed that short-term EMF treatment of silver birch seeds can potentially be used to improve seedling emergence and growth and increase the content of secondary metabolites, antioxidant capacity and photosynthetic pigments. Understanding of the impact of EMFs as well as the influence of genetic differences on tree responses can be significant for practical applications in forestry. Genetic selection of plant genotypes that exhibit positive response trends can open the way to improve the quality of forest stands.

Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (μsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4 + and CD8 + central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.

Electrical stimulation (ES) has been frequently used in different biomedical applications both in vitro and in vivo. Numerous studies have demonstrated positive effects of ES on cellular functions, including metabolism, proliferation, and differentiation. The application of ES to cartilage tissue for increasing extracellular matrix formation is of interest, as cartilage is not able to restore its lesions owing to its avascular nature and lack of cells. Various ES approaches have been used to stimulate chondrogenic differentiation in chondrocytes and stem cells; however, there is a huge gap in systematizing ES protocols used for chondrogenic differentiation of cells. This review focuses on the application of ES for chondrocyte and mesenchymal stem cell chondrogenesis for cartilage tissue regeneration. The effects of different types of ES on cellular functions and chondrogenic differentiation are reviewed, systematically providing ES protocols and their advantageous effects. Moreover, cartilage 3D modeling using cells in scaffolds/hydrogels under ES are observed, and recommendations on reporting about the use of ES in different studies are provided to ensure adequate consolidation of knowledge in the area of ES. This review brings novel insights into the further application of ES in in vitro studies, which are promising for further cartilage repair techniques.

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy.

Electroporation is an effective physical method for irreversible or reversible permeabilization of plasma membranes of biological cells and is typically used for tissue ablation or targeted drug/DNA delivery into living cells. In the context of cancer treatment, full recovery from an electroporation-based procedure is frequently dependent on the spatial distribution/homogeneity of the electric field in the tissue; therefore, the structure of electrodes/applicators plays an important role. This review focuses on the analysis of electrodes and in silico models used for electroporation in cancer treatment and gene therapy. We have reviewed various invasive and non-invasive electrodes; analyzed the spatial electric field distribution using finite element method analysis; evaluated parametric compatibility, and the pros and cons of application; and summarized options for improvement. Additionally, this review highlights the importance of tissue bioimpedance for accurate treatment planning using numerical modeling and the effects of pulse frequency on tissue conductivity and relative permittivity values., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Malyško-Ptašinskė, Staigvila and Novickij.)

Nanosecond pulsed electric fields (nsPEF) have been shown to exert anticancer effects; however, little is known about the mechanisms triggered in cancer cells by nanosecond-length pulses, especially when low, sub-permeabilization voltage is used. In this study, three human pancreatic cancer cell lines were treated with nsPEF and molecular changes at the cellular level were analyzed. Further, we assessed the efficacy of paclitaxel chemotherapy following nsPEF treatment and correlated that with the changes in the expression of multi-drug resistance (MDR) proteins. Finally, we examined the influence of nsPEF on the adhesive properties of cancer cells as well as the formation and growth of pancreatic cancer spheroids. Cell line response differed with the application of a 200 ns, 100 pulses, 8 kV/cm, 10 kHz PEF treatment. PEF treatment led to (1) the release of microvesicles (MV) in EPP85-181RDB cells, (2) electropermeabilization in EPP85-181RNOV cells and (3) cell shrinkage in EPP85-181P cells. The release of MV's in EPP85-181RDB cells reduced the membrane content of P-gp and LRP, leading to a transient increase in vulnerability of the cells towards paclitaxel. In all cell lines we observed an initial reduction in size of the cancer spheroids after the nsPEF treatment. Cell line EPP85-181RNOV exhibited a permanent reduction in the spheroid size after nsPEF. We propose a mechanism in which the surface tension of the membrane, regulated by the organization of actin fibers, modulates the response of cancer cells towards nsPEF. When a membrane's surface tension remains low, we observed some cells form protrusions and release MVs containing MDR proteins. In contrast, when cell surface tension remains high, the cell membrane is being electroporated. The latter effect may be responsible for the reduced tumor growth following nsPEF treatment., (© 2023. The Author(s).)

In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4 + T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4 + Tr1 cells, increased the expansion of splenic CD8 + T, CD4 + CD8 + T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Lin - population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy.

Pulsed electric fields (PEFs) are commonly used to facilitate the delivery of various molecules, including pharmaceuticals, into living cells. However, the applied protocols still require optimization regarding the conditions of the permeabilization process, i.e., pulse waveform, voltage, duration, and the number of pulses in a burst. This study highlights the importance of electrochemical processes involved in the electropermeabilization process, known as electroporation. This research investigated the effects of electroporation on human non-small cell lung cancer cells (A549) in potassium (SKM) and HEPES-based buffers (SHM) using sub-microsecond and microsecond range pulses. The experiments were performed using 100 ns - 100 μs (0.6-15 kV/cm) bursts with 8 pulses in a sequence. It was shown that depending on the buffer composition, the susceptibility of cells to PEF varies, while calcium enhances the cytotoxic effects of PEF, if high cell membrane permeabilization is triggered. It was also determined that electroporation with calcium ions induces oxidative stress in cells, including lipid peroxidation (LPO), generation of reactive oxygen species (ROS), and neutral lipid droplets. Here, we demonstrated that calcium ions and optimized pulse parameters could potentiate PEF efficacy and oxidative alternations in lung cancer cells. Thus, the anticancer efficacy of PEF in lung cancers in combination with standard cytostatic drugs or calcium ions should be considered, but this issue still requires in-depth detailed studies with in vivo models., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julita Kulbacka reports financial support was provided by Polish National Centre of Science. Vitalij Novickij reports financial support was provided by Research Council of Lithuania., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Electroporation is a pulsed electric field (PEF) induced phenomenon, which effectiveness varies dependent on pulse parameters. This work focuses on nano-electrochemotherapy with bleomycin and doxorubicin to derive protocols as effective as European Standard Operating Procedures on Electrochemotherapy (ESOPE), which employ conventional microsecond range pulses. As a model, murine Lewis lung carcinoma (LLC1) cell line was used. The effects of pulse duration (100-500 ns), PEF amplitude (6-10 kV/cm) and pulse repetition frequency (10 kHz, 100 kHz, 1 MHz) were studied. A total of 75 ns protocol variations have been used. For detection of cell permeabilization, Yo-Pro-1 and flow cytometry were employed. Cell viability was evaluated 24-, 48-, or 72-hours post-electroporation. Nanosecond parametric protocols resulting in comparable treatment efficiency as ESOPE (1.3 kV/cm × 100 μs × 8) have been proposed. It was shown that high-frequency nanosecond electrochemotherapy with bleomycin or doxorubicin could be an alternative for established ESOPE protocols., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20-200 kHz, with up to 20-30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-μs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3-5 %. nsEP bursts were compared with single "long" pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2-3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-μs pulse decreased to 57 ± 8 V/cm for a 100-μs burst of 100-ns, 0.25-MHz pulses (p < 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Electric pulses are widely used in biology, medicine, industry, and food processing. Numerous studies indicate that electroporation (EP) is a pulse-dependent process, and the electric pulse shape and duration strongly determine permeabilization efficacy. EP protocols are precisely planned in terms of the size and charge of the molecules, which will be delivered to the cell. In reversible and irreversible EP applications, rectangular or sine, polar or bipolar pulses are commonly used. The usage of pulses of the asymmetric shape is still limited to high voltage and low voltage (HV/LV) sequences in the context of gene delivery, while EP-based applications of ultra-short asymmetric pulses are just starting to emerge. This review emphasizes the importance and role of the pulse shape for membrane permeabilization by EP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Novickij, Rembiałkowska, Szlasa and Kulbacka.)

A high-intensity pulsed electromagnetic field (HI-PEMF) is a non-invasive and non-contact delivery method and may, as such, have an advantage over gene electrotransfer mediated by conventional electroporation using contact electrodes. Due to the limited number of in vitro studies in the field of gene electrotransfection by HI-PEMF, we designed experiments to investigate and demonstrate the feasibility of such a technique for the non-viral delivery of genetic material into cells in vitro. We first showed that HI-PEMF causes DNA adsorption to the membrane, a generally accepted prerequisite step for successful gene electrotransfection. We also showed that HI-PEMF can induce gene electrotransfection as the application of HI-PEMF increased the percentage of GFP-positive cells for two different combinations of pDNA size and concentration. Furthermore, by measuring the uptake of larger molecules, i.e., fluorescently labelled dextrans of three different sizes, we showed endocytosis to be a possible mechanism for introducing large molecules into cells by HI-PEMF.

Electroporation-based antitumor therapies, including bleomycin electrotransfer, calcium electroporation, and irreversible electroporation, are very effective on directly treated tumors, but have no or low effect on distal nodules. In this study, we aimed to investigate the abscopal effect following calcium electroporation and bleomycin electrotransfer and to find out the effect of the increase of IL-2 serum concentration by muscle transfection. The bystander effect was analyzed in in vitro studies on 4T1tumor cells, while abscopal effect was investigated in an in vivo setting using Balb/c mice bearing 4T1 tumors. ELISA was used to monitor IL-2 serum concentration. We showed that, similarly to cell treatment with bleomycin electrotransfer, the bystander effect occurs also following calcium electroporation and that these effects can be combined. Combination of these treatments also resulted in the enhancement of the abscopal effect in vivo. Since these treatments resulted in an increase of IL-2 serum concentration only in mice bearing one but not two tumors, we increased IL-2 serum concentration by muscle transfection. Although this did not enhance the abscopal effect of combined tumor treatment using calcium electroporation and bleomycin electrotransfer, boosting of IL-2 serum concentration had a significant inhibitory effect on directly treated tumors.