Development of High Power Square Wave Electroporators

High power microsecond and submicrosecond electric pulse generation and the devices for pulse generation (electroporators) development and application problem is focused in the dissertation. The electric field technologies, pulse forming circuits and circuit transient process compensation methods are investigated. The introduction presents the investigated problem, objects of research, importance of the dissertation, describes research methodology, scientific novelty and the defended statements. In the first chapter the scientific publications in the area of the high power electric pulses generation and application for biological cell permeabilization are overviewed. The influence of the pulse parameters on the biological effects is analysed. The requirements for the electroporators are identified. In the second chapter the prototypes of the high power square wave 5 μs – 10 ms up to 4 kV, 100 A and 200 ns – 5 μs up to 8 kV, 100 A electroporators are developed. The models for investigation of the transient processes in the circuits and the solutions for compensation are overviewed. The adequacy of the proposed models to the experimental results is analysed. The interdigitated microelectrodes structure for planar electroporation is proposed. The resultant electric field distribution and the cell medium temperature rise due to the Joule heating are investigated. The third chapter is focused on the experimental application of the developed high power microsecond and submicrosecond electroporators prototypes in biological experiments. The experimental results with different cell types are presented and conclusions are formed. Research results on the dissertation subject are published in 5 scientific articles: 3 articles – Thomson Reuters ISI Web of Science database journals with impact factor, 2 – publications referenced and abstracted in other international databases, 4 presentations have been made in international conferences in Lithuania, Netherlands, Germany and Japan

Invasive and non-invasive electrodes for successful drug and gene delivery in electroporation-based treatments

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

Computer Adaptive Testing Using Upper-Confidence Bound Algorithm for Formative Assessment

There is strong support for formative assessment inclusion in learning processes, with the main emphasis on corrective feedback for students. However, traditional testing and Computer Adaptive Testing can be problematic to implement in the classroom. Paper based tests are logistically inconvenient and are hard to personalize, and thus must be longer to accurately assess every student in the classroom. Computer Adaptive Testing can mitigate these problems by making use of Multi-Dimensional Item Response Theory at cost of introducing several new problems, most problematic of which are the greater test creation complexity, because of the necessity of question pool calibration, and the debatable premise that different questions measure one common latent trait. In this paper a new approach of modelling formative assessment as a Multi-Armed bandit problem is proposed and solved using Upper-Confidence Bound algorithm. The method in combination with e-learning paradigm has the potential to mitigate such problems as question item calibration and lengthy tests, while providing accurate formative assessment feedback for students. A number of simulation and empirical data experiments (with 104 students) are carried out to explore and measure the potential of this application with positive results.This article belongs to the Special Issue Smart Learnin

Electroporation and Cell Killing by Milli- to Nanosecond Pulses and Avoiding Neuromuscular Stimulation in Cancer Ablation

Ablation therapies aim at eradication of tumors with minimal impact on surrounding healthy tissues. Conventional pulsed electric field (PEF) treatments cause pain and muscle contractions far beyond the ablation area. The ongoing quest is to identify PEF parameters efficient at ablation but not at stimulation. We measured electroporation and cell killing thresholds for 150 ns–1 ms PEF, uni- and bipolar, delivered in 10- to 300-pulse trains at up to 1 MHz rates. Monolayers of murine colon carcinoma cells exposed to PEF were stained with YO-PRO-1 dye to detect electroporation. In 2–4 h, dead cells were labeled with propidium. Electroporation and cell death thresholds determined by matching the stained areas to the electric field intensity were compared to nerve excitation thresholds (Kim et al. in Int J Mol Sci 22(13):7051, 2021). The minimum fourfold ratio of cell killing and stimulation thresholds was achieved with bipolar nanosecond PEF (nsPEF), a sheer benefit over a 500-fold ratio for conventional 100-µs PEF. Increasing the bipolar nsPEF frequency up to 100 kHz within 10-pulse bursts increased ablation thresholds by \u3c 20%. Restricting such bursts to the refractory period after nerve excitation will minimize the number of neuromuscular reactions while maintaining the ablation efficiency and avoiding heating

Electrochemotherapy for head and neck cancers: possibilities and limitations

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

PRE-SOWING SEED TREATMENT WITH PHYSICAL STRESSORS INDUCES CHANGES IN AMOUNT OF SECONDARY METABOLITES IN NEEDLES OF PICEA ABIES SEEDLINGS

Tree resistance to pathogens is related to the general viability of trees, their growth energy, and ability to synthesize and to mobilize secondary metabolites (SMs), usually phenolic compounds, in tissues. The total phenolic content (TPC) in Norway spruce needles was determined during two vegetation seasons in order to compare the chemical background and ability of different Norway spruce half-sib families to synthesize TPC in response to seed treatment with physical stressors: cold plasma for 1 and 2 min (CP1, CP2) and electromagnetic field for 2 min (EMF2). TPC in seedling needles significantly differed between affected and control groups, but differences were stronger exerted during the first year of vegetation. In the first vegetation season, the strongest positive effects on TPC synthesis were induced by EMF2 treatment in 463 and 577 half-sib families, by CP1 treatment − in 457 half-sib family and by CP2 treatment − in 541 half-sib family; in the second vegetation season strongest effects were detected in CP1 treated 457 half-sib family and in CP2 treated 548 and 477 half-sib families. The half-sib families of Picea abies with higher TPC induced by seed treatment with different physical stressors could be more resistant to pathogens and diseases. The variance component of family for TPC variated from 14±10 % till 17±11 %, and was significant. The interaction of genotype and environment on TPC was as high as 49 ± 20%

Feasibility Evaluation of Cu-Nb Microcomposite Joints Formed by Magnetic Field Pressing for Pulsed Power Applications

In this work we have evaluated the feasibility of the magnetic field pressing technology for formation of Cu-Nb joints for further pulsed power applications. The electrical and mechanical properties of joints have been investigated. The structure of Cu-Nb conductors joints was investigated using optical and scanning electron microscopy. The mechanical characteristics have been evaluated using tensile tests. It has been determined that the maximum tensile strength of 350 MPa could be achieved, which is 30,4 % of a Cu-Nb wire strength. The applied pressing technology allowed minimizing the defects in the microcomposite structure due to solid-state joining process, however the resultant decrease of conductivity by 10 % influenced an increase of the Joule heating. After a 2 min 200 A current flow the difference of 54,4 °C between the conductor and the joint area was observed. It was concluded that the Cu-Nb joints formed by magnetic field pressing are applicable for pulsed magnet setups where non-destructive joints are required

Inactivation of Escherichia coli Using Nanosecond Electric Fields and Nisin Nanoparticles: A Kinetics Study

Nisin is a recognized bacteriocin widely used in food processing, however, being ineffective against gram-negative bacteria and in complex food systems. As a result, the research of methods that have cell wall–permeabilizing activity is required. In this study, electroporation to trigger sensitization of gram-negative bacteria to nisin-loaded pectin nanoparticles was used. As a model microorganism, bioluminescent strain of E. coli was introduced. Inactivation kinetics using nanosecond pulsed electric fields (PEFs) and nisin nanoparticles have been studied in a broad range (100–900 ns, 10–30 kV/cm) of pulse parameters. As a reference, the microsecond range protocols (100 μs × 8) have been applied. It was determined that the 20–30 kV/cm electric field with pulse duration ranging from 500 to 900 ns was sufficient to cause significant permeabilization of E. coli to trigger a synergistic response with the nisin treatment. The kinetics of the inactivation was studied with a time resolution of 2.5 min, which provided experimental evidence that the efficacy of nisin-based treatment can be effectively controlled in time using PEF. The results and the proposed methodology for rapid detection of bacteria inactivation rate based on bioluminescence may be useful in the development and optimization of protocols for PEF-based treatments

Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca\u3csup\u3e2+\u3c/sup\u3e and Ba\u3csup\u3e2+\u3c/sup\u3e Ions

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within \u3c1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than \u3e7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing

Electrochemotherapy Using Doxorubicin and Nanosecond Electric Field Pulses: A Pilot in Vivo Study

Pulsed electric field (PEF) is frequently used for intertumoral drug delivery resulting in a well-known anticancer treatment—electrochemotherapy. However, electrochemotherapy is associated with microsecond range of electrical pulses, while nanosecond range electrochemotherapy is almost non-existent. In this work, we analyzed the feasibility of nanosecond range pulse bursts for successful doxorubicin-based electrochemotherapy in vivo. The conventional microsecond (1.4 kV/cm × 100 µs × 8) procedure was compared to the nanosecond (3.5 kV/cm × 800 ns × 250) non-thermal PEF-based treatment. As a model, Sp2/0 tumors were developed. Additionally, basic current and voltage measurements were performed to detect the characteristic conductivity-dependent patterns and to serve as an indicator of successful tumor permeabilization both in the nano and microsecond pulse range. It was shown that nano-electrochemotherapy can be the logical evolution of the currently established European Standard Operating Procedures for Electrochemotherapy (ESOPE) protocols, offering better energy control and equivalent treatment efficacy