mPOR enables completely new research in the field of electroporation:

Long-term changes in transmembrane voltage after electroporation are
governed by the interplay between nonselective leak current and ion
channel activation

Electroporation causes a temporal increase in cell membrane permeability and leads to prolonged changes in transmembrane voltage (TMV) in both excitable and non-excitable cells. However, the mechanisms of these TMV changes remain to be fully elucidated. To this end, we monitored TMV over 30 min after exposing two different cell lines to a single 100 µs electroporation pulse using the FLIPR Membrane Potential dye. In CHO-K1 cells, which express very low levels of endogenous ion channels, membrane depolarization following pulse exposure could be explained by nonselective leak current, which persists until the membrane reseals, enabling the cells to recover their resting TMV. In U-87 MG cells, which express many different ion channels, we unexpectedly observed membrane hyperpolarization following the initial depolarization phase, but only at 33◦C and not at 25◦C. We developed a theoretical model, supported by experiments with ion channel inhibitors, which indicated that hyperpolarization could largely be attributed to the activation of calcium-activated potassium channels. Ion channel activation, coupled with changes in TMV and intracellular calcium, participates in various physiological processes, including cell proliferation, differentiation, migration, and apoptosis. Therefore, our study suggests that ion channels could present a potential target for influencing the biological response after electroporation.

The Effects of Interphase and Interpulse Delays and Pulse Widths on Induced Muscle Contractions, Pain and Therapeutic Efficacy in Electroporation-Based Therapies

Electroporation is used in medicine for drug and gene delivery, and as a nonthermal ablation method in tumor treatment and cardiac ablation. Electroporation involves delivering high-voltage electric pulses to target tissue; however, this can cause effects beyond the intended target tissue like nerve stimulation, muscle contractions and pain, requiring use of sedatives or
anesthetics. It was previously shown that adjusting pulse parameters may mitigate some of these effects, but not how these adjustments would affect electroporation’s efficacy. We investigated the effect of varying pulse parameters such as interphase and interpulse delay while keeping the duration and number of pulses constant on nerve stimulation, muscle contraction and assessing pain and electroporation efficacy, conducting experiments on human volunteers, tissue samples
and cell lines in vitro. Our results show that using specific pulse parameters, particularly short high-frequency biphasic pulses with short interphase and long interpulse delays, reduces muscle contractions and pain sensations in healthy individuals. Higher stimulation thresholds were also observed in experiments on isolated swine phrenic nerves and human esophagus tissues. However, changes in the interphase and interpulse delays did not affect the cell permeability and survival, suggesting that modifying the pulse parameters could minimize adverse effects while preserving therapeutic goals in electroporation.

The equivalence of different types of electric pulses for lectrochemotherapy with cisplatin − an in vitro study

Background. Electrochemotherapy (ECT) is a treatment involving the administration of chemotherapeutics drugs followed by the application of 8 square monopolar pulses of 100 μs duration at a repetition frequency of 1 Hz or 5000 Hz. However, there is increasing interest in using alternative types of pulses for ECT. The use of high-frequency short bipolar pulses has been shown to mitigate pain and muscle contractions. Conversely, the use of millisecond pulses is interesting when combining ECT with gene electrotransfer for the uptake of DNA-encoding proteins that stimulate the immune response with the aim of converting ECT from a local to systemic treatment. Therefore, the aim of this study was to investigate how alternative types of pulses affect the efficiency of the ECT.
Materials and methods. We performed in vitro experiments, exposing Chinese hamster ovary (CHO) cells to con ventional ECT pulses, high-frequency bipolar pulses, and millisecond pulses in the presence of different concentra tions of cisplatin. We determined cisplatin uptake by inductively coupled plasma mass spectrometry and cisplatin cytotoxicity by the clonogenic assay.
Results. We observed that the three tested types of pulses potentiate the uptake and cytotoxicity of cisplatin in an equivalent manner, provided that the electric field is properly adjusted for each pulse type. Furthermore, we quantified that the number of cisplatin molecules, resulting in the eradication of most cells, was 2−7 × 107 per cell.
Conclusions. High-frequency bipolar pulses and millisecond pulses can potentially be used in ECT to reduce pain and muscle contraction and increase the effect of the immune response in combination with gene electrotransfer, respectively.

Analysis of magnetic resonance contrast agent entrapment following reversible electroporation in vitro

Background. Administering gadolinium-based contrast agent before electroporation allows the contrast agent to enter the cells and enables MRI assessment of reversibly electroporated regions. The aim of this study was evaluation of contrast agent entrapment in Chinese hamster ovary (CHO) cells and comparison of these results with those determined by standard in vitro methods for assessing cell membrane permeability, cell membrane integrity and cell survival following electroporation.
Materials and methods. Cell membrane permeabilization and cell membrane integrity experiments were per formed using YO-PRO-1 dye and propidium iodide, respectively. Cell survival experiments were performed by assessing metabolic activity of cells using MTS assay. The entrapment of gadolinium-based contrast agent gadobutrol inside the cells was evaluated using T1 relaxometry of cell suspensions 25 min and 24 h after electroporation and confirmed by inductively coupled plasma mass spectrometry.
Results. Contrast agent was detected 25 min and 24 h after the delivery of electric pulses in cells that were reversibly electroporated. In addition, contrast agent was present in irreversibly electroporated cells 25 min after the delivery of electric pulses but was no longer detected in irreversibly electroporated cells after 24 h. Inductively coupled plasma
mass spectrometry showed a proportional decrease in gadolinium content per cell with shortening of T1 relaxation time (R2 = 0.88 and p = 0.0191).
Conclusions. Our results demonstrate that the contrast agent is entrapped in cells exposed to reversible electroporation but exits from cells exposed to irreversible electroporation within 24 h, thus confirming the hypothesis on which detection experiments in vivo were based.

Determination of the Impact of High-Intensity Pulsed
Electromagnetic Fields on the Release of Damage-Associated
Molecular Pattern Molecules

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.

 Dynamics of cell death due to electroporation using different pulse parameters as revealed by different viability assays

The mechanisms of cell death due to electroporation are still not well understood. Recent studies suggest that cell death due to electroporation is not an immediate all-or-nothing response but rather a dynamic process that occurs over a prolonged period of time. To investigate whether the dynamics of cell death depends on the pulse parameters or cell lines, we exposed different cell lines to different pulses [monopolar millisecond, microsecond, nanosecond, and high-frequency bipolar (HFIRE)] and then assessed viability at different times using different viability assays. The dynamics of cell death was observed by changes in metabolic activity and membrane integrity. In addition, regardless of pulse or cell line, the dynamics of cell death was observed only at high electroporation intensities, i.e., high pulse amplitudes and/or pulse number. Considering the dynamics of cell death, the clonogenic assay should remain the preferred viability assay for assessing viability after electroporation.

Muscle contractions and pain sensation accompanying high‑frequency electroporation pulses

To minimize neuromuscular electrical stimulation during electroporation-based treatments, the replacement of long monophasic pulses with bursts of biphasic high-frequency pulses in the range of microseconds was suggested in order to reduce muscle contraction and pain sensation due to pulse application. This treatment modality appeared under the term high-frequency electroporation (HF-EP), which can be potentially used for some clinical applications of electroporation such as electrochemotherapy, gene electrotransfer, and tissue ablation. In cardiac tissue ablation, which utilizes irreversible electroporation, the treatment is being established as Pulsed Field Ablation. While the reduction of muscle contractions was confrmed in multiple in vivo studies, the reduction of pain sensation in humans was not confrmed yet, nor was the relationship between muscle contraction and pain sensation investigated. This is the frst study in humans examining pain sensation using biphasic high-frequency lectroporation pulses. Twenty-fve healthy individuals were subjected to electrical stimulation of the tibialis anterior muscle with biphasic high-frequency pulses in the range of few microseconds and both, symmetric and asymmetric interphase and interpulse delays. Our results confrm that biphasic high-frequency pulses with a pulse width of 1 or 2 µs reduce muscle contraction and pain sensation as opposed to currently used longer monophasic pulses. In addition, interphase and interpulse delays play a signifcant role in reducing the muscle contraction and/or pain sensation. The study shows that the range of the optimal pulse arameters may be increased depending on the prerequisites of the therapy. However, further evaluation of the biphasic pulse protocols presented herein is necessary to confrm the efciency of the newly proposed HF-EP.

Immunogenic cell death in electroporation-based therapies depends on pulse waveform characteristics

Traditionally, electroporation-based therapies such as electrochemotherapy (ECT), gene electrotransfer (GET) and irreversible electroporation (IRE) are performed with different but typical pulse durations—100 microseconds and 1–50 milliseconds. However, recent in vitro studies have shown that ECT, GET and IRE can be achieved with virtually any pulse duration (millisecond, microsecond, nanosecond) and pulse type (monopolar, bipolar-HFIRE), although with different efficiency. In electroporation-based therapies, immune response activation can affect treatment outcome, and the possibility of controlling and predicting immune response could improve the treatment. In this study, we investigated if different pulse durations and pulse types cause different or similar activations of the immune system by assessing DAMP release (ATP, HMGB1, calreticulin). Results show that DAMP release can be different when different pulse durations and pulse types are used. Nanosecond pulses seems to be the most immunogenic, as they can induce the release of all three main DAMP molecules—ATP, HMGB1 and calreticulin. The least immunogenic seem to be millisecond pulses, as only ATP release was detected and even that assumingly occurs due to increased permeability of the cell membrane. Overall, it seems that DAMP release and immuneresponse in electroporation-based therapies can be controlled though pulse duration.

Efficient Gene Transfection by Electroporation—In Vitro and In Silico Study of Pulse Parameters

Gene electrotransfer (GET) is a widely used method for nucleic acids’ delivery into cells. We explored, evaluated, and demonstrated the potential use of different pulse durations for introducing plasmid DNA (pDNA) into cells in vitro and compared the efficiency and dynamics of transgene expression after GET. We performed experiments on cell suspensions of 1306 fibroblasts and C2C12 myoblasts with four ranges of pulse durations (nanosecond, high frequency bipolar (HF-BP), and micro- and millisecond). Six different concentrations of pDNA encoding green fluorescent protein were used. We show that GET can be achieved with nanosecond pulses with a low pulse repetition rate (10 Hz). The GET’s efficiency depends on the pDNA concentration and cell line. Time dynamics of transgene expression are comparable between millisecond, microsecond, HF-BP, and nanosecond pulses but depend greatly on cell line. Lastly, based on the data obtained in the experiments of pDNA concentration effect on GET the model of the probability of pDNA and cell membrane contact during GET was developed. The model shows that pDNA migration is dominated by diffusion for nanosecond and HF-BP pulses and by electrophoresis for micro- and millisecond pulses. Modeling results can provide valuable guidance for further experiments and interpretations of the results obtained by various pulse protocols.