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 immune response in electroporation-based therapies can be controlled though pulse duration.

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.

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.

Background/Objectives: Nanoparticles (NPs) were previously explored as enhancers in electroporation due to their potential to locally amplify electric fields near cell membranes, with gold nanoparticles (AuNPs) in particular showing promise in improving membrane permeability and gene electrotransfer (GET). In this study, we systematically investigated the influence of NP properties—including size, shape, surface functionalization, and material—on electroporation efficacy. Methods: A combined approach using theoretical modeling and experimental validation was employed, encompassing numerical simulations, membrane permeabilization assays, transmission electron microscopy, and GET efficiency measurements. Results: Numerical results revealed that the presence of NPs alters local electric field distributions, but the amplification is highly localized, regardless of NP conductivity or geometry. Experimentally, only two out of six tested NP types produced a statistically significant, yet modest, increase in membrane permeability at one electric field intensity. Similarly, GET improvement was observed with only one NP type, with no dependence on concentration or functionalization. Conclusions: Overall, our findings demonstrate that NPs, under tested conditions, do not substantially enhance cell membrane permeability or GET efficacy. These conclusions are supported by both computational modeling and in vitro experiments.

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.