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.

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.

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.

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

Cell death due to electroporation - A review.

Exposure of cells to high voltage electric pulses increases transiently membrane permeability through membrane electroporation. Electroporation can be reversible and is used in gene transfer and enhanced drug delivery but can also lead to cell death. Electroporation resulting in cell death (termed as irreversible electroporation) has been successfully used as a new non-thermal ablation method of soft tissue such as tumours or arrhythmogenic heart tissue. Even though the mechanisms of cell death can influence the outcome of electroporation-based treatments due to use of different electric pulse parameters and conditions, these are not elucidated yet. We review the mechanisms of cell death after electroporation reported in literature, cell injuries that may lead to cell death after electroporation and membrane repair mechanisms involved. The knowledge of membrane repair and cell death mechanisms after cell exposure to electric pulses, targets of electric field in cells need to be identified to optimize existing and develop of new electroporation-based techniques used in medicine, biotechnology, and food technology.

Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro.

Background Tumor cells can die via immunogenic cell death pathway, in which damage-associated molecular pattern molecules (DAMPs) are released from the cells. These molecules activate cells involved in the immune response. Both innate and adaptive immune response can be activated, causing a destruction of the remaining infected cells. Activation of immune response is also an important component of tumor treatment with electrochemotherapy (ECT) and irreversible electroporation (IRE). We thus explored, if and when specific DAMPs are released as a consequence of electroporation in vitro. Materials and methods In this in vitro study, 100 µs long electric pulses were applied to a suspension of Chinese hamster ovary cells. The release of DAMPs - specifically: adenosine triphosphate (ATP), calreticulin, nucleic acids and uric acid was investigated at different time points after exposing the cells to electric pulses of different amplitudes. The release of DAMPs was statistically correlated with cell permeabilization and cell survival, e.g. reversible and irreversible electroporation. Results In general, the release of DAMPs increases with increasing pulse amplitude. Concentration of DAMPs depend on the time interval between exposure of the cells to pulses and the analysis. Concentrations of most DAMPs correlate strongly with cell death. However, we detected no uric acid in the investigated samples. Conclusions Release of DAMPs can serve as a marker for prediction of cell death. Since the stability of certain DAMPs is time dependent, this should be considered when designing protocols for detecting DAMPs after electric pulse treatment.

Development of adaptive resistance to electric pulsed field treatment in CHO cell line in vitro.

Pulsed electric field treatment has increased over the last few decades with successful translation from in vitro studies into different medical treatments like electrochemotherapy, irreversible electroporation for tumor and cardiac tissue ablation and gene electrotransfer for gene therapy and DNA vaccination. Pulsed electric field treatments are efficient but localized often requiring repeated applications to obtain results due to partial response and recurrence of disease. While these treatment times are several orders of magnitude lower than conventional biochemical treatment, it has been recently suggested that cells may become resistant to electroporation in repetitive treatments. In our study, we evaluate this possibility of developing adaptive resistance in cells exposed to pulsed electric field treatment over successive lifetimes. Mammalian cells were exposed to electroporation pulses for 30 generations. Every 5th generation was analyzed by determining permeabilization and survival curve. No statistical difference between cells in control and cells exposed to pulsed electric field treatment was observed. We offer evidence that electroporation does not affect cells in a way that they would become less susceptible to pulsed electric field treatment. Our findings indicate pulsed electric field treatment can be used in repeated treatments with each treatment having equal efficiency to the initial treatment.

Cancellation effect is present in high-frequency reversible and irreversible electroporation.

It was recently suggested that applying high-frequency short biphasic pulses (HF-IRE) reduces pain and muscle contractions in electrochemotherapy and irreversible ablation treatments; however, higher amplitudes with HF-IRE pulses are required to achieve a similar effect as with monophasic pulses. HF-IRE pulses are in the range of a microseconds, thus, the so-called cancellation effect could be responsible for the need to apply pulses of higher amplitudes. In cancellation effect, the effect of first pulse is reduced by the second pulse of opposite polarity. We evaluated cancellation effect with high-frequency biphasic pulses on CHO-K1 in different electroporation buffers. We applied eight bursts of 1-10 µs long pulses with inter-phase delays of 0.5 µs - 10 ms and evaluated membrane permeability and cell survival. In permeability experiments, cancellation effect was not observed in low-conductivity buffer. Cancellation effect was, however, observed in treatments with high-frequency biphasic pulses looking at survival in all of the tested electroporation buffers. In general, cancellation effect depended on inter-phase delay as well as on pulse duration, i.e. longer pulses and longer interphase delay cause less pronounced cancellation effect. Cancellation effect could be partially explained by the assisted discharge and not by the hyperpolarization by the chloride channels.

Contactless electroporation induced by high intensity pulsed electromagnetic fields via distributed nanoelectrodes.

Pulsed electric fields (PEFs) can be used to transiently increase cell membrane permeability in procedures ranging from gene therapy to tumor eradication. Although very efficient, PEF-based therapies generally require the use of invasive electrodes, which cause pain and tissue damage. An emerging noninvasive, contactless alternative to PEFs are High Intensity Pulsed Electromagnetic Fields (HI-PEMF), whereby the electric field inside the tissue is induced remotely by external pulsed magnetic field. However, one of the current major drawbacks of HI-PEMFs is their inferior efficiency compared to PEFs. In this study we present the proof-of-concept that by adding highly conductive 5 and 20 nm gold nanoparticles (Au NPs), we can significantly potentiate the permeabilizing effect of HI-PEMFs, making it possible to permeabilize up to 80% of the cells with minimal or no effect on cell survival, compared to negligible percentage of permeabilized cells using HI-PEMF alone. Experiments, conducted on Chinese Hamster Ovary cells and Escherichia coli, suggest that Au NPs act as distributed nanoelectrodes, locally enhancing the electric field induced at the plasma membrane. Our findings open up an avenue of possibilities for combining naked as well as functionalized Au NPs with HI-PEMFs for noninvasive, remotely controlled smart drug delivery applications.