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.

Effect of Experimental Electrical and Biological Parameters on Gene Transfer by Electroporation: A Systematic Review and Meta-Analysis.

The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization.

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 confirmed in multiple in vivo studies, the reduction of pain sensation in humans was not confirmed yet, nor was the relationship between muscle contraction and pain sensation investigated. This is the first study in humans examining pain sensation using biphasic high-frequency electroporation pulses. Twenty-five 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 confirm 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 significant role in reducing the muscle contraction and/or pain sensation. The study shows that the range of the optimal pulse parameters may be increased depending on the prerequisites of the therapy. However, further evaluation of the biphasic pulse protocols presented herein is necessary to confirm the efficiency of the newly proposed HF-EP.

Gene transfer by electroporation with high frequency bipolar pulses in vitro.

High-frequency bipolar pulses (HF-BP) have been demonstrated to be efficient for membrane permeabilization and irreversible electroporation. Since membrane permeabilization has been achieved using HF-BP pulses we hypothesized that with these pulses we can also achieve successful gene electrotransfer (GET). Three variations of bursts of 2 µs bipolar pulses with 2 µs interphase delay were applied in HF-BP protocols. We compared transfection efficiency of monopolar micro and millisecond pulses and HF-BP protocols at various plasmid DNA (pDNA) concentrations on CHO - K1 cells. GET efficiency increased with increasing pDNA concentration. Overall GET obtained by HF-BP pulse protocols was comparable to overall GET obtained by longer monopolar pulse protocols. Our results, however, suggest that although we were able to achieve similar percent of transfected cells, the number of pDNA copies that were successfully transferred into cells seemed to be higher when longer monopolar pulses were used. Interestingly, we did not observe any direct correlation between fluorescence intensity of pDNA aggregates formed on cell membrane and transfection efficiency. The results of our study confirmed that we can achieve successful GET with bipolar microsecond i. e. HF-BP pulses, although at the expense of higher pDNA concentrations.

Water Pores in Planar Lipid Bilayers at Fast and Slow Rise of Transmembrane Voltage.

Basic understanding of the barrier properties of biological membranes can be obtained by studying model systems, such as planar lipid bilayers. Here, we study water pores in planar lipid bilayers in the presence of transmembrane voltage. Planar lipid bilayers were exposed to fast and slow linearly increasing voltage and current signals. We measured the capacitance, breakdown voltage, and rupture time of planar lipid bilayers composed of 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), 1-pamitoyl 2-oleoyl phosphatidylserine (POPS), and a mixture of both lipids in a 1:1 ratio. Based on the measurements, we evaluated the change in the capacitance of the planar lipid bilayer corresponding to water pores, the radius of water pores at membrane rupture, and the fraction of the area of the planar lipid bilayer occupied by water pores.planar lipid bilayer capacitance, which corresponds to water pores, water pore radius at the membrane rupture, and a fraction of the planar lipid bilayer area occupied by water pores. The estimated pore radii determining the rupture of the planar lipid bilayer upon fast build-up of transmembrane voltage are 0.101 nm, 0.110 nm, and 0.106 nm for membranes composed of POPC, POPS, and POPC:POPS, respectively. The fraction of the surface occupied by water pores at the moment of rupture of the planar lipid bilayer The fraction of an area that is occupied by water pores at the moment of planar lipid bilayer rupture is in the range of 0.1-1.8%.

Effect of electroporation and recovery medium pH on cell membrane permeabilization, cell survival and gene transfer efficiency in vitro.

Electroporation is a method which uses an adequate number of electric pulses of enough amplitude, duration and number applied to cells, thus inducing transient permeabilization of the cell membrane. Due to possibility that microenvironment in applications of in vivo electroporation is slightly acidic, we studied the effects of slightly acidic electroporation and recovery medium. We observed no difference in the permeabilization threshold, detected by propidium iodide, of cells which were electroporated and allowed to recover in growth (pH 7.8) or acidic (pH 6.5) medium. In contrast, statistically significant difference was observed in survival of cells that were exposed to pulse amplitudes greater than permeabilization threshold. Survival of cells was greater if acidic electroporation and recovery medium were used, but acidic extracellular pH decreased gene electrotransfer efficiency. We also observed differences in morphology between cells that were electroporated and left to recover in growth medium and cells that were electroporated and left to recover in acidic medium. Our results imply that slightly acidic extracellular pH allows more efficient repair of damage that is induced on cell membrane during electroporation with high pulse amplitudes.

Specific electrical capacitance and voltage breakdown as a function of temperature for different planar lipid bilayers.

The breakdown voltage and specific electrical capacitance of planar lipid bilayers formed from lipids isolated from the membrane of archaeon Aeropyrum pernix K1 as a function of temperature were studied and compared with data obtained previously in MD simulation studies. Temperature dependence of breakdown voltage and specific electrical capacitance was measured also for dipalmitoylphosphatidylcholine (DPPC) bilayers and bilayers formed from mixture of diphytanoylphosphocholine (DPhPC) and DPPC in ratio 80:20. The breakdown voltage of archaeal lipids planar lipid bilayers is more or less constant until 50°C, while at higher temperatures a considerable drop is observed, which is in line with the results from MD simulations. The breakdown voltage of DPPC planar lipid bilayer at melting temperature is considerably higher than in the gel phase. Specific electrical capacitance of planar lipid bilayers formed from archaeal lipids is approximately constant for temperatures up to 40°C and then gradually decreases. The difference with MD simulation predictions is discussed. Specific electrical capacitance of DPPC planar lipid bilayers in fluid phase is 1.75 times larger than that of the gel phase and it follows intermediated phases before phase transition. Increase in specific electrical capacitance while approaching melting point of DPPC is visible also for DPhPC:DPPC mixture.

Monovalent ions and water dipoles in contact with dipolar zwitterionic lipid headgroups-theory and MD simulations.

The lipid bilayer is a basic building block of biological membranes and can be pictured as a barrier separating two compartments filled with electrolyte solution. Artificial planar lipid bilayers are therefore commonly used as model systems to study the physical and electrical properties of the cell membranes in contact with electrolyte solution. Among them the glycerol-based polar phospholipids which have dipolar, but electrically neutral head groups, are most frequently used in formation of artificial lipid bilayers. In this work the electrical properties of the lipid layer composed of zwitterionic lipids with non-zero dipole moments are studied theoretically. In the model, the zwitterionic lipid bilayer is assumed to be in contact with aqueous solution of monovalent salt ions. The orientational ordering of water, resulting in spatial variation of permittivity, is explicitly taken into account. It is shown that due to saturation effect in orientational ordering of water dipoles the relative permittivity in the zwitterionic headgroup region is decreased, while the corresponding electric potential becomes strongly negative. Some of the predictions of the presented mean-field theoretical consideration are critically evaluated using the results of molecular dynamics (MD) simulation.

Molecular-level characterization of lipid membrane electroporation using linearly rising current.

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-µm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.

Feasibility study for cell electroporation detection and separation by means of dielectrophoresis.

Electroporation is a phenomenon during which exposure of a cell to high voltage electric pulses results in a significant increase in its membrane permeability. Aside from the fact that after the electroporation the cell membrane becomes more permeable, the cells' geometrical and electrical properties change considerably. These changes enable use of the force on dielectric particles exposed to non-uniform electric field (dielectrophoresis) for separation of non-electroporated and electroporated cells. This paper reports the results of an attempt to separate non-electroporated and electroporated cells by means of dielectrophoresis. In several experiments we managed to separate the non-electroporated and electroporated cells suspended in a medium with conductivity 0.174 S/m by exposing them to a non-uniform electric field at a frequency of 2 MHz. The behaviour of electroporated cells exposed to dielectrophoresis raises the presumption that in addition to conductivity, considerable changes in membrane permittivity occur after the electroporation.

The effect of high frequency electric pulses on muscle contractions and antitumor efficiency in vivo for a potential use in clinical electrochemotherapy.

Muscle contractions present the main source of unpleasant sensations for patients undergoing electrochemotherapy. The contractions are a consequence of high voltage pulse delivery. Relatively low repetition frequency of these pulses (1 Hz) results in separate muscle contractions associated with each single pulse that is delivered. It would be possible to reduce the number of unpleasant sensations by increasing the frequency of electric pulses above the frequency of tetanic contraction, provided that the antitumor efficiency of electrochemotherapy remains the same. These assumptions were investigated in the present paper by measuring the muscle torque at different pulse repetition frequencies and at two different pulse amplitudes in rats and studying the antitumor efficiency of electrochemotherapy at different pulse repetition frequencies on tumors in mice. Measurements of muscle torque confirmed that pulse frequencies above the frequency of tetanic contraction (>100 Hz) reduce the number of individual contractions to a single muscle contraction. Regardless of the pulse amplitude, with increasing pulse frequency muscle torque increases up to the frequency of 100 or 200 Hz and then decreases to a value similar to that after application of a 1 Hz pulse train. Electrochemotherapy in vivo with higher repetition frequencies inhibits tumor growth and is efficient at all pulse frequencies examined (1 Hz-5 kHz). These results suggest that there is a considerable potential for clinical use of high frequency pulses in electrochemotherapy.