The use of high-frequency short bipolar pulses in cisplatin electrochemotherapy in vitro

Mouse skin melanoma cell line B16-F1, obtained from the European Collection of Authenticated Cell Cultures (ECACC, cat. no. 92101203, Sigma Aldrich, Germany, mycoplasma free), was grown 2–4 days in 75 cm² cell culture flasks (TPP, Austria) until 80% confluency in Dulbecco’s Modified Eagle’s Medium (DMEM, cat. no. D5671, Sigma Aldrich, Germany) in an incubator (Kambič, Slovenia) at 37°C and humidified 5% CO². DMEM, used in this composition for all in vitro experiments, was supplemented with 10% fetal bovine serum (cat. no. F7524, Sigma Aldrich, Germany), 2 mM L-glutamine (cat. no. G7513, Sigma Aldrich, Germany) and antibiotics, 50 μg/ml gentamycin (cat. no. G1397, Sigma Aldrich, Germany), 1 U/ml penicillin-streptomycin (cat. no. P11-010, PAA, Austria).

Cell suspension was prepared by detaching the cells in the exponential phase of growth with 10x trypsin-EDTA (cat. no T4174, Sigma Aldrich, Germany), diluted 1:9 in Hank’s basal salt solution (cat. no. H4641, Sigma Aldrich, Germany). After no more than 3 minutes, trypsin was inactivated by adding DMEM, and cells were transferred to a 50 ml centrifuge tube. Then, the cells were centrifuged (5 min, 180 g, 21°C) and re-suspended in DMEM at concentration 5x10⁶ cells/ml (experiments to measure the optimal parameters of electroporation and resealing rate of cells), 5x10⁴ cells/ml (experiments to measure the cytotoxicity of cisplatin without electroporation) or 2.2x107cells/ml (experiments to measure the cytotoxicity of cisplatin with electroporation). We performed experiments with different cell densities due to different requirements for cell number and sensitivities of the chosen assays. Even at the highest concentration (2.2x10⁷ cells/ml) we were still well below the concentration where shielding of the electric field and decreased uptake were observed.39

Two types of pulses were applied – 100 μs long monopolar pulses (i.e classical electrochemotherapy) and bursts of short bipolar pulses (HF-EP pulses). They were applied between plate stainless-steel electrodes with 2 mm distance.40 Between pulses, electrodes were cleaned in potassium-phosphate buffer (KPB, 10 mM KH₂PO₄/K₂HPO₄ in ratio 40.5:9.5, 1 mM MgCl₂, 250 mM sucrose) and dried with sterile gauze. 100 μs long monopolar pulses (8 pulses, delivered at repetition frequency 1 Hz, Figure 1A) of different voltages (80, 120, 160, 200, 240 V) were delivered by the commercially available BetaTech pulse generator (Electro cell B10, BetaTech, France) or BTX Gemini X2 pulse generator (Harvard Apparatus, USA). Short bipolar pulses of different voltages (HF-EP protocol, 100 V to 1000 V with a step of 100 V, Figure 1B) were delivered by a laboratory prototype pulse generator (University of Ljubljana) based on H-bridge digital amplifier with 1 kV MOSFETs (DE275-102N06A, IXYS, USA).38, 39, 40, 41 Short bipolar pulses were delivered in 8 bursts at repetition frequency 1 Hz, each containing 50 short bipolar pulses of 1 μs positive and 1 μs negative pulse. The delay between short bipolar pulses and between positive and negative pulse was 1 μs. The on-time (the time when the voltage was different from zero) of the HF-EP pulses was 800 μs, equivalent to the duration of the eight 100 μs long monopolar pulses. The duration of one short bipolar pulse was chosen as it successfully permeabilized cell membranes as previously demonstrated by an increased uptake of a fluorescent dye.38 The voltage and the current were monitored in all experiments with an oscilloscope Wavesurfer 422, 200 MHz, a differential voltage probe ADP305 and a current probe CP030 or AP015, all from LeCroy, USA to ensure that delivered voltage and current were consistent at the same settings even if delivered with different generators.

Figure 1

In permeability experiments, just before pulse application, 60 μl of cell suspension was mixed with 6 μl of 1.5 mM propidium iodide (PI) (136 μM final concentration). In resealing experiments, PI was not added before pulse application but after electroporation. 60 μl of the cell suspension was electroporated, and 50 μl of the treated sample was transferred to a 1.5 ml centrifuge tube. In resealing experiments, 5 μl of PI (136 μM final concentration) was added to 50 μl of the treated sample 2 min, 5 min, 10 min or 20 min after pulse delivery. Two minutes after electroporation (permeability experiments) or PI addition (resealing experiments), the samples were diluted in 100 μl of KPB, and vortexed. The uptake of propidium was measured on the flow cytometer (Attune NxT; Life Technologies, Carlsbad, CA, USA). Cells were excited with a blue laser at 488 nm, and the emitted fluorescence was detected through a 574/26 nm band-pass filter. The measurement was finished when 10,000 events were acquired. Single cells were separated from all events by gating. Obtained data were analyzed using the Attune NxT software. The percentage of permeabilized cells was determined from the histogram of PI fluorescence.

60 μl of the cell suspension was electroporated, 50 μl was transferred to a 15 ml centrifuge tube, and two minutes after pulses delivery, the samples were diluted in 450 μl of DMEM and mixed with a pipette. When all the samples were finished, 5x10⁴ cells were transferred in each well on a 96-well plate in three technical repetitions. After 24 h of incubation at 37°C and humidified 5% CO₂, the survival assay was performed. 20 μl of MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS), Promega, USA) was added per well according to manufacturer’s instructions and left in an incubator for 2h. MTS assay was used to quantify the number of viable cells evaluating their metabolic activity by measuring the formazan absorbance at 490 nm. After 2 h, the absorbance was measured on a spectrofluorometer (Tecan Infinite 200; Tecan, Grödig, Austria). Cell survival was calculated by first subtracting the background (only DMEM and MTS) from all measurements and then normalizing the absorbance of the treated samples to the absorbance of the control samples.

On the first day, 5x10³ B16-F1 cells were seeded per well on a 96-well plate and left for one day in an incubator (Kambič, Slovenia) at 37°C and humidified 5% CO₂. On the second day (24 h after cell seeding), the 3.3 mM stock cisplatin (Accord HealthCare, Poland) was diluted in 0.9% NaCl (physiological solution) to obtain the 10x higher concentration of cisplatin than desired with the cells (1, 10, 100, 330 μM). Diluted cisplatin was then mixed with the DMEM in ratio 1:9 and cells were incubated in DMEM with cisplatin for 10 min, 1 h, 24 h or 48 h. After the indicated time, DMEM with cisplatin was substituted with DMEM only. On the fourth day (72 h after cell seeding), the MTS survival assay was performed as described in the subsection Cell survival following electroporation only.

We performed two types of experiments. We applied: 1) different electric fields at fixed cisplatin concentration (100 μM) to evaluate the effect of electric field on cell death; 2) fixed electric field (optimal value – long monopolar pulses E = 1.2 kV/cm and short bipolar (HF-EP) pulses E = 3 kV/cm) with different cisplatin concentrations to evaluate the effect of cisplatin concentration on cell survival. Optimal parameters of electroporation were determined with experiments described in the subsections Determination of permeability and resealing, and Cell survival following electroporation only and were chosen as those where the highest uptake of propidium iodide (i.e, highest cell membrane permeability) and the highest cell survival were obtained.

The 3.3 mM stock cisplatin was diluted in 0.9% NaCl to obtain the desired concentrations of cisplatin with the cells (1, 10, 100, 330 μM) in both experiments. The drug was prepared fresh for each experiment. Right before experiments, 120 μl of cell suspension was mixed with 13.3 μl of cisplatin. 60 μl of the cell suspension with added cisplatin was transferred between the electrodes, and long monopolar or short bipolar (HF-EP) pulses were delivered (electroporation+cisplatin). The remaining 60 μl was used as a control and was transferred between the electrodes, but no pulses were delivered (only cisplatin). 50 μl of the treated and control sample were transferred in a 15 ml centrifuge tube. 10 minutes after pulse delivery, the samples were diluted 40x in full DMEM and vortexed. 5.5x10³ cells were transferred in each well on a 96-well plate in triplicates. The survival assay was performed as described in the subsection Cell Survival after 72 hours as previously suggested.42

Statistical analysis was performed using the software SigmaPlot v11 (Systat Software, San Jose, CA). We performed the t-test or one sample t-test when comparing two groups or one group towards normalized control. We performed the 1-way or 2-way ANOVA if the normality test was passed or the ANOVA on ranks if the normality test failed with the post-hoc Tukey test. The details on the performed test and the obtained P-value are written in respective figure captions in the Results section. On figures, one asterisk (*) signifies P < 0.05, two (**) P < 0.01 and three (***) P < 0.001.

First, we performed experiments to determine the optimal parameters of electroporation to be later used in the experiments with cisplatin. As optimal parameters of electroporation were considered those where the highest cell membrane permeability and the highest cell survival were achieved. In Figure 2 we can observe the permeability curves (blue dashed line) and the survival curves (red solid line) as a function of electric field amplitude for (A) 100 μs long monopolar pulses and (B) bursts of short bipolar (HF-EP) pulses. In Figure 2A we can see that the threshold of electroporation was at 0.8 kV/cm and highest uptake and survival were achieved at 1.2 kV/cm which was considered as the optimal point of electroporation. In Figure 2B we can see that the threshold of electroporation was at 2 kV/cm, the threshold for irreversible electroporation at 4.5 kV/cm and the highest uptake and survival for HF-EP pulses were obtained at 3 kV/cm which was chosen as the optimal point of electroporation with short bipolar pulses. Electric pulses of 1.2 kV/cm with 100 μs monopolar pulses and 3 kV/cm in HF-EP protocol were thus considered to be equivalent and were used in further experiments.

Figure 2

With the optimal parameters of electroporation, we measured the resealing of cell membranes after electroporation. Figure 3 shows the permeability curves obtained as a function of different time of exposure to propidium iodide after electroporation delivering (A) long monopolar pulses at E = 1.2 kV/cm and (B) HF-EP pulses at E = 3 kV/cm. Figure 3A and Figure 3B show a peak of permeability at 0 min, i.e, right after the pulses are applied. Then, we can see a decrease in permeability that reaches a plateau after 10 min. We chose 10 min as the time after which cell membranes resealed. Accordingly, in the subsequent experiments, electroporated samples with cisplatin were diluted after 10 minutes.

Figure 3

We measured the cytotoxicity of cisplatin without electroporation at different cisplatin concentrations and incubation times on attached confluent cell monolayers (Figure 4). Cells were more affected if they were exposed to cisplatin for a longer time (24 h and 48 h incubation caused significantly higher cell death than 10 min and 1 h incubation). There was no difference if cells were incubated for 10 min vs 1 h and 24 h vs 48 h. There was no difference between 1 μM and 10 μM, but in general, cytotoxicity increased with higher cisplatin concentrations. After 10 min and 1 h of incubation (red solid and green dashed curve, respectively) there was a decrease in cell survival with increasing cisplatin concentration and at the highest tested concentration

Figure 4

(330 μM) we obtained 58.55% ± 14.90% and 48.12% ± 14.01% survival for 10 min and 1 h, respectively. After 24 h and 48 h (blue dotted and black dash-dot curve, respectively) of incubation, cell survival decreased rapidly to less than 10% already with 100 μM of cisplatin.

First, we measured the cytotoxicity of cisplatin with electroporation at different electric fields and selected cisplatin (CDDP) concentration of 100 μM. In Figure 5, we can observe cell survival as a function of applied electric field, on Figure 5A for long monopolar pulses and Figure 5B for HF-EP pulses. The solid green line shows cell survival after electroporation with cisplatin and red dashed line survival after only electroporation without cisplatin. The red dashed curves of Figure 5A and B are already shown in Figure 2A and B. We can see in both Figure 5A and B that the combination of electric pulses and cisplatin is more efficient in achieving cell death than applying only electric pulses or only cisplatin (100% survival at 100 μM cisplatin and 10 min incubation time, Figure 4) and that cytotoxicity of cisplatin increases with increasing electric field, starting at 0.8 kV/cm for 100 μs long monopolar pulses and 2 kV/cm for short bipolar pulses, which coincides with the thresholds for reversible electroporation (Figure 2). In Figure 5A we can see that at E = 1.2 kV/cm with cisplatin 32.16% ± 14.08% of cells survive while when we apply only electric pulses, all cell survive. Similarly, in Figure 5B at E = 3 kV/cm 25.33% ± 3.73% of cells survive electroporation with cisplatin opposed to 100% when only electric pulses are applied.

Figure 5

Then, we measured cytotoxicity of cisplatin with electroporation at a fixed electric field (optimal point of electroporation with the highest cell membrane permeability and lowest survival - long monopolar pulses at E = 1.2 kV/cm and HF-EP pulses at E = 3 kV/cm) and different cisplatin concentrations. In Figure 6 we can see two cell survival curves obtained by applying 1) only cisplatin (red dashed curve) and 2) cisplatin in combination with electroporation (solid green curve). From the red dashed curve in Figure 6A and B we can see that cell survival does not decrease with increasing cisplatin concentration due to short incubation time (see also Figure 4). From the solid green curve in Figure 6 A and B we can see that the cytotoxicity of cisplatin increases when electric pulses are applied with increasing cisplatin concentration. A similar trend in survival is observed for both types of pulses.

Figure 6

First, we determined the cytotoxic effects of cisplatin on a confluent monolayer of cells, because survival after longer exposure time was not possible to evaluate on cell suspension (Figure 4). At 100 μM, short exposure (1 hour or less) did not affect survival. We decided to perform experiments with electroporation at 100 μM cisplatin in order to see possible potentiation of the cytotoxic effect of cisplatin after electroporation. Namely, using higher concentration could already decrease survival without applying electric pulses and we could not asses, if electroporation increases cytotoxicity. In the experiments assessing survival after incubation with cisplatin as determined by the MTS assay, 24 h and 48 h time points were not different one from another and we assumed that also 72 h exposure (which was used in the electroporation experiments) would yield similar results. However, we did not make experiments also at 72 h exposure time.

We determined the optimal parameters for experiments with cisplatin and electric pulses, i.e, the optimal voltage of electric pulses, incubation time with cisplatin after pulse application and cisplatin concentration with a) 100 μs long monopolar pulses (ECT) and b) short bipolar pulses (HF-EP). In experiments with 8x100 μs monopolar pulses, the optimal electric field (highest uptake of propidium and the highest cell survival) was 1.2 kV/cm (Figure 2A) which is in agreement with other studies 44 and corroborates our existing data where cell permeabilization was detected via intracellular platinum measurements.45 Unfortunately, we could not apply voltages higher than 240 V (1.2 kV/cm) due to the current limitations of the pulse generator. We determined that the optimal electric field with HF-EP pulses was 3 kV/cm (Figure 2B). With bipolar pulses, we had to apply 2.5-times higher electric field than with monopolar pulses to obtain comparable effect, which is in agreement with the results reported by Sweeney et al. for propidium uptake38 and with the in vitro data on irreversible electroporation, where irreversible electroporation threshold increased 2.1-times, when 1 μs long pulses were applied in bursts instead as 100 μs long pulses.46

With the selected parameters of electroporation, we measured the resealing rate of cells after electroporation. We determined that after 10 min cell membrane is mostly resealed (Figure 3) and did all subsequent cisplatin experiments with 10 min incubation. Dilution of cells with permeable membranes would namely reduce or stop the influx too early or even cause efflux of cisplatin due to dilution and potential reversal of the direction of the concentration gradient.47 This time range is in agreement with the existing in vitro studies, where the incubation time ranges from 5 minutes23 to 60 minutes48 as well as with the in vivo standard operating procedures where the pulses are applied between 8 and 28 minutes after intravenous drug injection.19 With propidium iodide (PI) we could use shorter incubation times (2 minutes) as PI binds soon after entering the cell49, but with cisplatin, we do not know how fast it binds, and we have to wait until cell membranes are completely resealed before the dilution is made. PI was used as a model for cisplatin as its molecular weight is in the same range as of cisplatin (668 g/mol and 300 g/mol for PI and cisplatin, respectively). The similarity in the shape of the permeabilization curve (Figure 2) and cell death due to cisplatin uptake (Figure 5) is another indicator that PI is an appropriate molecule to assess the uptake of cisplatin. Also, experiments with PI and flow cytometry are fast and easy to perform, enable screening of a wide range of parameters quicker than assessing cell survival or platinum uptake via mass spectrometry and are thus usually used to determine optimal parameters of electric pulses for electrochemotherapy in vitro50, 51, 52

100 μM cisplatin concentration was chosen as we could (1) test several pulse parameters without reaching the limitations of the survival assay, (2) it is in a similar range as used in other in vitro studies.23 ,45 ,48 ,53 ,54 Other tested concentrations (1, 10, 100, 330 μM) were chosen as they were already used in previous in vitro experiments.23 ,45 (3) The IC50 value of cisplatin pooled together from several studies in53 was determined to be between 0.83 μM and 1000 μM without electroporation and 0.083 μM and 106 μM with electroporation. As we determined graphically from Figure 6, the IC50 value was in our study 85 μM for monopolar, and 45 μM for bipolar pulses, which is in agreement with the literature and close to the 100 μM.

In our study, different cell densities were used due to different requirements for cell number and sensitivities of the chosen assays. However, even at the highest concentration (2.2x10⁷ cells/ml) we were still well below the concentration where shielding of the electric field and decreased uptake were observed.39 72 h growth time after electrochemotherapy was chosen as it was shown that results of metabolic assays are highly dependent on evaluation time point and they correspond to the results of clonogenic assay better at later time points.42

We measured the cytotoxicity of cisplatin with electroporation at fixed cisplatin concentration of 100 μM and different electric fields (Figure 5). We were interested in the effect of electric field intensity on cisplatin cytotoxicity, as usually when treating tumors in vivo, the electric field distribution is inhomogeneous due to different dielectric properties of different tissues and various electrode configurations.56, 57 A similar tendency of cell survival as a function of the electric field was observed with monopolar as well as HF-EP pulses - we achieved greater cell death by applying cisplatin in combination with electric pulses than by only applying electric pulses. Survival decreased with increasing electric field. In Figure 5A, comparing the red curve with the green one, we can see that at E = 1.2 kV/cm cells die because of the cisplatin uptake and not due to irreversible electroporation. The survival after applying 1.2 kV/cm was still 100%, the survival with electric pulses and cisplatin dropped to 32.16% ± 14.08%. Similarly as with monopolar pulses, when applying bipolar pulses of E = 3 kV/cm, cells die due to the cisplatin uptake and not due to irreversible electroporation (Figure 5B). At E > 3 kV/cm cell death is due to the cytotoxic effect of cisplatin as well as irreversible electroporation. As expected and in accordance with previously published results for propidium iodide, we needed to deliver 2.5-times higher electric field with the HF-EP pulses to achieve a comparable effect.38

Interestingly, the shape of the permeabilization curve to propidium (Figure 2) corresponds perfectly to the shape of the survival curve after electrochemotherapy (Figure 5). The onset of membrane permeabilization is at 0.8 kV/cm for long monopolar pulses (Figure 2A) and at 2 kV/cm for HF-EP pulses (Figure 2B), which corresponds to the onset of the decrease in survival after electrochemotherapy (Figure 5). The plateau of membrane permeabilization for HF-EP pulses is reached at 3–3.5 kV/cm (Figure 2B) which corresponds to the reached plateau of survival (Figure 5B). Thus at our specific conditions, membrane permeability to propidium is a good indicator of cytotoxicity of cisplatin.

In Figure 6, we measured cytotoxicity of cisplatin with electroporation at a fixed electric field (monopolar pulses E = 1.2 kV/cm and short bipolar pulses E = 3 kV/cm) and different cisplatin concentrations. Namely, in tissues, inhomogeneous cisplatin concentration is expected, also initial cisplatin concentration is usually inhomogeneous after intratumoral injection.45 Both (A) monopolar pulses at E = 1.2 kV/cm and (B) HF-EP pulses at E = 3 kV/cm show a similar behavior. In both Figures 6 A and B, the cytotoxicity of cisplatin increases more with cisplatin in combination with electric pulses than using only cisplatin.23 ,25 Indeed, without electric pulses application, a high dose of cisplatin and/or longer incubation times need to be used to achieve a decrease in cell survival (Figure 4). However, applying 330 μM cisplatin with long monopolar pulses only 14.28% ± 5.84% of cell survived and with short bipolar pulses (HF-EP) only 8.45% ± 5.22% of cell survived. We must keep in mind, that with short bipolar pulses, 2.5-times higher electric field was applied to achieve a similar effect. From the red dashed curve in Figure 6A and B we can see that cell survival did not decrease with increasing cisplatin concentration. This result should be the same as in Figure 4 considering only the 10 min curve, but in Figure 4 cell survival slightly decreases with increasing cisplatin concentration. The reasons for this discrepancy could be the differences in the protocols: attached cell monolayers to measure the cytotoxicity of cisplatin without electroporation and cells in suspension to measure the cytotoxicity of cisplatin in combination with electroporation. Also, the attached cells were diluted much less with fresh DMEM after exposure to cisplatin than cells in suspension. Besides, cell survival was measured after 48h for the attached cell and after 72 h for the cell in suspension.

HF-IRE pulses were reported to reduce muscle contractions in comparison with classic 100 μs pulses which was observed in several studies in vivo. For example, muscle contractions with HF-IRE pulses were much less noticeable than with 100 μs long monopolar pulses in experiments on rabbit liver.33 ,57 ,58 Even in the absence of cardiac synchronization and paralytics, only minor muscle twitch was recorded in one out of 24 cases59 ,60 when treating porcine liver. Sano et al observed that HF-IRE waveforms reduced the intensity of muscle contractions in comparison with traditional IRE pulses on ex-vivo porcine model34 and in in vivo murine tumor.46 Arena et al observed that HF-IRE pulses eliminated muscle contractions when electric pulses were applied to the brain of rats37 and achieved blood-brain-barrier disruption without inducing local or distal muscle contractions.61 Latouche et al. observed no evidence of muscle or nerve excitation or cardiac arrhythmia during any pulse delivery when treating intracranial meningioma in dogs.35 In a first human study on high-frequency irreversible electroporation of prostate cancer, only a small amount of muscle relaxant was needed, and there were no visible muscle contractions during the pulse delivery process.36 Additionally, the histological analysis in in vivo porcine experiments indicates that with HF-IRE rapid and reproducible ablation in the liver can be achieved, while preserving gross vascular/biliary architecture.60 The mechanism for decreased muscle contractions is still unknown. However, different possible explanations were offered. It was suggested that (1) stimulation threshold raises faster than the threshold for irreversible electroporation with decreasing pulse length62 which is a consequence of geometrical differences between nerve fibers and tumor cells.63 (2) At around 1 μs there is an overlap of the depolarization threshold and electroporation threshold on the strength-intensity curve.41 (3) The short negative pulse delivered after a positive pulse accelerated the passive repolarization and swamped the regenerative response, thus abolishing the action potential.64 The pain was not yet evaluated, but promising results regarding muscle contractions indicate that we can expect less pain with HF-EP than with classical 100 μs pulses.

Before transfer to the clinical setting, more experiments in vitro as well in vivo need to be performed. In the scope of the current study, experiments with bleomycin are not feasible due to organizational reasons. However, we are planning to perform, in the future, experiments using bleomycin with HF-EP, as bleomycin is frequently used for ECT in the clinics. So, cytotoxicity of bleomycin and HF-EP needs to be assessed, and experiments determining intratumoral cisplatin/bleomycin concentration should be performed. The electric field needed to achieve cell death is with HF-EP higher than in classical EP, and thus the effect of high voltage on important structures in the vicinity of the tumors should be investigated, similarly as in60 for hepatic veins. Also, temperature increase due to Joule heating has to be minimized for example by introducing a delay between bursts36 ,59, limiting electric current or number of bursts36 ,46 ,61 and avoiding increased temperature by optimizing treatment parameters.35 ,37 ,58 ,61 The influence of HF-EP on muscle contractions, pain and heart rhythm should also be studied, as is being done for high-frequency irreversible electroporation. Currently, pulses in the published studies are being applied with laboratory prototypes - a clinical generator of bipolar pulses needs to be designed and certified before clinical use. However, electrode geometry could be the same as those used with the longer monopolar pulses, but electrical isolation of the wiring and stray capacitance should be re-evaluated.

Applying HF-EP pulses comes at the expense of delivering considerably higher pulse amplitude. However, we need to take into account that in our study, we focused on eight bursts in total on-time of 800 μs to enable comparison with the standard ECT protocol and be consistent with previous studies.38 To obtain a good effect while keeping the applied voltage low, we could apply more bursts, longer pulses than 1 μs or asymmetrical bipolar pulses34 ,65, although it was indicated that muscle contractions are increased with the asymmetrical waveforms. Also of importance is that with pulses in the range of a few microseconds, we are already in the range of the so-called cancellation effect which could be partially responsible for decreased effect of shorter pulses in comparison to longer pulses.38 ,66 We can nevertheless conclude that HF-EP pulses can be successfully used in electrochemotherapy treatments in vitro, however, at the expense of delivering electric pulses of higher amplitudes.38

Although still at the in vitro testing stage, we believe that the use of HF-EP pulses for electrochemotherapy in the clinics could potentially decrease the discomfort connected with muscle contractions and pain, simplifying the treatment procedure by lowering dose of muscle relaxants and anesthesia, and avoid synchronization with the electrocardiogram, while potentially achieving more homogeneous electric field distribution67 and reducing the electrolytic contamination.68