Radiol Oncol 2022; 56(3): 326-335. doi: 10.2478/raon-2022-0028 326 research article Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses Angelika Vizintin1, Stefan Markovic2, Janez Scancar2, Jerneja Kladnik3, Iztok Turel3, Damijan Miklavcic1 1 Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia 2 Department of Environmental Sciences, Jožef Stefan Institute, Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2022; 56(3): 326-335. Received 7 June 2022 Accepted 19 June 2022 Correspondence to: Prof. Damijan Miklavčič, Ph.D., Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, SI-1000 Ljubljana, Slovenia. E-mail: Damijan.Miklavcic@fe.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. Nanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 μs pulses and to investigate the effects of electric pulses on cisplatin structure. Materials and methods. Chinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Results. Nanosecond pulses equivalent to 8 × 100 μs pulses were established in vitro based on membrane permea- bilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and ac- cumulating cisplatin intracellularly as 8 × 100 μs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin. Conclusions. Equivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 μs pulses. Key words: electroporation; electrochemotherapy; nanosecond pulses; cisplatin Introduction Electrochemotherapy (ECT) is a local cancer treat- ment. The dominant mechanism of ECT is in- creased cellular uptake of impermeant or low permeant anticancer drugs with high intrinsic cytotoxicity - most commonly bleomycin and cis- diaminedichloroplatinum(II) (cisplatin) - due to transiently increased membrane permeability of cells/tumors after exposure to short high-voltage electric pulses.1 Over the past ten years, the number of ECT treatments performed for superficial tumors has increased dramatically and new indications have been added, such as treatment of skin metastases from visceral or hematological malignancies, vul- var cancer, deep-seated malignancies, and some noncancerous skin lesions.2 ECT has become Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 327 broadly accepted mainly because of its simplicity (it is easy to master) and versatility (it allows treat- ing a variety of cancers). Its efficacy, tolerability, and high patient satisfaction have been demon- strated in several studies, but also some side effects have been reported. According to the reports, the main side effects are unpleasant sensations, which can be painful, and muscle contractions triggered by applied high voltage electric pulses.3,4 Most commonly, electric pulses are administrated as trains of eight monophasic pulses with a duration of 100 μs at 1 Hz or 5 kHz pulse repetition rate. Nanosecond pulses have shown potential ad- vantages over micro- and millisecond pulses in electroporation-based applications. The use of pulses with high electric field strength, but very short duration (i.e., in the nanosecond range) re- sults in low energy transfer by the pulses to the treated volume, resulting in a low heating5,6 and thereby minimizing the possibility of thermal dam- age to the tissue, which is very important for spar- ing delicate structures in and around the treated area.7 In addition, nanosecond pulses limit elec- trochemical reactions at the electrode-electrolyte interface8 which may affect the treated medium or cells/tissues.9-11 Although a much higher electric field strength is required to achieve a comparable biological effect, excitation thresholds appear to be higher than the electroporation thresholds with na- nosecond pulses12-16, implying that shortening the pulse duration to nanosecond pulses could also reduce neuromuscular stimulation in electropora- tion-based applications. Recently, nanosecond pulses have been ex- plored in ECT and calcium electroporation and have shown promising results – either tumor re- gression in vivo or a decrease in cell survival in vit- ro.8,17-21 We have previously reported that nanosec- ond pulses of an appropriately chosen amplitude in combination with cisplatin decreased cell sur- vival in in vitro assays to the same extent as stand- ard 8 × 100 μs pulses.8 The aim of our present work was to investigate the underlying mechanisms of ECT with nanosecond pulses and cisplatin in vitro on Chinese hamster ovary CHO and mouse skin melanoma B16F1 cells. Two nanosecond pulse protocols (1 × 200 ns pulse at 12.6 kV/cm and 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate) were compared with 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate stand- ardly used in ECT. Accumulation of cisplatin and cell survival after in vitro ECT were measured and effects of high voltage electric pulses on the cispl- atin molecular structure were investigated by nu- clear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). Materials and methods Cell culture of Chinese hamster ovary (CHO) cells and in vitro cell survival after ECT experiment protocols were described previously.8 Mouse skin melanoma cell line B16F1 (European Collection of Authenticated Cell Cultures, cat. no. 92101203, Sigma Aldrich, Germany, mycoplasma free) was cultured in the same way as CHO cells except that Dulbecco’s Modified Eagle Medium (DMEM, cat. no. D5671, Sigma-Aldrich, Missouri, United States) supplemented with 10% FBS (cat. no. F9665, Sigma- Aldrich), 2.0 mM L-glutamine, 1 U/ml penicillin/ streptomycin and 50 μg/ml gentamycin was used instead of Nutrient Mixture F-12 Ham. Briefly, cis- platin (Cisplatin Kabi, 1 mg/mL, Fresenius Kabi, Germany or Cisplatin Accord, 1 mg/ml, Accord, UK) diluted in saline was added to cells suspended in complete growth medium DMEM just before electroporation so that the final concentration was 4 × 106 cell/ml and 0, 10, 30 or 50 μM cisplatin. The cell suspension was exposed to monophasic rectan- gular pulses (1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 μs at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate) or no pulses (non-electropo- rated controls). Cell survival was determined by the clonogenic assay. For determination of cellular cisplatin, 125 μl of the treated cell suspension was diluted 40–100 times in complete growth medium Ham F-12 (CHO) or complete growth medium DMEM (B16F1) 25 min after electroporation (or addition of cisplatin/saline for non-electroporated con- trols) and centrifuged at 900 g for 5 min at 23°C in 15 ml centrifuge tubes. The supernatant was separated from the cell pellet and the pellet was washed with 2 ml saline and centrifuged again. After centrifugation, saline was discarded, and the cell pellet was kept at −20°C until digestion. For digestion, 0.1 ml H2O2 and 0.1 ml HNO3 (both from Merck, Germany) were added to the cell pel- lets, and the tubes were closed and sealed with Teflon tape and left overnight at 80°C. After diges- tion, 1.8 ml of Milli-Q water (18.2 MΩ obtained from a Direct-Q 5 Ultrapure water system, Merck Millipore, Massachusetts, USA) was added and samples were measured by inductively coupled plasma mass spectrometry (7900 ICP-MS Agilent Technologies, Japan) with 193Ir used as an inter- Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 328 nal standard during the measurement. The ex- periments were repeated 4–7 times. The number of cisplatin molecules per cell was calculated by first dividing the measured total mass of Pt in the cell pellet by the number of cells in the pellet, then subtracting the average mass of Pt per cell of non- electroporated cell pellets that were not incubated with cisplatin, and finally calculating the number of cisplatin molecules per cell from the difference of the mass of Pt per cell in samples (assuming 1 mol of Pt is equivalent to 1 mol of cisplatin). Cell survival and amount of Pt data (after outli- ers, defined using the interquartile range method, were removed) were analyzed using the Kruskal– Wallis test and p-values were adjusted with the post-hoc Holm method test (α = 0.05) because the Shapiro-Wilk normality test failed (α = 0.05). The Spearman correlation coefficient was calculated to test the correlation between the number of cisplatin molecules per cell and cell survival. The data were processed and visualized using Microsoft Excel 2016 and R 3.6.1.22 Potential structural changes of cisplatin in the so- lution treated with high voltage electric pulses were investigated by NMR spectroscopy and HRMS. For practical reasons, both microsecond and nanosec- ond pulses were delivered to electroporation cu- vettes with 2 mm gap with the laboratory proto- type pulse generator based on an H-bridge digital amplifier for this set of experiments. For microsec- ond pulses, 8 × 100 μs at 1.1 kV/cm at 1 Hz pulse repetition rate were delivered (same pulse protocol as in cellular electrochemotherapy experiments). For nanosecond pulses, 25 × 400 ns at 2.2 kV/cm at 10 Hz repetition rate were delivered – the elec- tric field strength for this pulse protocol was lower than in cellular electrochemotherapy experiments because of the technical limitations of the prototype pulse generator. 1 × 200 ns pulse was not applied because the pulse generator used is not capable of generating such short pulses. 1H NMR spectra were obtained on NMR Bruker AscendTM 600 MHz spectrometer at room temperature at 600 MHz. Chemical shifts, reported in ppm, are referenced to residual peaks of D2O at 4.79 ppm. Spectra were re- corded in D2O (with and without NaCl) as well as in 90% H2O/10% D2O (with or without NaCl) using water suppression (WATERGATE) method. NMR data were processed with MestReNova 11.0.4. To approximately 1–2 mg of cisplatin (Sigma Aldrich) 1 mL of a) D2O, b) D2O containing 154 mM NaCl, c) 90% H2O/10% D2O or d) 90% H2O/10% D2O con- taining 154 mM NaCl was added. The obtained suspension was filtered through Minisart NML Cellulose Acetate Syringe Filter (28 mm, 0.2 μL). 1H NMR spectra were recorded immediately after the filtration when not treated with any pulse pro- tocol or directly after microsecond or nanosecond pulse application. HRMS spectra were recorded on Agilent 6224 Accurate Mass Time of Flight (TOF) Liquid Chromatography-Mass Spectrometry (LC- MS) instrument using water-acetonitrile solution (80:20, v/v) as the mobile phase. Fragmentor volt- age was set to 150.0 V. To approximately 1–2 mg of cisplatin (Sigma Aldrich) 1 mL of distilled water or saline was added and obtained suspension was filtered through Minisart NML Cellulose Acetate Syringe Filter (28 mm, 0.2 μL). Filtered solutions underwent a) no pulses, b) microsecond pulses, or c) nanosecond pulses application as mentioned above, followed by immediate injection of such so- lutions into the LC-MS. Results CHO and B16F1 cells were electroporated in pres- ence of 10, 30 and 50 μM cisplatin with: 1 × 200 ns pulse at 12.6 kV/cm; 25 × 400 ns pulses at 3.9 kV/ cm, 10 Hz pulse repetition rate; or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse rep- etition rate. The electric field strengths for specific pulse parameters were selected based on survival- permeabilization curves (refer to Vižintin et al.8 for graphs for CHO cells and to Figure S1 in the Supplementary material for graphs for B16F1 cells). Cell survival results after ECT determined by the clonogenic assay are shown in Figure 1. Survival data of CHO cells were combined from the previ- ous8 (for non-electroporated cells and cells elec- troporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (additional non-electropo- rated cells and cells electroporated with 1 × 200 ns pulse). As intended, electroporation alone (i.e., in the absence of cisplatin) did not decrease cell survival in both cell lines compared with the non- electroporated control for any of the pulse proto- cols tested. For the non-electroporated cells treated with cisplatin, a statistically significant decrease in cell survival was observed only for CHO cells at the highest (50 μM) cisplatin concentration tested. On the other hand, electroporation in the presence of cisplatin decreased cell survival except for B16F1 cells treated with 1 × 200 ns pulse. For CHO cells, 1 × 200 ns, 25 × 400 ns, and 8 × 100 μs pulse pro- tocols were all equally effective at decreasing cell survival at all the three tested cisplatin concentra- tions (Figure 1A). In B16F1 cells, 25 × 400 ns and 8 Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 329 × 100 μs pulses were equally effective, whereas 1 × 200 ns pulse protocol was less effective (Figure 1B). The amount of Pt in the cells was determined by measuring the total mass of Pt in the cell pellets by ICP-MS. Electroporation increased the cellular Pt amount. For both cell lines, there were no sta- tistically significant differences in the measured Pt amount in cells electroporated with 25 × 400 ns or 8 × 100 μs pulses at the same cisplatin concentration. For CHO cells, the amount of Pt in cells electropo- rated with 1 × 200 ns pulse was statistically signifi- cantly lower compared to the amount of Pt in cells electroporated with 25 × 400 ns and 8 × 100 μs pulse incubated only at 50 μM cisplatin (Figure 2A). For B16F1 cells, lower cellular Pt was measured after application of 1 × 200 ns pulse compared to 25 × 400 ns and 8 × 100 μs pulses at all tested cisplatin concentrations (Figure 2B). From the measured Pt content, the number of cisplatin molecules per cell was calculated and plotted against the cell survival data. The num- ber of cisplatin molecules per cell and cell sur- vival were more strongly correlated for B16F1 cells (Spearman’s correlation coefficient: ρ = −0. 85, p < 0.001 for CHO and ρ = −0. 92, p < 0.01 for B16F1). In the case of CHO cells, at the same number of cisplatin molecules per cell, notably lower cell sur- vival was measured for electroporated cells com- pared to non-electroporated cells (Figure 3A). For example, cell survival of 98% was achieved for non-electroporated cells with 9.4 × 106 cisplatin molecules per cell, whereas cell survival of 68.5% FIGURE 1. Cell survival of (A) CHO and (B) B16F1 cells at different cisplatin concentrations determined by the clonogenic assay for non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the survival of non-electroporated cells without cisplatin. Survival data were combined from the previous8 (for non-electroporated cells and cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse). FIGURE 2. Pt amount in cell pellets of (A) CHO and (B) B16F1 cells after 25 min incubation at different extracellular cisplatin concentrations in non-electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/ cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation, asterisks (*) show statistically significant differences (p < 0.05) to the measured number of cisplatin molecules in non-electroporated cells at the same extracellular cisplatin concentration. A A B B Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 330 was measured for cells electroporated with 1 × 200 ns pulse with 8.2 × 106 cisplatin molecules per cell, cell survival of 54.8% was measured for cells elec- troporated with 25 × 400 ns pulses with 9.5 × 106 cisplatin molecules per cell, and cell survival of 33.7% was measured for cells electroporated with 8 × 100 μs pulses with 9.2 × 106 cisplatin molecules per cell. From the data acquired, it could not be concluded if also in B16F1 cells a lower number of cisplatin molecules per cell causes a larger decrease in cell survival because the range of the number of cisplatin molecules in electroporated and non-elec- troporated cells did not overlap and thus survival could not be compared at approximately the same number of cisplatin molecules per cells (Figure 3B). Cisplatin has been widely investigated for its biospeciation in aqueous solutions due to its di- verse stepwise ligand displacement reactions.23 Therefore, 1H NMR spectroscopy was applied to investigate potential structural changes of cis- platin due to high voltage electric pulses. First, spectra of cisplatin in D2O and D2O with 154 mM NaCl (corresponding to physiological saline 0.9% NaCl) not exposed to electric pulses were recorded (Figure 4A–B). Weak broadened peaks for hydro- gen atoms of amino ligands (NH3) were found at approximately 4.08 ppm. Similarly, also repre- sentative peaks of cisplatin after treatment with 8 × 100 μs pulses at 1.1 kV/cm at 1 Hz pulse repetition rate or 25 × 400 ns pulses at 2.2 kV/cm at 10 Hz repetition rate remained at the same shift. The only major difference was observed in the spectrum of cisplatin recorded in D2O with 154 mM NaCl af- ter treatment with microsecond pulses (Figure 4B), where the broad peak for hydrogens of cisplatin disappeared. This can be attributed to the fast hy- drogen-deuterium (H/D) exchange of deuterium from D2O with hydrogen atoms of NH3 ligands.24 However, when spectra of cisplatin were recorded in 90% H2O/10% D2O solution containing 154 mM NaCl acquiring water suppression (to minimize the intensity of water signal to obtain a stronger signal of the NH3 ligand) no such disappearance of the peak was observed (Figure 4D). Comparable spectra with peaks at 4.08 ppm were obtained al- so when no electric pulses or nanosecond pulses were applied. Similarly, the hydrogen peak of NH3 was observed in the samples recorded in a 90% H2O/10% D2O solution without NaCl (Figure 4C). It is also important to note that no new peaks ap- peared in other regions of the NMR spectra. High-resolution mass spectrometry (HRMS), which can also provide abundant information on molecular structure, was also performed to inves- tigate possible newly formed cisplatin species. In some reports, authors detected hydrolysis prod- ucts corresponding to mono-, di- and trimeric spe- cies, by mass spectrometry.25-28 Therefore, HRMS was used in our structural investigation of cisplatin in water and saline (0.9% NaCl) exposed to micro- and nanosecond pulses. First, cisplatin in H2O was investigated and on the full-scan positive-ion mass spec- trum (mass range of m/z 100–1100) presented in Figure S2 in Supplementary Material. It can be observed that the most abundant peaks occur in the mass range of m/z 280–330, where the fol- lowing fragments were observed: [Pt(NH3)2(N2) FIGURE 3. Cell survival as a function of the number of cisplatin molecules per cell for (A) CHO cells and (B) B16F1 cells in non- electroporated (non-EP) cells (black circles) and cells electroporated with 25 x 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate (dark blue squares), 1 × 200 ns pulse at 12.6 kV/cm (light blue diamonds) or 8 × 100 μs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz pulse repetition rate (orange triangles). Bars represent standard deviation. Survival data were combined from the previous8 (for non-electroporated CHO cells and CHO cells electroporated with 25 × 400 ns and 8 × 100 μs pulses) and the present study (for B16F1 cells, additional non-electroporated CHO cells and CHO cells electroporated with 1 × 200 ns pulse). A B Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 331 Cl]+ (m/z 292.9909), [M+NH4]+ (M – indicates mo- lecular formula for cisplatin, i.e. [Pt(NH3)2Cl2]) (m/z 317.9872) (both Figure S3), [M+H]+ (m/z 300.9601) (Figure S4), [Pt(NH3)2(CH3CN)Cl]+ (m/z 306.0101) (Figure S5) and [M+Na]+ (m/z 322.9425) (Figure S6). Additionally, three lower abundant clusters can be found in the mass range of m/z 540–590. Two of them were identified as [Pt(NH3)2Cl2∙Pt(NH3)Cl]+ (m/z 547.9121) and [Pt(NH3)2Cl2∙Pt(NH3)2Cl]+ (m/z 564.9378) (Figure S7). Additionally, one cluster at m/z 610–630 with the main ion fragment at m/z 617.9408 belongs to [2M+NH4]+ (Figure S8). Similar fragments have been observed when the samples were treated with micro- and nanosecond pulses (Figure S9–10 and Figure S11–S12). The species ob- served are in agreement with those reported in the literature.26 Figure S19 represents the spectrum of water from the electroporation cuvette without the application of electric pulses. No differences were observed between the solutions treated with either nanosecond or microsecond pulses or untreated control. HRMS experiments have been further per- formed in saline, where more extensive fragmenta- tion was observed throughout the mass range of m/z 100–1100 (Figure S13). However, these peaks are comparable to the ones in the spectrum of sa- line from electroporation cuvette without the ap- plication of electric pulses (Figure S20). Similarly to spectra without NaCl, peaks of [Pt(NH3)2(N2)Cl]+ fragment and sodium [M+Na]+ adduct were identi- fied on zoom-scan spectrum (Figure S14). Again, spectra recorded in saline that was not treated with electric pulses are comparable with the spec- tra where cisplatin in saline solutions were treated with micro- and nanosecond pulses (Figures S15– 16 and Figures S17–18, respectively). Overall, NMR, as well as HRMS investigations, point to cisplatin remaining structurally compara- ble after the exposure to high voltage electric puls- es similar to those used in in vitro ECT experiments with respect to its aqueous solutions without elec- tric pulses. Discussion ECT has been shown to be a safe and effective can- cer treatment, requiring much lower doses of the chemotherapeutic agent than conventional chemo- therapy. However, pain and muscle contractions were reported as a drawback. Nanosecond pulses and high-frequency biphasic pulses of a few mi- crosecond duration (H-FIRE)29-31 were suggested to limit neuromuscular stimulation and contrac- tions.15,16 Additionally, with nanosecond pulses, the possibility of thermal damage to the tissue is minimized5,6 due to low energy being transferred to the treated area and electrochemical reactions are reduced.8 ECT with nanosecond pulses has shown promising results8,17-19, but the underlying mechanisms of the observed decrease in cell sur- vival and tumor regression remain to be explained. In this study, we measured cell survival and cisplatin accumulation after in vitro ECT with 8 × 100 μs pulses, which are standardly used in ECT procedures, and equivalent nanosecond pulses, i.e. pulse protocols that have an equivalent biological effect on cell survival and cell membrane permea- bilization. The electric field strength was chosen for each pulse protocol at a value that resulted in the highest permeabilization (determined as the percentage YO-PRO1 fluorescing cells) of the cell membrane without a decrease in cell survival (measured by the metabolic MTS assay). In the case of 8 × 100 μs pulses, 1.1 kV/cm was selected for CHO cells, but the survival for B16F1 cells was around 55% at this electric field strength, thus a lower (i.e. 0.9 kV/cm) electric field strength was used for electroporating B16F1 cells with this pulse proto- col. For 25 × 400 ns pulses, the same electric field strength (3.9 kV/cm) was determined to be optimal for both cell lines. For 1 × 200 ns pulse, we used the highest experimentally achievable electric field FIGURE 4. 1H NMR spectra of cisplatin, showing the signals for hydrogens of NH3 ligands labeled with asterisks (*). Spectra were recorded in a) D2O, b) D2O containing 154 mM NaCl, c) 90% H2O/10% D2O and d) 90% H2O/10% D2O containing 154 mM NaCl treated with 25 × 400 ns pulses (blue), 8 × 100 µs pulses (green) or no pulses (red). A B C D Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 332 strength (i.e. 12.6 kV/cm), which did not decrease the cell survival in either cell line. Electroporating both cell lines with 8 × 100 μs or 25 × 400 ns pulses at the selected electric field strengths resulted in > 95% permeabilization (optimal for ECT), while for the 1 × 200 ns pulse at 12.6 kV/cm the permeabiliza- tion was 85% for CHO and only 42% for B16F1 cells (suboptimal for ECT). However, 1 × 200 ns pulse protocol was also included in the study based on results of cell survival of CHO cells after ECT de- termined by the metabolic MTS assay that showed that this pulse protocol was as effective in decreas- ing cell survival in ECT with cisplatin as the 25 × 400 ns protocol at all cisplatin concentrations.8 The aim was to test whether the combination of permeabilizing electric pulses (that alone do not cause a decrease in cell survival) and cisplatin re- sults in increased cellular cisplatin accumulation (compared to non-electroporated cells) and wheth- er the amount of cellular cisplatin is correlated to cell survival due to the increase of intracellular accumulation of the chemotherapeutic agent be- ing one of the main mechanisms of action of ECT. To exert its cytotoxic effect, cisplatin must enter the cell. The exact mechanisms of cisplatin uptake have not been fully elucidated. Cisplatin is only slightly permeant; thus, it only partially enters the cell through passive diffusion across the cell mem- brane. Recent studies pointed out active transport mechanisms such as facilitated diffusion involved in cisplatin uptake - and LRRC8 volume-regulat- ed anion channels (VRAC), copper transporter 1 (CTR1), and organic cation transporters (OCTs) were shown to be involved in cisplatin uptake.32,33 Electroporation makes the cell membrane non-se- lectively permeable, allowing a larger quantity of cisplatin to enter the cell (Figure 5). As expected, the measured amount of Pt was higher in electroporated cells when compared to non-electroporated cells incubated at the same cisplatin concentration, although the differences were not always statistically significant (Figure 2). These results indicate that the application of elec- tric pulses indeed increases the intracellular accu- mulation of cisplatin. Overall, the amount of Pt in B16F1 was lower than in CHO cells exposed to the same cisplatin concentration, with or without elec- troporation, which also correlates with the higher cell survival of B16F1 cells (Figure 1). A compari- son of cell survival of CHO and B16F1 cells with a similar number of cisplatin molecules per cell (Figure 3) reveals that a higher number of cisplatin molecules is needed to decrease the cell survival of B16F1 cells compared to CHO. There were no statistically significant differ- ences in the cell survival and amount of cellular Pt obtained in cells electroporated with 25 × 400 ns and 8 × 100 μs pulses at the same cisplatin concen- tration when comparing within the same cell line. Thus, it can be assumed that by using equivalent nanosecond pulses, it is possible to achieve the same decrease in cell survival and same cisplatin accumulation in cells and the as with the standard 8 × 100 μs pulses; in other words, equivalent nano- second pulses are equally effective in ECT as 8 × 100 μs pulses. The 1 × 200 ns pulse in combination with cispl- atin did not decrease cell survival in B16F1 cells. This could be explained by the fact that 1 × 200 ns pulse permeabilizes less than half of the cell pop- ulation of B16F1 and is also consistent with the measured Pt amount which was not significantly higher as in non-electroporated cells (Figure 2B). Application of 1 × 200 ns pulse alone (i.e., in the absence of cisplatin) seemed to even slightly pro- mote cell growth (although the cell survival was not statistically significantly higher compared to the non-electroporated control). More interesting- ly, however, is that application of 1 × 200 ns pulse to CHO cells resulted in a lower amount of Pt in cells electroporated with 1 × 200 ns pulse as with 25 × 400 ns or 8 × 100 μs pulses, but the same decrease in cell survival was achieved with the 1 × 200 ns pulse as with 25 × 400 ns or 8 × 100 μs pulses. The lower amount of cisplatin in CHO cells electropo- rated with 1 × 200 ns could be explained per se by the fact that this pulse protocol achieved subop- timal cell membrane permeabilization compared to the 25 × 400 ns and 8 × 100 μs pulse protocols. FIGURE 5. The mechanism of cisplatin uptake into cells is not completely elucidated. In non-electroporated cells, cisplatin enters partially through passive diffusion and facilitated diffusion through ion channels including LRRC8 volume-regulated anion channels (VRAC) and membrane transporters like copper transporter 1 (CTR1) and organic cation transporters (OCTs). In electroporated cells, more cisplatin can enter through the permeabilized cell membrane (pore is a symbolic presentation of increased membrane permeability even though the mechanisms behind electroporation are more complex – refer to34). Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 333 Nevertheless, a comparable decrease in cell sur- vival was achieved, suggesting that increased accu- mulation of cisplatin into cells may not be the only cause of cell death in ECT. Figure 2A indicates that in electroporated CHO cells, a lower number of cis- platin molecules per cell is required to decrease cell survival to the same extent as in non-electroporated cells. Similar results have been reported previously in the literature35,36, but not discussed. There may be a synergistic effect of cisplatin and electropora- tion, i.e., the observed decrease in cell survival in ECT is not the sum of the decrease in cell survival caused by electric pulses and cisplatin alone, but electroporation appears to make cells more suscep- tible to cisplatin. The results of survival and number of internal- ized cisplatin molecules for B16F1 cells, however, do not show a similar synergistic effect of cisplatin and electroporation. Contrary to CHO cells, the number of cisplatin molecules per cell seems to lin- early correlate with the logarithm of cell survival for B16F1 cells (Figure 3). Nonetheless, as men- tioned above, lower cellular cisplatin was consist- ently measured for the B16F1 cell line and there is only one experimental point from the electroporat- ed cells (cells electroporated with 1 × 200 ns pulse at 10 μM cisplatin) that falls in the range of the num- ber of molecules of the non-electroporated cells. A similar number of internalized cisplatin molecules was measured for non-electroporated cells at 30 μM cisplatin and for cells electroporated with 1 × 200 ns pulse at 10 μM cisplatin, but the cell survival was even slightly higher for the latter. As discussed above, however, the 1 × 200 ns pulse protocol did not effectively permeabilize B16F1 cells. More data (from non-electroporated cells incubated at higher cisplatin concentrations) would thus be needed to determine if also in the case of B16F1 cells a lower number of internalized cisplatin molecules is need- ed to decrease cell survival in electroporated cells. To test whether electric pulses could affect cis- platin by modifying the structure of the molecule as proposed in theoretical studies37, we used NMR spectroscopy and HRMS spectrometry and found that the structure of cisplatin remains comparable after the application of electric pulses to either its saline or water solution (representing a simplified extra- and intracellular environment, respectively). Thus, high voltage electric pulses did not affect the structure of the studied complex under the condi- tions used in our experiments. Therefore, the rea- son for the observed increased susceptibility of the electroporated CHO cells to cisplatin is probably a consequence of the effect of electroporation on the cells. The cytotoxicity of cisplatin is thought to be mediated primarily by the formation of DNA ad- ducts and the resulting impairment of transcrip- tional and/or DNA replication mechanisms. It was shown that electroporation increases the amount of cisplatin bound to the DNA, which could increase cisplatin cytotoxicity in electroporated cells.35,38 However, additional mechanisms play an impor- tant role in exerting the toxic effects of cisplatin, including generation of ROS, mitochondrial dys- function, increase in intracellular Ca2+ concentra- tion, and activation of signal transduction path- ways.39 Electric pulses can also lead to generation of intracellular reactive oxygen species (ROS)40,41, damage mitochondria42,43, and disrupt calcium ho- meostasis through the entry of Ca2+ from the extra- cellular space or intracellular stores.44,45 It has been shown that an increase in ROS enhances the effica- cy of cisplatin and vice versa.46,47 Moreover, an in- crease in intracellular Ca2+ concentration enhances cisplatin-mediated ROS production and increases cisplatin cytotoxicity.48-50 This type of potentiation of cisplatin cytotoxicity may be responsible for the enhanced cisplatin cytotoxicity in electroporated cells, but it yet needs to be elucidated. Michel et al.51 observed an increased immunoreactivity with SOD-2 (an enzyme that clears mitochondrial ROS) in cells subjected to ECT with cisplatin. To the best of our knowledge, this is the only report that meas- ured ROS after ECT with cisplatin. Our study also has limitations. Two different pulse generators and electrode geometries (i.e., electroporation cuvettes with 2 or 4 mm gap) were used in the cell experiments because of the techni- cal limitations of the pulse generators used. Also in cell experiments, we did not directly measure the amount of cisplatin in cell pellets, but Pt was measured instead and assumed that cisplatin most likely accounts for the majority of the measured amount of Pt in cells incubated with cisplatin. This assumption is supported by the fact that the amount of Pt in non-electroporated cells that were not incubated with cisplatin was 2–3 orders of magnitude lower than in samples incubated with cisplatin or even below the detection limit. We also do not know whether the measured Pt was located inside the cells or was e.g. bound to the surface of the cell membrane. However, the for- mation of reactive hydrolyzed cisplatin products that would bind immediately and irreversibly to cell membrane phospholipids is not expected be- cause the electroporation medium used has a high concentration of chloride ions so cisplatin should be stable in it and the measured Pt most probably Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 334 comes from intracellular cisplatin.52 Additionally, in experiments investigating the effects of electric pulses on cisplatin structure, the conditions be- fore the measurements by NMR spectroscopy and HRMS spectrometry could not be fully matched with the conditions in the cell experiments due to several reasons. First, it was namely not possible to record spectra of cisplatin in growth media due to many species present in the growth medium which interfere with cisplatin signals; thus, pulses were delivered to cisplatin dissolved in water or saline for NMR spectroscopy and HRMS spectrometry. Second, because of the limitations of the pulse generator used for NMR spectroscopy and HRMS spectrometry experiments, 25 × 400 ns pulses were delivered at lower amplitudes than in the cell ex- periments. Third, because of the difference in con- ductivity, electric pulses delivered to H2O and D2O had a notably different shape than pulses delivered to saline or cells in growth medium; due to the low conductivity of the load, they resembled an expo- nentially decaying rather than a rectangular pulse shape. In conclusion, we have shown that by using equivalent nanosecond pulses in ECT, the same decrease in cell survival is achieved and the same amount of cisplatin accumulates in the cells as with the standard 8 × 100 μs pulses, i.e., that in ECT, equivalent nanosecond pulses are equally efficient as 8 × 100 μs pulses. By investigating the under- lying mechanisms in nanosecond pulse ECT, we discovered that electroporated CHO cells are more susceptible to cisplatin than non-electroporated cells (regardless of the pulse protocol). The electric pulses used for electroporation do not appear to alter the structure of the cisplatin molecule, so the observed increased susceptibility is likely a conse- quence of the effect of electroporation on the cells. The use of nanosecond pulses in ECT is promis- ing as it was demonstrated to be effective with the potential to mitigate muscle contractions. Because extensive preclinical data and solid evidence of mechanisms of action have been the basis for intro- ducing ECT into clinical practice, further studies of nanosecond pulse ECT in vivo are necessary to en- able translation into clinical trials. Acknowledgements The study was funded by Pulse Biosciences and the Slovenian Research Agency (ARRS) (research core funding No. P2-0249 and P1-0175). The work was partially performed within the network of research and infrastructural centres of University of Ljubljana, which is financially supported by Slovenian Research Agency through infrastruc- tural grant IP-0510. A.V. was granted a scholarship from the University Foundation of ing. Lenarčič Milan. We would like to acknowledge dr. Damijana Urankar for HRMS analyses. References 1. Miklavčič D, Mali B, Kos B, Heller R, Serša G. Electrochemotherapy: from the drawing board into medical practice. BioMed Eng Online 2014; 13: 1-20. doi: 10.1186/1475-925X-13-29 2. Campana LG, Miklavčič D, Bertino G, Marconato R, Valpione S, Imarisio I, M, et al. Electrochemotherapy of superficial tumors – Current status: basic principles, operating procedures, shared indications, and emerging applications. Semin Oncol 2019; 46: 173-91. doi: 10.1053/j.seminon- col.2019.04.002 3. Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, Occhini A, Quaglino P, et al. Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases. Acta Oncol 2018; 57: 874-82. doi: 10.1080/0284186X.2018.1454602 4. Kendler M, Micheluzzi M, Wetzig T, Simon JC. Electrochemotherapy under tumescent local anesthesia for the treatment of cutaneous metastases. Dermatologic Surg 2013; 39: 1023-32. doi: 10.1111/dsu.12190 5. Schoenbach KH, Beebe SJ, Buescher ES. Intracellular effect of ultrashort electrical pulses. Bioelectromagnetics 2001; 22: 440-8. doi: 10.1002/ bem.71 6. Pliquett U, Nuccitelli R. Measurement and simulation of Joule heating during treatment of B-16 melanoma tumors in mice with nanosecond pulsed electric fields. Bioelectrochemistry 2014; 100: 62-8. doi: 10.1016/j. bioelechem.2014.03.001 7. Cornelis FH, Cindrič H, Kos B, Fujimori M, Petre EN, Miklavčič D, et al. Peri- tumoral metallic implants reduce the efficacy of irreversible electroporation for the ablation of colorectal liver metastases. Cardiovas Intervent Radiol 2020; 43: 84-93. doi: 10.1007/s00270-019-02300-y 8. Vižintin A, Marković S, Ščančar J, Miklavčič D. Electroporation with nano- second pulses and bleomycin or cisplatin results in efficient cell kill and low metal release from electrodes. Bioelectrochemistry 2021; 140: 107898. doi: 10.1016/j.bioelechem.2021.107798 9. Saulis G, Rodaite R, Rodaitė-Riševičienė R, Dainauskaitė VS, Saulė R. Electrochemical processes during high-voltage electric pulses and their importance in food processing technology. In: Rai VR, editor. Advances in food biotechnology. First Edition. Wiley Online Books, John Wiley & Sons Ltd; 2016. p. 575-92. 10. Kotnik T, Miklavčič D, Mir LM. Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses: Part II. Reduced electrolytic contamination. Bioelectrochemistry 2001; 54: 91-5. doi: 10.1016/S1567- 5394(01)00115-3 11. Loomis-Husselbee JW, Cullen PJ, Irvine RF, Dawson AP. Electroporation can cause artefacts due to solubilization of cations from the electrode plates. Aluminum ions enhance conversion of inositol 1,3,4,5-tetrakisphosphate into inositol 1,4,5-trisphosphate in electroporated L1210 cells. Biochem J 1991; 277(Pt 3): 883-5. doi: 10.1042/bj2770883 12. Long G, Shires PK, Plescia D, Beebe SJ, Kolb JF, Schoenbach KH. Targeted tissue ablation with nanosecond pulses. IEEE Trans Biomed Eng 2011; 58: 2161-7. doi: 10.1109/TBME.2011.2113183 13. Rogers WR, Merritt JH, Comeaux JA, Kuhnel CT, Moreland DF, Teltschik DG, et al. Strength-duration curve an electrically excitable tissue extended down to near 1 nanosecond. IEEE Trans on Plasma Sci 2004; 32: 1587-99. doi: 10.1109/TPS.2004.831758 14. Pakhomov AG, Pakhomova ON. The interplay of excitation and electropo- ration in nanosecond pulse stimulation. Bioelectrochemistry 2020; 136: 107598. doi: 10.1016/j.bioelechem.2020.107598 Radiol Oncol 2022; 56(3): 326-335. Vizintin A et al. / Nanosecond pulses are equally effective in electrochemotherapy 335 15. Gudvangen EK, Kondratiev O, Redondo L, Xiao S, Pakhomov AG. Peculiarities of neurostimulation by intense nanosecond pulsed electric fields: how to avoid firing in peripheral nerve fibers. Int J Mol Sci 2021; 22: 1763. doi: 10.3390/ijms22137051 16. Gudvangen EK, Novickij V, Battista F, Pakhomov AG. Electroporation and cell killing by milli-to nanosecond pulses and avoiding neuromuscular stimula- tion in cancer ablation. Sci Rep 2022; 12: 1-15. doi: 10.1038/s41598-022- 04868-x 17. Silve A, Leray I, Mir LM. Demonstration of cell membrane permeabiliza- tion to medium-sized molecules caused by a single 10 ns electric pulse. Bioelectrochemistry 2012; 87: 260-4. doi: https://doi.org/10.1016/j.bioel- echem.2011.10.002 18. Tunikowska J, Antończyk A, Rembiałkowska N, Jóźwiak Ł, Novickij V, Kulbacka J. The first application of nanoelectrochemotherapy in feline oral malignant melanoma treatment – case study. Animals 2020; 10: 556. doi: 10.3390/ani10040556 19. Novickij V, Malyško V, Želvys A, Balevičiūte A, Zinkevičiene A, Novickij J, et al. Electrochemotherapy using doxorubicin and nanosecond electric field pulses: a pilot in vivo study. Molecules 2020; 25: 4601. doi: 10.3390/mol- ecules25204601 20. Kiełbik A, Szlasa W, Novickij V, Szewczyk A, Maciejewska M, Saczko J, et al. Effects of high-frequency nanosecond pulses on prostate cancer cells. Sci Rep 2021; 11: 1-10. doi: 10.1038/s41598-021-95180-7 21. Kulbacka J, Rembiałkowska N, Szewczyk A, Moreira H, Szyjka A, Girkontaitė I, et al. The impact of extracellular Ca2+ and nanosecond electric pulses on sensitive and drug-resistant human breast and colon cancer cells. Cancers 2021; 13: 3216. doi: 10.3390/cancers13133216 22. R Core Team R. A language and environment for statistical computing. [internet]. 2018. Available at: https://www.r-project.org/ 23. Berners-Price SJ, Appleton TG. The chemistry of cisplatin in aqueous solu- tion. In: Kelland LR, Farrell NP, editors. Platinum-based drugs in cancer therapy. Cancer drug discovery and development. Totowa, NJ: Humana Press; 2000. p. 3-35. doi: 10.1007/978-1-59259-012-4_1 24. Chen Y, Guo Z, Sadler PJ. 195Pt- and 15N-NMR spectroscopic studies of cis- platin reactions with biomolecules. In: Lippert B, editor. Cisplatin. Chemistry and biochemistry of a leading anticancer drug. Wiley Online Library. p. 293- 318. doi: 10.1002/9783906390420.ch11 25. Cui M, Mester Z. Electrospray ionization mass spectrometry coupled to liq- uid chromatography for detection of cisplatin and its hydrated complexes. Rapid Commun Mass Spectrom 2003; 17: 1517-27. doi: 10.1002/rcm.1030 26. Feifan X, Pieter C, Jan VB. Electrospray ionization mass spectrometry for the hydrolysis complexes of cisplatin: implications for the hydrolysis process of platinum complexes. J Mass Spectrom 2017; 52: 434-41. doi: https://doi. org/10.1002/jms.3940 27. Du Y, Zhang N, Cui M, Liu Z, Liu S. Investigation on the hydrolysis of the anticancer drug cisplatin by Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun Mass Spectrom 2012; 26: 2832-6. doi: https://doi.org/10.1002/rcm.6408 28. Cui M, Ding L, Mester Z. Separation of cisplatin and its hydrolysis products using electrospray ionization high-field asymmetric waveform ion mobility spectrometry coupled with ion trap mass spectrometry. Anal Chem 2003; 75: 5847-53. doi: 10.1021/ac0344182 29. Arena CB, Sano MB, Rossmeisl Jr JH, Caldwell JL, Garcia PA, Rylander MN, et al. High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction. BioMed Eng Online e 2011; 10: 102. doi: 10.1186/1475-925X-10-102 30. Scuderi M, Reberšek M, Miklavčič D, Dermol-Černe J. The use of high- frequency short bipolar pulses in cisplatin electrochemotherapy in vitro. Radiol Oncol 2019; 53: 194-205. doi: 10.2478/raon-2019-0025 31. Pirc E, Miklavčič D, Uršič K, Serša G, Reberšek M. High-frequency and high-voltage asymmetric bipolar pulse generator for electroporation based technologies and therapies. Electronics 2021; 10: doi: 10.3390/electron- ics10101203 32. Makovec T. Cisplatin and beyond: molecular mechanisms of action and drug resistance development in cancer chemotherapy. Radiol Oncol 2019; 53: 148-58. doi: 10.2478/raon-2019-0018 33. Hucke A, Ciarimboli G. The role of transporters in the toxicity of chemother- apeutic drugs: focus on transporters for organic cations. J Clin Pharmacol 2016; 56(Suppl 7): S157-72. doi: 10.1002/jcph.706 34. Kotnik T, Rems L, Tarek M, Miklavčič D. Membrane electroporation and electropermeabilization: mechanisms and models. Annu Rev Biophys 2019; 48: 63-91. doi: 10.1146/annurev-biophys-052118-115451 35. Ursic K, Kos S, Kamensek U, Cemazar M, Scancar J, Bucek S, Kranjc S, Staresinic B, Sersa G. Comparable effectiveness and immunomodula- tory actions of oxaliplatin and cisplatin in electrochemotherapy of murine melanoma. Bioelectrochemistry 2018; 119: 161-71. doi: 10.1016/j.bioel- echem.2017.09.009 36. Zakelj MN, Prevc A, Kranjc S, Cemazar M, Todorovic V, Savarin M, et al. Electrochemotherapy of radioresistant head and neck squamous cell carcinoma cells and tumor xenografts. Oncol Rep 2019; 41: 1658-68. doi: 10.3892/or.2019.6960 37. Zhang L, Ye Y, Zhang X, Li X, Chen Q, Sun JCW. Cisplatin under oriented external electric fields: a deeper insight into electrochemotherapy at the molecular level. Int J Quantum Chem 2020; 121: e26578. doi: 10.1002/ qua.26578 38. Cemazăr M, Miklavcĭc ̆D, S̆căncăr J, Dolzăn V, Golouh R, Sersă G. Increased platinum accumulation in SA-1 tumour cells after in vivo electrochemo- therapy with cisplatin. Br J Cancer 1999; 79: 1386-91. doi: 10.1038/ sj.bjc.6690222 39. Florea AM, Büsselberg D. Cisplatin as an anti-tumor drug: cellular mecha- nisms of activity, drug resistance and induced side effects. Cancers 2011; 3: 1351-71. doi: 10.3390/cancers3011351 40. Pakhomova ON, Khorokhorina VA, Bowman AM, Rodaite-Riševičiene R, Saulis G, Xiao S, et al. Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media. Arch Biochem Biophys 2012; 527: 55- 64. doi: 10.1016/j.abb.2012.08.004 41. Szlasa W, Kiełbik A, Szewczyk A, Rembiałkowska N, Novickij V, Tarek M, et al. Oxidative effects during irreversible electroporation of melanoma cells – in vitro study. Molecules 2021; 26: doi: 10.3390/molecules26010154 42. Batista Napotnik T, Wu Y-H, Gundersen MA, Miklavčič D, Vernier PT. Nanosecond electric pulses cause mitochondrial membrane permeabiliza- tion in Jurkat cells. Bioelectromagnetics 2012; 33: 257-64. doi: 10.1002/ bem.20707 43. Nuccitelli R, McDaniel A, Connolly R, Zelickson B, Hartman H. Nano-pulse stimulation induces changes in the intracellular organelles in rat liver tumors treated in situ. Lasers Surg Med 2020; 52: 882-9. doi: 10.1002/lsm.23239 44. Semenov I, Xiao S, Pakhomov AG. Primary pathways of intracellular Ca2 + mobilization by nanosecond pulsed electric field. Biochim Biophys Acta - Biomembr 2013; 1828: 981-9. doi: 10.1016/j.bbamem.2012.11.032 45. Frandsen SK, Gissel H, Hojman P, Tramm T, Eriksen J, Gehl J. Direct thera- peutic applications of calcium electroporation to effectively induce tumor necrosis. Cancer Res 2012; 72: 1336-41. doi: 10.1158/0008-5472.CAN-11- 3782 46. Marullo R, Werner E, Degtyareva N, Moore B, Altavilla G, Ramalingam SS, et al. Cisplatin induces a mitochondrial-ros response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS ONE 2013; 8: 1-15. doi: 10.1371/journal.pone.0081162 47. Kleih M, Böpple K, Dong M, Gaißler A, Heine S, Olayioye MA, et al. Direct impact of cisplatin on mitochondria induces ROS production that dictates cell fate of ovarian cancer cells. Cell Death Dis 2019; 10: 31-59. doi: 10.1038/ s41419-019-2081-4 48. Kawai Y, Nakao T, Kunimura N, Kohda Y, Gemba M. Relationship of intra- cellular calcium and oxygen radicals to cisplatin-related renal cell injury. J Pharmacol Sci 2006; 100: 65-72. doi: 10.1254/jphs.FP0050661 49. Al-Taweel N, Varghese E, Florea A-M, Büsselberg D. Cisplatin (CDDP) triggers cell death of MCF-7 cells following disruption of intracellular calcium ([Ca2+] i) homeostasis. J Toxicol Sci 2014; 39: 765-74. doi: 10.2131/jts.39.765 50. Gualdani R, de Clippele M, Ratbi I, Gailly P, Tajeddine N. Store-operated calcium entry contributes to cisplatin-induced cell death in non-small cell lung carcinoma. Cancers 2019; 11: 2023. doi: 10.3390/cancers11030430 51. Michel O, Kulbacka J, Saczko J, Mączyńska J, Błasiak P, Rossowska J, et al. Electroporation with cisplatin against metastatic pancreatic cancer: in vitro study on human primary cell culture. Biomed Res Int 2018; 2018: 7364539. doi: 10.1155/2018/7364539 52. Speelmans G, Sips WHHM, Grisel RJH, Staffhorst RWHM, Fichtinger- Schepman AMJ, Reedijk J, et al. The interaction of the anti-cancer drug cisplatin with phospholipids is specific for negatively charged phospholipids and takes place at low chloride ion concentration. Biochim Biophys Acta - Biomembr 1996; 1283: 60-6. doi: 10.1016/0005-2736(96)00080-6