When a cell is exposed to a sufficiently high electric field, the permeability of the cell membrane rapidly increases due to membrane electroporation. This transiently increased membrane permeability allows for the exchange of ions and molecules between inside and outside of the cells.1, 2, 3, 4,If cells recover and survive, electroporation is called reversible. If the damage is too extensive, resealing too slow, cells cannot restore the homeostasis, and they die, electroporation is called irreversible. Electroporation depends on the characteristics of the cells (shape, size, cytoskeleton structure, membrane composition) and the electrical parameters (amplitude, duration, number of electrical pulses and repetition frequency). Electroporation is used in medicine5, 6, 7, 8, 9, 10, (electrochemotherapy, gene therapy, irreversible electroporation as an ablation technique and transdermal drug delivery), in biotechnology11, 12,, (inactivation of microorganisms, extraction of biomolecules from microorganisms and plants, genetic transformation of microorganisms) and food processing.13, 14,
Electrochemotherapy (ECT) is used in clinics to treat patients with various types of cancer (
Mostly, H-FIRE pulses have been used to achieve irreversible electroporation. However, they can also be used to increase the uptake of molecules into cells38 which could be applied in achieving reversible electroporation to treat tumors with electrochemotherapy. Thus, this study aimed to determine whether H-FIRE pulses could also be used in electrochemotherapy which we call high-frequency electroporation (HF-EP).
We delivered 8 bursts of 50 bipolar pulses, each consisting of 1 μs long positive and negative pulse, with a 1 μs delay between them with electric field from 0.5–5 kV/cm. We compared HF-EP to classic eight monopolar 100 μs long pulses, delivered at frequency 1 Hz, with electric field from 0.4–1.2 kV/cm. Cisplatin concentration was from 1 μM to 330 μM. We showed that HF-EP pulses indeed cause higher cytotoxicity of cisplatin
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
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 (
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
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
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
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.
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,
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
(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
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.
We aimed to determine whether it is possible to use bursts of short bipolar pulses (HF-EP) in
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,
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
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 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
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
Before transfer to the clinical setting, more experiments
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
Although still at the
In conclusion, with long monopolar and short bipolar pulses (HF-EP), we achieved similar efficiency of electrochemotherapy with cisplatin