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

Abstract

Background In electrochemotherapy (ECT), chemotherapeutics are first administered, followed by short 100 μs monopolar pulses. However, these pulses cause pain and muscle contractions. It is thus necessary to administer muscle relaxants, general anesthesia and synchronize pulses with the heart rhythm of the patient, which makes the treatment more complex. It was suggested in ablation with irreversible electroporation, that bursts of short high-frequency bipolar pulses could alleviate these problems. Therefore, we designed our study to verify if it is possible to use high-frequency bipolar pulses (HF-EP pulses) in electrochemotherapy. Materials and methods We performed in vitro experiments on mouse skin melanoma (B16-F1) cells by adding 1-330 μM cisplatin and delivering either (a) eight 100 μs long monopolar pulses, 0.4-1.2 kV/cm, 1 Hz (ECT pulses) or (b) eight bursts at 1 Hz, consisting of 50 bipolar pulses. One bipolar pulse consisted of a series of 1 μs long positive and 1 μs long negative pulse (0.5-5 kV/cm) with a 1 μs delay in-between. Results With both types of pulses, the combination of electric pulses and cisplatin was more efficient in killing cells than cisplatin or electric pulses only. However, we needed to apply a higher electric field in HF-EP (3 kV/cm) than in ECT (1.2 kV/cm) to obtain comparable cytotoxicity. Conclusions It is possible to use HF-EP in electrochemotherapy; however, at the expense of applying higher electric fields than in classical ECT. The results obtained, nevertheless, offer an evidence that HF-EP could be used in electrochemotherapy with potentially alleviated muscle contractions and pain.

Keywords: cell survival; cisplatin; drug uptake; electrochemotherapy; electroporation; high-frequency bipolar pulses.

Figure 1

Scheme of the applied pulses. (A) 100 μs long monopolar pulses of amplitude ΔU (80 V – 240 V in a step of 40 V) were applied with a repetition frequency of 1 Hz. (B) Short bipolar pulses (HF-EP). Above: 8 bursts were applied with a repetition frequency of 1 Hz. Down left: One burst was 200 μs long and consisted of 50 bipolar pulses. Below right: One bipolar pulse of amplitude ΔU (100 V – 1000 V in a step of 100 V) consisted of 1 μs long positive pulse, 1 μs long negative pulse (both of voltage ΔU) with a 1 μs long delay between pulses.

Figure 2

Cell membrane permeability and cell survival as a function of electric field for (A) 8 x 100 μs long monopolar pulses, delivered at repetition frequency 1 Hz; (B) 8 bursts of short bipolar pulses (HF-EP) of 1-1-1-1 μs, delivered at repetition frequency 1 Hz. Each data point was repeated 3–4 times (mean ± standard deviation). In the control sample, no pulses were applied. Note different scales on the x-axes. On (A), the threshold of electroporation was at 0.8 kV/cm (P = 0.029, t-test) and survival did not decrease in comparison with control (one-sample t-test). On (B) the threshold of electroporation was at 2 kV/cm (P = 0.022, t-test), while the survival decreased at 4.5 kV/cm (P = 0.004, one-sample t-test). In Figure 2B, blue asterisks refer to permeability curve and red asterisks to the survival curve.

Figure 3

Cell membrane permeability as a function of different time of propidium iodide administration after electroporation for (A) 8 x 100 μs long monopolar pulses, delivered at a repetition frequency 1 Hz; (B) 8 bursts of short bipolar pulses (HF-EP) of 1-1-1-1 μs, delivered at repetition frequency 1 Hz. Each data point was repeated 4 times (mean ± standard deviation). We performed a 1-way ANOVA on ranks. For both types of pulses, there was a significant difference between 0 min vs 10 min and 20 min (P < 0.05), other pairwise comparisons were not significant.

Figure 4

Cytotoxicity of cisplatin without electroporation at different concentrations and time of incubation. Each data point was repeated 4 times (mean ± standard deviation) and is normalized to the control sample in which cisplatin was substituted by 0.9% NaCl. A 2-way ANOVA was performed. 10 min or 1 h of incubation was different from 24 h or 48 h (P < 0.001) while there was no difference between 10 min vs 1 h and 24 h vs 48 h. 330 μM cisplatin was more cytotoxic than other tested concentrations (P < 0.001). There was no significant difference between 1 μM and 10 μM cisplatin; all other comparisons were significantly different (P < 0.001).

Figure 5

Cytotoxicity of cisplatin in combination with electroporation (EP) at fixed value of cisplatin (CDDP) 100 μM as a function of electric field: (A) 8 x 100 μs long monopolar pulses (ECT) were delivered at repetition frequency 1 Hz; (B) 8 bursts of short bipolar pulses (HF-EP) of 1-1-1-1 μs were delivered at repetition frequency 1 Hz. Each data point was repeated 3–6 times (mean ± standard deviation). Results are normalized to the control sample without an electric field and with 100 μM cisplatin. We performed a (A) 2-way ANOVA or (B) 2-way ANOVA on ranks. (A) At 0.8 kV/cm (P = 0.036) and 1 kV/cm and 1.2 kV/cm (P < 0.001) EP samples were significantly different from CDDP+EP samples. (B) At electric fields equal to or higher than 2 kV/cm EP samples were significantly different from CDDP+EP samples (P < 0.001).

Figure 6

Cytotoxicity of cisplatin at different concentration of cisplatin (CDDP) and electroporation (EP) at a fixed value of electric field (A) 1.2 kV/cm, 8x100 μs long monopolar pulses, delivered at repetition frequency 1 Hz; (B) 3 kV/cm, 8 bursts of short bipolar pulses (HF-EP) of 1-1-1-1 μs, delivered at repetition frequency 1 Hz. Each data point was repeated 3-7 times (mean ± standard deviation). Each data was normalized to the control sample electroporated and with 0.9% NaCl instead of cisplatin. We performed a 2-way ANOVA. For both types of pulses, at 100 μM and 330 μM the CDDP samples were significantly different from the CDDP+EP samples (P < 0.001).