Background/Objectives: Electrochemotherapy (ECT) is a safe and efficient method of targeted drug delivery using pulsed electric fields (PEF), one that is based on the phenomenon of electroporation. However, the problems of electric field homogeneity within a tumor can cause a diminishing of the treatment efficacy, resulting only in partial response to the procedure. This work used gold nano-particles for electric field amplification, introducing the capability to improve available elec-trochemotherapy methods and solve problems associated with field non-homogeneity. Methods: We characterized the potential use of gold nanoparticles of 13 nm diameter (AuNPs: 13 nm) in combination with microsecond (0.6–1.5 kV/cm × 100 μs × 8 (1 Hz)) and nanosecond (6 kV/cm × 300–700 ns × 100 (1, 10, 100 kHz and 1 MHz)) electric field pulses. Finally, we tested the most prominent protocols (microsecond and nanosecond) in the context of bleomycin-based electrochemotherapy (4T1 mammary cancer cell line). Results: In the nano-pulse range, the synergistic effects (improved permeabilization and electrotransfer) were profound, with increased pulse burst frequency. Addi-tionally, AuNPs not only reduced the permeabilization thresholds but also affected pore resealing. It was shown that a saturated cytotoxic response with AuNPs can be triggered at significantly lower electric fields and that the AuNPs themselves are non-toxic for the cells either separately or in combination with bleomycin. Conclusions: The used electric fields are considered sub-threshold and/or not applicable for electrochemotherapy, however, when combined with AuNPs results in successful ECT, indicating the methodology’s prospective applicability as an anticancer treatment method.

The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca²⁺ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3–7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca²⁺ electrochemotherapy (B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals.

Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells’ plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.

Bipolar cancellation (BPC) is an efficiency-limiting phenomenon in bipolar nanosecond pulsed electric field (nsPEF) exposures, in which the second, opposite-polarity phase reduces or partially reverses the electroporation induced by the first phase. Nisin, a cationic antibiotic peptide, has been reported to interact with lipid membranes in bacterial systems and artificial bilayer models, where it may contribute to membrane destabilization and increased permeability during pulsed electric field exposure. This study investigated whether nisin may enhance the efficacy of bleomycin electrochemotherapy (ECT) in the presence of bipolar nanosecond pulses, which are typically associated with pronounced BPC effects. Pulsed electric field (PEF) parameters and drug concentrations were selected based on preliminary viability and Yo-Pro-1 uptake experiments in CLS-354 human squamous cell carcinoma cells. To evaluate the effect of nisin, cell viability and membrane permeabilization were assessed following exposure to 300 ns pulses across a range of bipolar PEF protocols, with or without nisin, while identical unipolar pulses were used for comparison. Nisin (50 µg/mL) increased membrane permeabilization across the tested range of field amplitudes (9–15 kV/cm) and burst repetition frequencies (0.1–1.66 MHz). The presence of nisin was also associated with increased efficacy of bleomycin-based ECT under both unipolar and symmetrical bipolar PEF conditions. Under the optimized parameters tested (13 kV/cm; 150 pulses of 300 ns at 1.66 MHz), bipolar nsPEFs in combination with nisin reached levels of efficacy comparable to those observed with unipolar waveforms, suggesting a potential attenuation of bipolar cancellation effects.

Electroporation can be used as an effective non-viral gene delivery method, while the application of conductive nanoparticles (NPs) with pulsed electric fields (PEFs) may increase treatment efficacy due to local field amplification in close proximity to the cell plasma membrane. In this work, we have employed 100 ns and 300 ns pulses (9–17 kV/cm), which were delivered in bursts (n = 100) and predefined inter-pulse delays (100–900 ns), which enabled successful gene delivery (4.7 kbp; p-EGFP-N1) using pulses as short as 100 ns, which previously was considered impossible. As a model, a murine breast cancer cell line (4T1) was used. It was shown that sub-microsecond pulses (i.e., 300 ns) can be effective for gene delivery, whereas 100 ns pulses are several-fold inferior, yet still trigger successful gene transfer (>10% of cells being electrotransfected). In order to increase the efficacy of the treatment, we used gold nanoparticles (AuNPs; the diameter of 13 nm), which allowed us to achieve electrotransfection efficacy several-fold for both sub-microsecond and microsecond protocols (1.2 kV/cm × 100 µs × 8 pulses at 1 Hz). The results suggest high potential applicability of conductive nanoparticles in future translational or clinical research involving electroporation and gene transfer.