## 4 Discussion  
The application of electric field pulses to cancer cells results in the permeabilization of their plasma membrane. This technique specifies the application of short high-voltage pulses to penetrate the barrier of the cell membrane. Electroporation can be a very efficient method for introducing molecules into cells, including various non-permeant molecules, which could significantly enhance the cytotoxicity [37]. Optimizing electroporation parameters is crucial for the future application and can be easily performed in cell suspension using, e.g., propidium iodide [38; 39] or lucifer yellow [40]. The electric field intensity is the key parameter in electrochemotherapeutic protocols as it should lead to the pore formation without simultaneous cell destruction. In our study the following electroporation parameters were applied: 8 square pulses, 100 us width, delivered at 1 Hz frequency, which corresponded to ESOPE protocol [34]. Our previous study demonstrated that the application of electric pulses increases cell membrane permeability to propidium iodide in human breast adenocarcinoma cells [14; 32; 41; 42]. The dose of DOX with no cytotoxic effect was determined using a metabolic activity assay. There are few papers discussing electroporation in combination with DOX. However, these reports indicate that DOX causes a dose-dependent decrease in cell viability. The available studies of other research teams have shown that the use of electroporation using DOX in vitro in MCF-7 cells increases the absorption of the drug and causes an increased cytotoxicity [24]. Satisfactory results were obtained by DOX loading in biodegradable micelles in a mouse hepatocellular carcinoma xenograft model, using two electroporation variants: reversible for parameter 500 V/cm and irreversible for parameter 2500 V/cm. A three-fold increase in DOX uptake in the cell nucleus was observed when using micelles and irreversible electroporation compared to the micelles alone. Authors observed in these cells an increase in reactive oxygen species, abnormalities in mitochondria, and an increase in intracellular calcium [43]. In the case of clinical research it was revealed that 5 uM was the maximum drug concentration observed in the plasma of patients after intravenous administration [43]. The energy levels necessary for the electroporation-supported therapy are similar to the intensities used for ECT in vivo [44]. For the successive clinical electrochemotherapy of human breast cancer with bleomycin, [PERSON] et al. applied pulses of intensity 1400 V/cm, 100 us duration [45]. [PERSON] et al., showed that low voltage pulses 200 V/cm with longer duration (20 and 40 ms, 8 pulses, 1 Hz) are also effective towards permeabilization in MCF-7 breast cancer cell in vitro [46]. [PERSON] et al. proved that the intensity of 800-1000 V/cm (pulse duration 100 us, 8 pulses, 1 Hz) was the most appropriate for electroporation of MCF-7/WT cancer cells in vitro [47]. [PERSON] et al. reported on the effects of a combination of local electroporation with ionizing radiation and DOX on subcutaneous solid tumor murine fibrosarcoma. These results suggest that the antitumor effects of gamma radiation and a low concentration of DOX can be enhanced by combination with electroporation [48]. Several studies have shown that intracellular accumulation and location of cytostatics have a significant impact on their cytotoxic properties and pharmacological characteristics [49; 50; 51]. Here, we have also demonstrated DOX location inside cells by LSCM studies. Cells incubated with DOX showed high drug accumulation in cell nuclei in both tested cell lines. DOX fluorescence intensity in sensitive cells increased proportionally to the increase in incubation time, whereas in resistant cells the fluorescence intensity was at a similar, constant level. Similar research results were obtained by other research groups [52; 53]. The analysis of the distribution and elimination of DOX from the cells was performed in tests with and without the application of electrical impulses.