## 3 Results  
The obtained results indicated that both cell lines, the sensitive one to doxorubicin and the resistant one, showed sensitivity to doxorubicin as the concentration increased (Figure 1). Mitochondrial activity test was used for the assessment of cell viability. The viability of the sensitive cells after 24 and 72 h of incubation was not strongly affected as the subsequent increase was visible after 120 h of incubation. No cytotoxic effect for both cell lines was observed using 1.7 uM DOX and this concentration was selected for further study. An enhanced doxorubicin efficacy was observed in MCF-7/DOX cells when it was combined with electroporation (Figure 2). The most significant difference in cell viability was observed following 120 h incubation after ECT with 1.7 uM DOX concentration and electric field strength, with respect to the untreated control. The response of the resistant cell line indicates that it can be more sensitive to EP. The fluorescence intensity of DOX and DOX solutions subjected to electrical pulses is shown Figure 3. The fluorescence intensity of the DOX solution was at the level of 1.3-1.4 \(\times\) 10\({}^{6}\), and its max was 1.425 \(\times\) 10\({}^{6}\). After the application of electrical pulses, the fluorescence intensity of DOX was about 1.1-1.2 \(\times\) 10\({}^{6}\), and its max was 1.254 \(\times\) 10\({}^{6}\). The difference in absorption is related to the photosensitive properties of DOX and the application of the electric pulses causing decrease in the fluorescent signal of DOX.. In order to validate if the doxorubicin's structure was affected by the electric field used in the in vitro experiments, mass spectrometry analysis was performed. The spectra of DOX without (Figure 4a) and with the application of the electric pulses (Figure 4b) used in the electroporation protocol do not differ from each other. In both cases, the only present compound in the solution was doxorubicin (100% precision). Figure 1: The results of the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT) assay following cytotoxicity tests with doxorubicin (DOX) at a range of 0.017–170 uM concentration on human breast adenocarcinoma cell lines sensitive (MCF-7/WT) and resistant to doxorubicin (MCF-7/DOX). After being incubated for 10 min with DOX, cells were incubated in complete culture medium 24, 72 and 120 h. Statistically significant differences between DOX-treated cells and nontreated control (\({}^{*}p\leq 0.05\); \({}^{**}p\leq 0.005\); \({}^{***}p\leq 0.001\)). The relative fluorescence of the DOX solutions with and without the application of the electric pulse indicate a statistically significant difference between the cell lines (Figure 5). Interestingly, it was observed that MCF-7/WT cells were losing the doxorubicin fluorescent signal after electroporation. The effect of EP was not so apparent for MCF-7/DOX cells. According to Figure 5b, EP had no statistically significant impact on doxorubicin fluorescence in resistant cells. Considering the time-dependence of DOX efflux, it could be observed that the statistical differences occur only in shorter (2, 3, 6 h) incubation times and the differences diminish after 24 h of incubation. Electroporated MCF-7/DOX did not differ statistically from the control cells. However, electroporated MCF-7/WT cells released less DOX to the solution. Intracellular distribution of DOX is presented in Figure 6 for sensitive cell line (MCF-7/WT) and in Figure 7 for resistant cell line (MCF-7/DOX). In both cell lines, DOX signal was stronger in non-electroporated cells and the drug localized mainly in the cell nucleus. The DOX fluorescence intensity increased proportionally to the time of drug incubation in both cell lines, and MCF-7/WT cells exhibited a stronger signal than MCF-7/DOX. Only individual MCF-7/DOX cells showed a significant DOX signal after 15 h of incubation. The morphological changes manifested by the irregular shape of the nuclei were observed for MCF-7/WT 18 h after ECT application.