## 3 Results
Figure 3: Fluorescence intensity of doxorubicin solutions without and after application of electrical pulses. Figure 2: The results of MTT assay following the application of electric pulses ((EP+) 8 pulses, duration of 100 μs, electric field intensity 1000 V/cm, 1 Hz), DOX alone (DOX+) and following electrochemotherapy (ECT) (EP+ DOX+) on human breast adenocarcinoma cell lines sensitive (MCF-7/WT) and resistant to doxorubicin (MCF-7/DOX) cell lines after 24, 72 and 120 h of incubation. Statistically significant differences between cells exposed to EP/DOX treated/ECT and nontreated control (\({}^{*}p\leq 0.05\); \({}^{**}p\leq 0.005\); \({}^{***}p\leq 0.001\)). The combination of DOX with electroporation resulted in a reduction of the DOX fluorescent signal and altered drug distribution. DOX was dispersed throughout the cytoplasm and in cellular compartments in both cell lines. The disintegration of cell structure and cell death was observed after at least 12 h of ECT, but the toxic effect was stronger in MCF-7/WT than MCF-7/DOX. Additionally, the total nuclei lysis in MCF-7/WT was noted. MCF-7/DOX morphology was changed by ECT i.e., cell membrane was disrupted and nuclei showed irregular shape, but damages were not as significant as for MCF-7/WT. Electroporation is a physical process, which seems not to be dependent on the resistance of the cell line to a certain drug. Therefore, combining these results with the DOX fluorescence studies (Figure 5), there could be stated, that electroporation decreases the concentration of the drug in the cytoplasm of both cell lines. However, the decrease in MCF-7/DOX is simultaneous and the drug  
Figure 4: Mass spectrometry spectra of doxorubicin (**A**) and after application of 8 electric pulses, duration of 100 \(\upmu\)s, 1000 V/cm each (**B**). Figure 5: Relative fluorescence of doxorubicin in the supernatant of the (**a**) MCF-7/WT and (**b**) MCF-7/ DOX cells without and after the application of electrical impulses. concentration in the supernatant is not affected by the application of an electric field. One of the reasons for that could be the increased activity of DOX-metabolizing enzymes. Such enzymes could include specific MCF-7/DOX efflux pumps, which could simultaneously release the drug from the cells. In this research features of Nomarski contrast and the dark field observation (Figure 8 MCF-7/WT, Figure 9 MCF-7/DOX) were used. Nomarski contrast provides the incident light phase detection based on the interference phenomena. The resulting image can reveal the difference between two closely spaced points. The image has the appearance of a three-dimensional object under very oblique illumination, causing strong light and dark shadows. This type of microscopy that can capture images of transparent objects without the need for chemical staining are of a significant importance. A key feature is the use of a long focal length and non-contact metallurgical objectives which avoid the need for coverslips. The morphological changes manifested by the irregular shape of the cells (bulges) were observed for both cell lines within 24 h after electroporation, consistent with prior research. Cells exposed to electroporation were more flattened than the not treated samples, in particular the nucleus, and this resulted in significant alternations of cell morphology. The ultrastructural morphology of human breast adenocarcinoma doxorubicin-sensitive MCF-7/WT are presented in Figure 10a-d and doxorubicin-resistant MCF-7/DOX in Figure 11a-d. Untreated MCF-7/WT cells (Figure 10a) presented normal morphological structures. We observed a regularly shaped nucleus with nuclear envelope invaginations, mitochondria, RER and lysosomes. After exposure to DOX (Figure 10b) extended rough endoplasmic reticulum occurred. Figure 6: Intracellular distribution of doxorubicin (DOX = 1.7 \(\upmu\)M) in MCF-7/WT cells treated with chemotherapy only (CT) or electropermeabilized with DOX (ECT, EP-DOX) (EP = 1000 V/cm, 100 \(\upmu\)s, 8 pulses, 1 Hz) after different incubation times.