## 4 Discussion
Even though the results suggested that pDNA electrophoretic mobility was not altered, other studies revealed that sub-millimolar levels of Ca\({}^{2+}\) can induce conformational changes in DNA molecules. Calcium ions can negatively affect not only electrotransfection efficiency [18] but also gene transcription and translation, and they can even entail epigenetic mechanisms that can significantly alter gene expression [30; 31]. These findings may explain the main result obtained in our study: a Ca\({}^{2+}\)-induced decrease in GFP-encoded plasmid electrotransfer efficiency. Even though our results revealed that cell death was not solely responsible for the decreased level of GET, an observed significant cytotoxic effect of the Ca\({}^{2+}\) and pDNA combination could not be discriminated. Our data proposed that the increasing CaCl\({}_{2}\) was related to an increasing rate of pDNA-induced cell death and showed a maximum effect at the 0.25 mM concentration of Ca\({}^{2+}\) (Figure 4A,B). This allowed us to consider the other possible hypothesis: an increased potency of pDNA electrotransfer associated with the presence of calcium that could lead to an increase in the rate of cell death. This hypothesis was adopted from a study published by [PERSON] et al. [32], who investigated Ca\({}^{2+}\)-mediated DNA adsorption to yeast cells. Subsequent studies have also suggested that Ca\({}^{2+}\) may operate as a bridge and reduce electrostatic repulsion between the negatively charged lipid bilayer and pDNA [33]. Furthermore, computer simulations have revealed that calcium ions can stabilize DNA-membrane complexes [34]. Theoretically, more stable DNA-lipid complexes are formed, more DNA molecules enter the cell, and the transfection efficiency is higher. However, another study [33] showed that even though Mg\({}^{2+}\) induced the increased adsorption of pDNA on the cell membrane, the resultant efficiency of gene electrotransfer was decreased. The authors suggested that fewer DNA molecules could enter the cell due to strong Mg\({}^{2+}\)-induced interaction between DNA and the lipid bilayer. Assuming this was also the case with Ca\({}^{2+}\), pDNA would not be able to translocate through the membrane after DNA-membrane complex formation, and in addition, the pores would not be able to reseal normally, which potentially would lead to higher rates of cell death (as we observed in our study). Calcium electroporation alone was tested as a novel anticancer treatment both in vitro and in vivo and showed a significant decrease in cell viability [35; 36; 37; 38; 39; 40; 41]. Our results also revealed a dramatic decrease in the cell viability when higher concentrations (0.5-1 mM) of CaCl\({}_{2}\) were used (Figure 4A,B). Successively, we decided to evaluate the importance of Ca\({}^{2+}\) to the dynamics of pore resealing (Figure 5A,B). Experiments were performed with no pDNA in the sample because PI intercalated with pDNA before electroporation and it was impossible to distinguish the signal that arose from PI interaction with pDNA and its interaction with genomic DNA. In agreement with our previous study [24], the results revealed an increase in the rate of pore resealing with an increasing Ca\({}^{2+}\) concentration, and the measurement of PI-positive cells 15 min after the incubation revealed that the pores had resealed for all the CaCl\({}_{2}\) concentrations applied. This observation was also in concordance with our previous study [42], which demonstrated that secondary processes took place after electroporation and that pore resealing was only one of the factors that affected the cell viability. Indeed, calcium electroporation might induce ATP depletion leading to pronounced cell death and subsequent disturbances in natural processes such as gene expression, metabolism, and proliferation [43; 44]. This may explain why higher (0.5-1 mM) CaCl\({}_{2}\) concentrations failed to reduce the efficiency of pDNA transfection.