## 4 Discussion
In the case of the two other peptides, even in the highest CR used, no significant morphological changes were observed when the incubation of CPPs with the plasmid took place at room temperature (RT). However, increasing the temperature during complex formation led to a cytotoxic effect at a CR of only 1:2. Due to the characteristics of pHCAECs, we were unable to directly indicate the cytotoxicity of CPPs or CPPs-plasmid complexes using the MTT assay. We observed huge discrepancies in the MTT results (data not shown) and morphology of cells, which may be connected with their high sensitivity to changes in cell to extracellular matrix adhesion. Another problem is the way CPPs internalize the cell. The two main mechanisms of cell membrane penetration are direct translocation and endocytosis. Endocytosis is limited as the entrapment of CPPs in the endocytic follicle leads to their degradation. However, in some cases, methods aimed to intensify the phenomenon of molecules escaping from early endocytic vesicles before lysis have been used. Studies have shown that the primary mechanism for the transduction of the cell membrane by Pep-1 is direct translocation [25]. KALA enters the cell mainly through endocytosis. However, it can escape from the early endocytic vesicles [24]. In turn, the model of cell membrane penetration by TAT probably changes with temperature. Direct translocation occurs at lower temperatures, while endocytosis becomes the main penetration mechanism at higher temperatures [26]. The disadvantage of non-covalent binding of CPPs remains the impact of many factors on process efficiency. These factors are: (I) the type of cell line, (II) the type of transported molecule and its concentration, (III) the properties of the penetrating peptide, (IV) the temperature and incubation time, (V) the CR used, and (VI) the length of the culture period with complexes. Another variable that may influence the efficiency of CPP-cargo complex formation is the pH of incubation. As the study already involved a large number of variables, adding another one would make it extremely complicated, and thus, it was not considered. Applying many variables complicates the optimization process since optimization involves many variants. It extends the time of experiments and significantly increases their costs. It also makes it impossible to specify a universal protocol for the use of CPPs. Despite these adversities, there are many reports regarding effective cell penetration by CPPs in non-covalent interactions with other molecules like siRNA [20], proteins [27], and plasmids [28]. So far, however, no similar tests have been performed on endothelial cells. Unfortunately, the introduction of pmaxGFP expression plasmids in non-covalent combinations with CPPs into pHCAEC cells did not bring satisfactory results. Despite the varied conditions of complex formation and prolonged time of culture or the use of different CPPs, efficiency similar to the results obtained with electroporation was not achieved. This may be due to the characteristics of endothelial cells. Tight intercellular connections may not allow the internalization of weak CPPs and plasmid complexes. According to our current state of knowledge, this is the only study on the introduction of CPPs in non-covalent junctions into endothelial cells. The solution, in this case, is probably the use of covalent bonds. However, this prevents the transport of plasmids as they do not contain free ends capable of forming a connection. Additionally, two NLS sequences were used to compare transduction efficiency. NLS is a group of short cationic peptides containing a polylysine, polyarginine, or polyproline fragment that allows the molecule to be transported to the cell nucleus through nuclear pores. Some NLSs can also penetrate the cell membrane [29]. Studies by [PERSON] et al. showed that the interleukin 1\(\alpha\) precursor (pIL-1\(\alpha\)) can internalize the cell with similar efficiency as TAT [29].