## 4 Discussion
This suggests that more time is necessary to create non-covalent interactions between the protein and the plasmid. There was no significant effect of prolonged incubation on changes in cell morphology. In turn, an increase in the incubation temperature of CPPs with pmaxGFP intensified the cytotoxic effect, especially for Pep-1 and TAT. This could be due to inefficient binding of plasmid DNA by CPPs under these conditions. Research has shown that free CPPs are characterized by higher cytotoxicity than those associated with the transported molecule [19]. It also confirms the observed decrease in the number of GFP-positive cells when the incubation temperature increased. Based on the obtained data, it can be concluded that room temperature is optimal to create non-covalent interactions between selected CPPs and pmaxGFP. This is consistent with the previously obtained results [20]. High temperature may also be a factor that slightly changes protein activity. Optimization of the binding process should therefore be based on increasing the incubation time of CPPs with the transported molecule rather than increasing the temperature. The complexes were formed in an aqueous environment, which is also one of the standard methods. However, different approaches may be used since the complexes may be formed in various buffers [21] or culture media [22]. According to the current state of knowledge, the efficiency of using these environments has not been compared so far. However, due to the formation of electrostatic interactions, the presence of ions and other particles in the environment may be crucial. The amount of plasmid used was also important. Incubation of 1 ug of the plasmid with CPPs showed only single cells characterized by GFP expression. A better effect was obtained for 2 ug of the plasmid. Therefore, only this amount was used in the later stages of the experiment. Literature data confirm the satisfactory transduction level of 1 ug plasmid conditioning GFP expression combined with TAT [22]. However, the optimal amount due to the use of transcriptional machinery of the host cell may closely depend on the type of cell line used. The incubation time of the pHCAEC with selected peptides was also assessed. The effectiveness of the process was tested after 24 or 48 h of culture in a medium containing CPPs. Prolonged incubation intensified the effect. Research confirms the effective internalization of protein molecules introduced with CPPs within 5 min. However, for nucleic acids, the maximum efficiency is observed much later [13]. This time is necessary not only for the internalization but also for the expression of the introduced gene. However, shorter incubation times are also used. In the case of CRISPR/Cas9 system elements introduced into HEK293T (human embryonic kidney cells), HeLa, embryonic stem cells, and embryonic tumor cells using synthetic peptide cells were cultured in the presence of complexes only for 4 h. Cell observation was carried out after another 24 h of culture in pure medium and confirmed effective internalization [23]. Reduction of the exposure time of cells could potentially limit cytotoxicity. However, most studies do not confirm the negative effect of low CPP concentrations on cells in the case of either 24 or 48 h culture. Penetrating peptides are generally considered safe to use. Most studies have not demonstrated any cytotoxicity or immunogenicity. The results obtained in the presented study show an increase in cytotoxic effect with an increased CR value and incubation temperature. For KALA, changes in the cell morphology can already be observed at CR 1:8, whereas CR 1:16 caused a significant toxic effect. This is in line with the literature reports. [PERSON] et al. confirmed the minimal cytotoxicity of KALA in CR as between 1:1 and 1:10 and demonstrated its significant toxic effect at higher ratios [24].