## 3 Results  
The results were presented in the form of extensive photographic documentation. Cells with the expression of plasmid are characterized by green fluorescence. For control cells, no positive signal was observed (Figure 1). This confirms the assumption that the plasmid alone is not able to penetrate the cell membrane. Figures 2-22. Variations in the conditions of complex formation and the time of incubation with cells affected the efficiency of the transduction process. For CPP complexes with 1 ug plasmid, only single cells expressing GFP were observed either after 24 h (Figures 2 and 3) or 48 h culture (Figures 4 and 5). For this reason, further tests were carried out using 2 ug of pmaxGFP. In addition, CR 1:16 caused a significant cytotoxic effect. This was especially visible in the case of the KALA protein and contributed to the reduction of CR used in subsequent stages of the study (Figures 2-5). Extending the incubation time of CPPs with the plasmid led to an increase in the number of GFP positive cells (Figures 10-13 and 18-21). In turn, increasing the temperature during complex formation enhanced cytotoxicity, particularly for Pep-1 and TAT (Figures 8, 9, 12, 13, 16, 17, 20 and 21). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Type of** & **CR 1** & \multicolumn{3}{c}{**Amount of**} & \multicolumn{3}{c}{**Time of**} & \multicolumn{3}{c}{**Time of**} & **Temperature of** \\
**CPP/NLS** & **CR 2** & **Plasmid [ug]** & **Incubation [min]** & **Culture [h]** & **Incubation [°C]** \\ \hline \multirow{4}{*}{TAT} & 1:1 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:2 & 2 & 15 & 30 & 24 & 48 & 21 & 21 \\  & 1:4 & 1 & 2 & 15 & 24 & 24 & 48 & 21 & 37 \\  & 1:8 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:16 & & 1 & 15 & 30 & 24 & 48 & 21 & 37 \\ \hline \multirow{4}{*}{Pep-1} & 1:1 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:2 & 2 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:4 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 21 \\  & 1:8 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:16 & & 1 & 15 & 30 & 24 & 48 & 21 & 37 \\ \hline \multirow{4}{*}{KALA} & 1:1 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 21 \\  & 1:2 & 2 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:4 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:8 & 1 & 2 & 15 & 30 & 24 & 48 & 21 & 21 \\  & 1:16 & & 1 & 15 & 30 & 24 & 48 & 21 & 37 \\ \hline \multirow{4}{*}{NLS} & 1:1 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:2 & 2 & 15 & 30 & 24 & 48 & 21 & 21 \\
**[47–55]** & 1:4 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:8 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\ \hline \multirow{4}{*}{NLS} & 1:1 & 2 & 15 & 30 & 24 & 48 & 21 & 21 \\  & 1:2 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\
**[47–56]** & 1:4 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\  & 1:8 & 2 & 15 & 30 & 24 & 48 & 21 & 37 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Conditions of CPP-cargo complexes preparation. Figure 4: Results of 48 h culture in the presence of complexes in CRs of 1:1, 1:4, 1:8 and 1:16. The complexes were formed using incubation of 1 \(\upmu\)g pmaxGFP with chosen CPPs at 21 \({}^{\circ}\)C for 15 min. Bar = 50 \(\upmu\)m. CR, charge ratio. Figure 5: Effect of 48 h culture in the presence of complexes in CRs of 1:1, 1:4, 1:8 and 1:16. The complexes were formed using incubation of 1 \(\upmu\)g pmaxGFP with chosen CPPs at 21 \({}^{\circ}\)C for 15 min. CR, charge ratio. (**A**) Transfection efficiency. (**B**) Percent of cells with abnormal morphology. Data are presented as means, numbers in parentheses represent standard deviations. Figure 3: Effect of 24 h culture in the presence of complexes in CRs of 1:1, 1:4, 1:8 and 1:16. The complexes were formed using incubation of 1 \(\upmu\)g pmaxGFP with chosen CPPs at 21 \({}^{\circ}\)C for 15 min. CR, charge ratio. (**A**) Transfection efficiency. (**B**) Percent of cells with abnormal morphology. Data are presented as means, numbers in parentheses represent standard deviations. Figure 6: Results of 24 h culture in the presence of complexes in CRs of 1:1, 1:2, 1:4 and 1:8.