## 4 Discussion
For this purpose, a chimeric protein containing a cGPK inhibitor peptide in combination with TAT was synthesized. In addition, in this case, a covalent bond was used. The chimeric protein was administered to 8 mice by continuous 72-h infusion into the internal jugular vein. Effective internalization of peptides associated with cGPK inhibitors was confirmed by the measurements of systolic and diastolic blood pressure, which values increased in the study group. The presence of inhibitors in cardiac tissues has also been confirmed by Western blotting. Changes in blood pressure were not observed in any of the control groups. The researchers did not mention any side effects of CPPs, and the administration of unbound TAT molecules did not have any effect on blood pressure [12]. In turn, Pep-1 was used to introduce the chimeric Pep-1-EGFP protein into HUVECs (human umbilical vein endothelial cells) [13]. For this purpose, two expression plasmids, pET15b-EGFP (expressing only EGFP) and pET15b-PEP-1-EGFP (expressing both EGFP and Pep-1 bound covalently), were introduced into _E.coli_ cells. The fusion protein and EGFP were isolated by affinity chromatography. Purified EGFP protein was not transduced into HUVEC cells. In turn, the Pep-1-EGFP fusion protein internalized into the cells in a time and dose-dependent manner. EGFP fluorescence was observed after 27 h. Additionally, the fusion peptide did not show a cytotoxic effect in the MTT test, even at the highest dose of 200 \(\upmu\)mol/L [13]. So far, no clinical trials have been carried out using CPPs to transport molecules to vascular endothelium in humans. However, they were used to introduce an anti-c-Myc antisense molecule called AVI-5126 into vascular smooth muscle cells. Researchers wanted to determine whether graft immersed in a solution containing AVI-5126 in combination with CPP rich in arginine before bypass surgery would reduce the degree of inner layer growth and thus the risk of vascular occlusion at the intervention site (NCT00451256) [14]. Unfortunately, the studies were suspended in phase II due to insufficient efficacy [15]. A better approach could be the acceleration of endothelialization in the intervention site instead of inhibiting neeointina growth. Perhaps the opportunity for this will also be the use of CPPs. With proven effective internalization of vascular endothelial cells in in vivo studies, it is probably only a matter of time before they are used in clinical trials. Although the method of creating covalent bonds between a penetrating and transported molecule is still the most widespread, the formation of non-covalent interactions arouses more and more interest. Studies have confirmed that simple incubation of CPPs with selected molecules in the appropriate CR leads to bonding. Literature reports have confirmed the ability of TAT [16], Pep-1 [13], and KALA [17] to form non-covalent bonds. Moreover, each of the selected peptides contains sequences of a cationic character capable of producing electrostatic interactions with negatively charged phosphate residues in plasmid DNA. The binding efficiency strongly depends on the ratio of these charges. By default, low CR values between 1:1 and 1:10 are used. However, in some cases, the CR with optimal efficiency can be as high as 1:100 [18]. Selected penetrating peptides in CR 1:16 led to severe damage to endothelial cells, which justifies the subsequent reduction of the ratio used to a maximum value of 1:8. However, the most effective CR differed depending on the type of CPP used. The efficiency of generating non-covalent interactions between CPPs and selected molecules depends not only on CR but also on the conditions of the complex formation. The study used two variable incubation parameters for selected CPPs and pDNA: time and temperature. Both of the times (15 and 30 min) applied in the study are commonly used in laboratory practice [3; 12; 13]. However, for pmaxGFP, the extension of the incubation time from 15 to 30 min led to an increase in the number of GFP positive cells.