## 4 Discussion  
Drugs containing proteins or nucleic acids are a promising form of therapy for many diseases, including those considered incurable at the current level of medicine development. The structure of the cell membrane, however, does not allow direct cell penetration. The crucial issue is the choice of the appropriate method of transport to the cells' interior. The viral and non-viral systems created so far have many significant disadvantages. Therefore, attempts are still underway to optimize the transport of macromolecules across the cell membrane. The results published so far indicate that chimeric molecules composed of CPPs and various molecules can internalize cells both in vitro and in vivo [6,7]. Insufficient understanding of the internalization mechanism is holding back their commercialization. However, some of the best-known penetrating peptides have been included in clinical trials with moderate success [8,9]. Nevertheless, studies regarding the effectiveness of the production conditions of chimeric particles composed of CPPs and selected therapeutic molecules on the cytotoxicity and process efficiency are still necessary. This will allow for improvement of the methodology to maximize benefits. Endothelium obtained from a 70 kg male body would weigh over \(1\) kg, cover an area of almost 3 m\({}^{2}\), and contain about a trillion cells. These specialized cells build a layer that is the first barrier between blood components and other body tissues. For this reason, the endothelium is constantly exposed to harmful substances transported within liquid tissues. At the same time, maintaining the proper functioning of the endothelium is pivotal for the organisms [10]. Endothelial cells participate in such important processes as blood pressure regulation, coagulation, fibrinolysis, angiogenesis, and inflammatory reactions. Abnormalities in the functioning of endothelial cells are the cause of many diseases, e.g., atherosclerosis and hypertension. These disorders are also associated with stroke, venous thrombosis, peripheral vascular disease, chronic renal failure, cancer development, and vascular complications in diabetes [10]. Methods of proven efficacy in maintaining normal endothelial function include a Mediterranean diet, weight reduction, stopping smoking, or exercise [11]. Unfortunately, changes that have already appeared cannot be removed by such simple methods. In addition to many conventional approaches, such as invasive and non-invasive procedures and pharmacotherapy, the use of peptides penetrating cells to introduce molecules that restore or support endothelial function is a promising field of research. Effective internalization of CPPs in combination with various cargo has also been confirmed for endothelial cells. The first report on this subject came from 2000 when penetrating was used to transfer the caveolin-1 scaffold domain to mouse endothelial cells [7]. The study aimed to determine the role of this domain in the regulation of signal transduction. The result was an effective uptake of the constructed chimeric protein, which was manifested in the inhibition of NO production and a reduction in the degree of acetylcholine-induced vasodilation. Furthermore, peptide administration was associated with the inhibition of  
Figure 22: Results of pHCAECs’ transfection by nucleofection. Bar = 50 \(\upmu\)m.  
acute inflammation and vascular leakage to a similar extent as the use of glucocorticoids such as dexamethasone or endothelial NO synthase inhibitor. Penetrin was attached to the molecule via covalent binding and administered intraperitoneally to mice at 0.3 and 1 mg/kg three times for 24 h. The administration did not significantly affect animal body functions, such as blood pressure or heart rate [7]. The ability of cell-penetrating peptides to internalize endothelial cells has also been used to determine the role of cyclic guanosine monophosphate (cGMP) cGMP protein kinase (cGPK) in nitric oxide-dependent vasodilitation and blood pressure regulation [12].