Synthesis, characterization, and in vitro evaluation of short cationic peptides for gene delivery vehicle candidate

Background: Amongst gene delivery vehicles, peptide-based vectors have drawn the intensive attraction of experts globally due to their simplicity and many advantages due to ease in design, biocompatibility, and safety. Rationally designed peptides are capable of condensing DNA molecules efficiently and facilitating gene expression in the target cells. Objective: This study aims to design, synthesize and evaluate short cationic peptides composed of several positively charges amino acids of lysine (K) and arginine (R) for gene delivery vehicle can­didates. Methods: The short cationic peptides of PKKKRKV (P1), CHSPKKKRKV (P2), and YGRK- KRRQRRR (P3) were synthesized using a solid-phase method on 2-chlorotrityl chloride resin. The crude peptides were purified using RP-HPLC and characterized by HR-TOF-ESI-MS and¹H-NMR. The capability of the peptides to condense DNA was evaluated by ethidium bromide exclusion assay. Cytotoxicity study of the peptides was carried out in HEK-293T, CHO-K1, and HepG2 cells using MTT assay. Gene expression facilitated by the peptides was determined in the HEK-293T. Results: The peptides were successfully synthesized with high purity (> 90%) and in a high consistency with the synthetic products, as shown by the spectroscopic data. Physicochemical and biological evaluation showed that the cationic peptides are capable of condensing DNA mole­cule and have low cytotoxicity to the cells of HEK-293T, CHO-K1, and HepG2. Moreover, the cat­ionic peptides facilitated gene delivery of green fluorescence protein more efficiently compared to PLL. Conclusion: The short cationic peptides rich in lysine and arginine have been successfully synthe­sized using solid-phase peptide synthesis method. They were found to be capable of condensing DNA, have low cytotoxicity, and facilitate gene delivery. However, structure modification or for­mulation of cationic peptide with lipid components to form cationic liposome is still needed to en­hance transgene expression by these peptides.