Characterization and In-vitro Evaluation of Positively Charged Oligopeptide as Gene Delivery Vehicle Candidate on BHK-21 Cells

Peptide-based transfection agents have gathered significant interest and are being investigated globally as a possible gene delivery method. The preparation and in-vitro evaluation of oligopeptides composed of GRKKRRQRRR (TAT) and PKKKRKV (NLS) functionalized with the CKKHH sequence as a potential transfection agent is the main goal of this study. Solid-phase peptide synthesis was applied to generate the positively charged of the GRKKRRQRRR-PKKKRKV (OP-1, MW of ~ 2260 Da) and CKKKHH-YGRKKRRQRRR-PKKKRKV (OP-2, MW of ~ 3057 Da) sequences. The oligopeptide's capacity to condense DNA molecules was assessed using the ethidium bromide exclusion and DNA mobility retardation assays. Transmission electron microscopy (TEM) image examination and a particle size analyser were performed to further characterise the oligopeptide/DNA complexes. The MTT assay was used to determine the cytotoxicity, while transgene expression was carried out in BHK-21 cells using a gene encoding green fluorescence protein. Both oligopeptides of OP-1 and OP-02 were able to condense DNA molecules into compacted particles with a size range of 150–220 nm. The transfection agent based on TAT-NLS (OP-1) that functionalized with CKKHH (OP-2) was more efficient in condensing and compressing DNA molecules. Both oligopeptides were comparatively less hazardous to BHK-21 cells than those of poly-L-lysine (PLL), according to the MTT assay result. The oligopeptides' ability to promote GFP-encoding gene transgene expression further emphasises their effectiveness as potential transfection agent candidates. To further increase the positively charged oligopeptide efficacy, more development on the oligopeptide-based transfection agent needs to be explored.