Haleh Feyzyab, Azam Bolhassani, Mehrdad Hashemi, Alireza Milani. Evaluation of physicochemical properties of pEGFP-N1/KALA nanoparticles. The 5th International and 13th National Biotechnology Congress, Tehran-Iran; 07/2023. (Article)
Abstract: Cell-penetrating peptides can combine with DNA and efficiently deliver cargo by producing nanoparticles.Nanoparticles increase the stability and improve the transport of encapsulated cargoes. KALA is a modifiedCPP from GALA that contains lysine residues. Due to its properties, KALA can form a stable complex withDNA. The pEGFP-N1 is used as a reporter which encodes a green fluorescent protein (GFP) and can forma complex with KALA. The formation of pEGFP-N1/KALA nanoparticles and their stability wereevaluated in vitro. The pEGFP-N1 was purified from E.coli strain by plasmid DNA extraction minikit(FavorGen, Taiwan). The pEGFP-N1/KALA nanoparticles were prepared in different ratios of N/P, andthe ratio of complex formation was determined by gel retardation electrophoresis assay. The stability ofpEGFP-N1/KALA nanoparticles was investigated in the presence of DNase I endonuclease and serumprotease (fetal bovine serum; FBS). Moreover, the zeta potential of nanoparticles, and their morphologyand size were investigated by Zetasizer and SEM microscopy. The presence of the purified pEGFP-N1plasmid band was confirmed in agarose gel electrophoresis. KALA peptide showed a band of ~ 3.13 kDain SDS-PAGE 15%. The lack of migration of the pEGFP-N1/KALA nanoparticles from N/P=1:1 ratio onagarose gel indicates complex formation. The presence of plasmid DNA band on agarose gel indicated theprotection of DNA by complex formation with KALA peptide in the presence of endonuclease DNase Iand serum protease. The zeta potential of nanoparticles, and their size were about - 2.6 mV and 80-100 nm,respectively. The results suggested that KALA forms a stable complex with pEGFP-N1 due to the presenceof positive charges of lysine residues.
Keywords: Nanoparticle, KALA, Cell penetrating peptide
