Covalent conjugates of granulin-epithelial precursor-siRNA with arginine-rich peptide for improved stability and intracellular delivery in hepatoma cells

Backgrounds: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. However, little is known about the molecular mechanisms of HCC development, progression, or effective therapeutic approaches. Recently, granulin-epithelin precursor (GEP) has been suggested as a novel factor that can control HCC cell proliferation. Thus, we aimed to develop new cell-permeable and stable genetic agents that can control GEP expression in HepG2 cells. Methods: Small interfering RNA-GEP (GEP siRNA) was used for the development of therapeutic agents for HCC treatment. GEP siRNA was first modified by adding polyethylene glycol (PEG) at the terminus. Arginine-rich peptide (ARP) was chemically conjugated with PEGylated GEP siRNA. Chemical conjugates of GEP siRNA-PEG-ARP were evaluated for cytotoxicity and bioavailability, including stability against DNase I enzymatic activity and GSH. The conjugate was further evaluated for intracellular distribution and cell proliferation effects. Results: The optimal conjugation ratio of GEP siRNA to ARP was a 1:10 molar ratio, and stability was confirmed using DNase I and glutathione stability assays. Conjugates showed increased intracellular uptake and distribution in HepG2 cells. Interestingly, HepG2 cells treated with conjugates showed significantly decreased cell proliferation. Conclusion: These results provide insights into the importance of chemical modification of unstable genetic therapeutics for HCC treatment. Conjugation of PEGylated GEP siRNA with ARP may be a potential HCC therapeutic agent.