Characterization and evaluation of a peptide-based siRNA delivery system in vitro

Since its inception more than a decade ago, gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases. However, the use of siRNA is hampered by its rapid degradation and poor cellular uptake in vitro and in vivo. Recently, peptide-based carriers have been applied to siRNA delivery, as an alternative to the traditional delivery systems. Here, a histidine-containing amphipathic amino acid pairing peptide, C6M3, which can form complexes with siRNA, was used as a new siRNA delivery system. This peptide exhibited a high affinity for siRNA and ability to efficiently deliver siRNA into the cells. The interaction of C6M3 with siRNA was investigated to determine the loading capacity of C6M3 at different peptide/siRNA molar ratios. At C6M3/siRNA molar ratio of 10/1, siRNA molecules were entirely associated with C6M3 as indicated by a gel electrophoretic assay and further confirmed by zeta potential analysis. The particle size distribution of the C6M3-siRNA complexes was studied using dynamic light scattering, which showed an intensity-based size distribution peaked approximately at 100 nm in RNase-free water and 220 nm in the Opti-MEM medium. C6M3 adopted a helical secondary structure in RNase-free water and became more so after forming complexes with siRNA. The interaction of siRNA with C6M3 is an entropy-driven spontaneous process, as determined by isothermal titration calorimetry (ITC) study. The efficiency of cellular uptake of the siRNA complexes at different C6M3/siRNA molar ratios was evaluated, and the results showed that C6M3 promoted efficient cellular uptake of siRNA into cells. Furthermore, a significant level of GAPDH gene silencing efficiency (69%) was achieved in CHO-K1 cells, with minimal cytotoxicity.