Synthesis, characterization, and in vitro cytotoxicity of fatty acyl-CGKRK-chitosan oligosaccharides conjugates for siRNA delivery

In this studies, three fatty acyl derivatives of CGKRK homing peptides were coupled successfully to chitosan oligosaccharides (COS) using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (sulfo-SMCC). The COS-SMCC was prepared by direct coupling between COS and sulfo-SMCC in PBS (pH 7.5) at RT for 48 h. The structure of COS-SMCC and the three fatty acyl-CGKRK-SMCC-COS conjugates were characterized by FT-IR, 13C NMR, and SEM. The ability of three conjugates to condense siRNA into nanosized polyplexes and their efficacy in protecting siRNA from serum nucleases degradation were investigated. Among the investigated derivatives, S-CGKRK-COS showed higher siRNA binding affinity as compared to the P-CGKRK-COS and O-CGKRK-COS, respectively. At a ratio of 10:1, complete protection for siRNA from early enzymatic degradation was achieved. The polymers and the polymer/siRNA polyplexes showed negligible cytotoxicity on human breast cancer cell line MDA-MB-231 at all investigated ratios. However, the polyplexes prepared with palmitoyl and oleoyl derivatives at polymer concentration 10 μg/mL reduced the cell viability by 21.5% and 35%, respectively. The results of this study revealed the potential use of fatty acyl-CGKRK-COS as a siRNA carrier and confirmed the importance of incorporating a hydrophobic moiety into chitosan to improve its capacity in complexing with siRNA and protection from degradation.