Synthesis, Characterization, and In Vitro Cytotoxicity of Fatty Acyl-CGKRK-chitosan Oligosaccharides Conjugates for siRNA Delivery

Abstract 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, 13 C 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.