Enhanced gene delivery by chitosan-disulfide-conjugated LMW-PEI for facilitating osteogenic differentiation

Chitosan-disulfide-conjugated LMW-PEI (CS-ss-PEI) was designed to combine the biocompatibility of chitosan and the gene delivery ability of polyethylenimine (PEI) using bio-reducible disulfide for bone morphogenetic protein (BMP2) gene delivery in mediating osteogenic differentiation. It was prepared by conjugating low molecular weight PEI (LMW-PEI) to chitosan through oxidization of thiols introduced for the formation of disulfide linkage. The structure, molecular weight and buffer capacity were characterized by Fourier transform infrared (FTIR), light scattering and acid-base titration, respectively. The reduction in molecular weight of CS-ss-PEI by the reducing agent indicated its bio-reducible property. With the increment in the LMW-PEI component, the copolymer showed increased DNA binding ability and formed denser nanocomplexes. CS-ss-PEI exhibited low cytotoxicity in COS-1, HepG2 and 293T cells over the different weight ratios. The transfection efficiency of CS-ss-PEI4 was significantly higher than that of PEI 25k and comparable with Lipofectamine in mediating luciferase expression. Its application for BMP2 gene delivery was confirmed in C2C12 cells by BMP2 expression. For inducing in vitro osteogenic differentiation, CS-ss-PEI4 mediated BMP2 gene delivery showed a stronger effect in MG-63 osteoblast cells and stem cells in terms of alkaline phosphatase activity and mineralization compared with PEI25k and Lipofectamine. This study provides a potential gene delivery system for orthopedic-related disease.