Folate Modified Stearic Acid-G-Chitosan Oligosaccharide As A Gene Delivery Vector

Chitosan is a naturally occurring cationic polysaccharide with good biocompatibility, biodegradation, low immunogenicity and non-cytotoxicity. At present, more and more countries and research institutions have attached importance to the chitosan as a new Non-viral gene vector. However, low specificity and low transfection efficiency of chitosan need to be overcome prior to clinical trial. It is necessary to develop a new gene delivery system using chitosan derivatives to gain high transfection efficiency. In recent years, folate-folate receptor targeting is a new popular anti-tumor mechanisms. Targeted gene is targeting transported via the folate receptor, based on the fact that folate receptor can be overexpressed on the surface of some kinds of tumor cells but scarcely expressed on normal cells. Folate can be selected to chemically modify the chitosan derivatives to improve the targeting ability and the transfection efficiency of the gene transfection.Stearic acid grafted chitosan oligosaccharide copolymer (CSO-SA) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction of stearic acid (SA) and low-molecular-weight chitosan (CSO, Mw=5.0 KDa). Utilizing the carboxyl group of folic acid (FA) and remained amino groups of CSO-SA, the FA modified CSO-SA (FA-CSO-SA) was obtained. Substitute degrees of amino groups (SD%) means the graft number of SA groups per 100 amino groups of CSO, which was measured by TNBS method. The critical micelle concentration (CMC) of copolymers in water was determined by fluorescence measurement using pyrene as a probe. The micellar size distribution and Zeta-potential, measured by light scattering and eletrophoretic mobility, was investigated. SKOV3 (FR=93%) and A549 (FR= (-)) were chosen as typical tumor cells, the cellular uptakes of FA-CSO-SA against the cell lines were estimated. The Z-average diameter and Zeta potential of FA-CSO-SA and FA-CSO-SA/pDNA complex nanoparticles were measured by dynamic light scattering using a Zetasizer. The capability that micellar compact the plasmid DNA and protect pDNA from enzymatic degradation by ribozyme were study. The in vitro transfection efficiency of micelles were investigated by using plasmid DNA (pEGFP-C1), LipofectamineTM2000 as a positive control. Transfection efficiency was investigated using the pGL-3 luciferase reporter gene. Folic Acid Competition Study showed the roles of folic acid modification for enhancement of transfection efficency in vitro.In this study, CSO-SA was synthesized by the coupling reaction of SA and CSO (5.0 KDa). The substitute degrees of amino groups (SD%) was determined as 19.94%, and the CMC value of CSO-SA was determined to be 147.1μg/ml in DI water. Then FA-CSO-SA was synthesized by EDC mediated coupling reaction. The CMC value of FA-CSO-SA was determined to be 105.2μg/ml, which showed that the modification of FA impoved self-assembly ability of FA-CSO-SA in water. It was found that the size of FA-CSO-SA micelle was bigger than the size of CSO-SA, where the zeta potential of FA-CSO-SA was lower. Using SKOV3 and A549 cells as model tumor cells, the cellular uptake of FA-CSO-SA and CSO-SA micelles were earried out. It was found that cellular uptakes of FA-CSO-SA in SKOV3 cells which with higher FA receptor expression were faster than that in A549 in the short incubation time.The pEGFP-C1 was selected as the reported gene. The micelles could compact the plasmid DNA to form micelle/DNA complexes nanoparticles(100-200 nm), which can efficiently protect the condensed DNA from enzymatic degradation by DNase I. The IC50 value of the CSO-SA and FA-CSO-SA micelles against SKOV3 cells was 115.15μg/mL and 217.18μg/mL respectively. And the IC50 values of the micelles against A549 cells were 201.91 ug/mL and 298.46μg/mL respectively. The cytotoxicities of micelles were much lower than that of LipofectamineTM2000. In vitro gene transfection tests demonstrated that the transfection efficinency with FA-CSO-SA/DNA complexes was increased with the post-transfection time (in 96 h). The gene transfection of CSO-SA/DNA could be further enhanced by modifying of FA. Folic Acid Competition Study showed that the transfection of FA-CSO-SA/DNA complexes was decreased in presence of folic acid in the culture medium. This may due to the competitive displacement of the nanoparticles on the folate receptors by excess of free folic acid.