Studies On Gene Delivery System Mediated By Stearic Acid Grafted Chitosan/Proteins/DNA Ternary Complexes

Stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection and excellent cellular internalization, CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, the proteins, bovine serum albumin (BSA) with different isoelectric point value (4.7,6.0and9.3), was incorporated into CS-SA based gene delivery system and CS-SA/BSA/DNA ternary complexes were then prepared. The study was designed to enhance the gene release thus obtain high gene transfection.Firstly, chitosan with the molecular weight of18.0kDa was prepared by enzymatic degration. Stearic acid (SA) grafted chitosan (CS) copolymer (CS-SA) was synthesized via the reaction of carboxyl groups of SA with primary amino groups of CS catalyzed by1-ethyl-3-(3-dimethylamin-opropyl) carbodiimide (EDC). The physiochemical properties were investigated. The chemical composition and stearic acid graft ratio were comfirmed by NMR and determined by2,4,6-trinitrobenzene sulfonic acid (TNBS) method. The critical micelle concentration in aqueous medium was determined by by pyrene fluorescence method. The particle size and zeta potential were measured by dynamic light scattering and the morphology was observed by TEM. Secondly, BSA of different pI was obtained by blocking carboxyl with methanol catalyzed by acetyl chloride and the isoelectric point was determined by the pH value of BSA solution at which the zeta-potential is approximately zero. CS-SA/DNA and CS-SA/BSA/DNA complexes were prepared and characterized. Gel retardation assay was performed for examination of the condensation ability of polymer with DNA by electrophoresis. The effect of proteins on the gene transfection efficiency mediated by CS-SA was invesgated using HEK293as model cells. The trasfection capability was evaluated by inverted fluorescence microscope and flow cytometry. The cellular uptake and intracellular trafficking were observed by a confocal laser scanning microscope (CLSM). The study focused on the influence of three kinds of BSA of different pI on the gene delivery mediated by CS-SA micelles and investigated the possible mechanism in intracellular trafficking of CS-SA/BSA/DNA complexes. At last, survivin was chosen as RNA interference targets and the real-time PCR method was established. RNA interference gene delievery system was prepared and then transfected the model cells, MCF-7and MCF-7/ADR, the enhanced chemosensitivities of MCF-7and MCF-7/ADR cells to PTX was investigated by MTT assay.The results showed that stearic acid was successfully grafted onto the chitosan chain. The actual substitution degree (SD) of stearic acid was7.4%. The CMC value of CS-SA micell was81.0μg/L. The size and zeta potential was36.4±0.9nm and32.7±1.2mV.CS-SA binary complexes were prepared and DNA can be condensed tightly to form stable nanoparticles. BSA of different pI was obtained and was referred to as BSA (4.7), BSA (6.0) and BSA (9.3) respectively. CS-SA/BSA/DNA ternary complexes were prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However referring to the transfection activity, BSA of different isoelectric point value (pI) had distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA (4.7)/DNA and CS-SA/BSA (6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA (4.7)/DNA complexes showed the highest transfection efficiency. On the contrary, CS-SA/BSA (9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA (4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake.Survivin was chosen as the target gene for the success of gene silencing. Real-time PCR method was established and employed to determine survivn mRNA level. CS-SA based pshSUR delivery system was prepared and transfected MCF-7and MCF-7/ADR cells. The pre-gene deliver of CS-SA/BSA/pshSUR can enhanced the chemosensitivity of MCF-7/ADR cells to paclitaxel (PTX), and reversed the drug resistance (reversal power was about9).