The Effect Of Carboxyl Groups On Gene Delivery System Mediated By Stearic Acid Grafted Chitosan

In order to enhance gene transfection efficiency, anion was chemical grafted or physical incorporated with chitosan-g-stearic acid (CSO-SA) to promote gene transfection efficiency and explore its mechanism in this study.The small molecule anions-cis-aconitate was chemical grafted to on CSO-SA by EDC to get CA-CSO-SA.1H-NMR confirmed the chemical composition of CA-CSO-SA. As measured by the TNBS, the amino substitution degrees of CSO-SA and CA-CSO-SA were4.03%and6.31%, critical micelle concentration were80.3μg/mL and78.4μg/mL. the average number diameter of CSO-SA and CA-CSO-SA micelles were55.3±7.6nm and60.0μ7.1nm, the Zetal potential were38.3μ2.8mV and30.8μ1.0mV. Transmission electron microscopy showed that both micelles have irregular spherical, small particle size, and uniform distribution.After the cis-aconitate modification, the CA-CSO-SA micelles could also compact DNA to form nanocomplexes. However, the DNA binding ability of CA-CSO-SA is slightly reduced compared with CSO-SA. CA-CSO-SA could offer efficacious protection to pDNA to avoid enzymatic digestion and its nanocomplexes have good stability in serum. In vitro transfection assay sμggested CA-CSO-SA/DNA nanocomplexes mediated37.2%, which is a2-fold as compared to CSO-SA/DNA nanocomplexes in transfecting capability. We examined cell uptake, endocytosis pathway and intracellular trafficking. Althoμgh the modification reduced cellular uptake kinetics, but the amount of cellular uptake tends to be the same at24h. Endocytosis inhibitors experiment results show that the internalization of CA-CSO-SA/DNA in HEK293cells including clathrin mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis. The modification doesn’t change endocytic pathways of CSO-SA based gene delivery system. Intracellular trafficking observation conducted by confocal laser scanning microscopy (CLSM) provided evidence that CA-CSO-SA/DNA can escape faster from the endo-lysosomes,Negatively charged polyglutamic acid (PGA) was physical incorporated in CSO-SA/DNA complex to obtain CSO-SA/DNA/PGA ternary complex. With the amount of incorporated PGA increasing, the size of nanocomplexes increased appreciably while their polydispersity index and zeta potential value decreased noticeably. Green fluorescent protein plasmid (pEGFP-Cl) was used as reporter gene, transfected cells was measured by flow cytometry. The results showed that the percentage of transfected cells of CSO-SA/DNA/PGA ternary complex was increasing with the concentration of PGA increased at first and then decreased; The transfection efficiency of CSO-SA/DNA/PGA ternary complex at N/P/C=10/1/4reached27.4%, which is twice that of CSO-SA/DNA binary complex, and close to LipofectamineTM2000. The evaluation of gene expression was also conducted using reporter gene pGL-3. Transfection efficiency was expressed as relative light units (RLU/mg protein). CSO-SA/DNA/PGA ternary complex at N/P/C=10/1/4meditated the highest expression of luciferase, reached5.32*105RLU/mg Protein, which is about10times that of binary complex. Intracellular trafficking observation provided evidence that CSO-SA/DNA complexes were trapped in the endo-lysosomes, but CA-CSO-SA/DNA/PGA complexes were more widely distributed in the cytosol. Bafilomycin Al inhibition assay results showed that PGA also have proton buffering capacity, promote the CSO-SA/DNA complex to transport from endo-lysosome to cytoplasm, contributing to the high efficient gene transfection of CSO-SA/DNA/PGA ternary complex.The results show that negative charged anion, chemical grafted or physical incorporated with CSO-SA, could enhance CSO-SA-mediated gene transfection, wherein the protonated carboxyl groups may promote endo-lysosome escape.