A Novel Pullulan-based Non-virus Cationic Gene Deliver Vetor

The development of efficient and safe gene delivery systems is one of the most important factors for the gene therapy. In the present study, a novel non-virus gene delivery vector was successfully synthesized by grafting low-molecular branched PEI (1000Da) onto the backbone of pullulan with succinic acid as a spacer. Folic acid was further introduced onto P-PEI for targeting the cells with the expression of folic acid receptor (FR).1. Synthesis and characterization of P-PEI and P-PEI-FAA novel non-virus gene delivery vector (P-PEI) was successfully synthesized by grafting low-molecular branched PEI (1000Da) onto the backbone of pullulan with succinic acid as a spacer. Another vector was synthesized by introducing folic acid onto P-PEI for targeting FR positive cells.’H NMR and FTIR demonstrated that P-PEI and P-PEI-FA had been successfully synthesized. TEM and FESEM displayed that P-PEI and P-PEI-FA are spherical or approximately spherical. Buffering capacity tests demonstrated that P-PEI and P-PEI-FA show good buffering ability.2. Studies on P-PEI/pDNA and P-PEI-FA/pDNA complexesThe size of P-PEI/pDNA and P-PEI-FA/pDNA is between30nm to250nm and the C-potential is12mV to35mV at different N/P. Gel electrophoresis retardation assay demonstrated that P-PEI and P-PEI-FA can wrap pDNA with electrostatic interaction excellently even in the presence of heparin, and protect pDNA from the degradation by DNase I and serum. Compared with PEI/pDNA complexes, the P-PEI/pDNA and P-PEI-FA/pDNA complexes showed relative lower cytotoxicity against HeLa, MCF-7, COS-7and Hep G2cells, even at high N/P (up to12.5). In vitro transfection experiment displayed that both P-PEI and P-PEI-FA could delivery pEGFP and pGL3into HeLa cells. Transfection efficiency reached the highest at N/P=6.25. No obvious influence on transfection efficiency in the present of low concentration serum was observed. The transfection efficiency of P-PEI-FA/pDNA polyplexes depended on the folic acid concentration in culture medium for folic acid receptor-positive Hela cells. EGFP and Luciferase expression level markedly decreased with increasing the folic acid concentration.3. Studies on P-PEI/siRNA and P-PEI-FA/siRNA complexesGel electrophoresis retardation assay demonstrated that P-PEI and P-PEI-FA can also wrap siRNA with electrostatic interaction excellently. The investigation of the endocytosis pathway and location in the cell of the complexes showed that a large portion of them escaped from endo/lysosome and the FAM-siRNA were separated from the P-PEI and P-PEI-FA vector. Concentration of folic acid in the medium plays a pivotal role in the achievement of significant gene silencing. P-PEI-FA displays a good target of folic acid.