Cationization Of Inulin Via Atom Transfer Radical Polymerization(ATRP) For Gene Delivery

Vital movements are regulated by corresponding genes, several diseases are related to gene mutation. Gene therapy has been widely used in clinical trails. Gene therapy is a fundamental way to treat diseases. A key element for gene therapy is requiring for an effective and safe gene vector that can carry genes into target cells without side effects. Gene delivery vectors are divided into virus vector and non-virus gene vector. Although the transfection efficiency of virus vector is very high, but its application is limited by its high toxicity and high cost. But natural polysaccharide has low toxicity, good biocompatibility and easy to targeting modification, scientists are turning to modify polysaccharides in the designing of biomedical materials. Among them, inulin is a neutral polysaccharide with low cytotoxicity and good biocompatibility, but it can’t condense DNA into nanoparticle, so it is of crucial importance to cationization of inulin in the preparation of inulin derivatives as gene vector. In this thesis, several works have been done as follows:It is of crucial importance to modify inulin-based polysaccharides in the designing of biomedical materials. In this work, a new degradable cationic polymer named PDIN was synthesized by grafting poly(DMAEMA) onto the backbone of inulin oligosaccharide via atom transfer radical polymerization. Three inulin-based non-virus gene delivery vectors were synthesized by changing the mole ratio of DMAEMA to inulin.Multifunctional oligosaccharide initiator I-Br was synthesized by grafting BMPA onto the backbone of inulin. And then PDIN was synthesized by grafting poly(DMAEMA) onto the backbone of inulin oligosaccharide via ATRP. The structure of PDIN was confirmed by1H NMR. Transmission electron microscope and scanning electron microscope indicated that PDIN cationic polymer was well-distributed with size of30nm and microstructure was spherical. Buffering capacity tests showed that PDIN may deliver DNA into target cells.For three kinds of PDIN/pDNA complexes at different N/P, the size range from30nm to130nm, the ζ-potential range from2mV to12mV. These vectors show good ability condense DNA into nanoparticles even in high concentration of heparin. They can also prevent pDNA from the degradation by DNase I. The results of MTT assay displayed that the all PDIN/pDNA complexes showed comparable cytotoxicity against four kinds of cells with that of Lipofectamine2000/pDNA complex. PDIN showed good biocompatibility due to its low hemolytic rate. PDIN vector can delivery both pEGFP and pβgal into COS-7cells. The activity of β-galactosidase expressed in COS-7cells revealed that the highest transfection efficiency [(3.36±0.74) U/mg] of PDIN as a new gene delivery carrier was obtained at N/P ratio of1, which was comparable with that of Lipofectamine2000[(4.33±0.77) U/mg]. In the present of low concentration serum, there is a certain impact of PDIN2/siRNA on the expression of green fluorescent protein (EGFP) and β-galactosidase, but PDIN can still express green fluorescent protein (EGFP) and β-galactosidase.PDIN2exhibited good ability to electrostatic interact with siRNA. RNAi experiments showed that PDIN2/siRNA complexe can effectively inhibit the expression of green fluorescent protein (EGFP), with the increase of N/P, the effect of gene silencing was better. In the present of10%concentration serum was observed, there is a certain impact of PDIN2/siRNA in the inhibition expressing of green fluorescent protein (EGFP), but PDIN2can still inhibit the expression of green fluorescent protein (EGFP). Cell uptake experiment demonstrated that PDIN/FAM-siRNA complexe can enter into cells by endocytosis pathway.The above results illuminated that PDIN can delivery pDNA and siRNA into cells to transfect cells or silence specific proteins. So it is of crucial importance to modify inulin-based polysaccharides in the designing of biomedical materials. These studies provide a feasible approach to design a novel non virus gene delivery vector, which would promote the application of gene delivery system.