Preparation Of Lactobionic Acid Modified Curdlan Nanoparticles And Activity Studies Of Its Cell Targeting Delivery Of DNA/siRNA

As a new type of medical technology,gene therapy has been applied on treatment of diseases such as inherited disease and cancer.Meanwhile,with ideal carriers,specifically delivering therapeutic gene into diseased tissue and cells is the precondition and key to gene clinical therapy.Currently,construction of gene vectors with ideal safety,high efficiency and cell-specificity is one of the hottest fields to researchers.In this thesis,curdlan and L-ornithine were used as primary raw materials.According to the mechanism of Huisgen-Click reaction,we prepared polymers of triornithine conjugated curdlan(CTO)firstly,then the series of ligand functionalized conjugates(named as CTOL5%,CTOL10%and CTOL20%)were synthesized by modification of curdlan-triornithine grafted polymers with lactobionic acid in different chemical ratios.The structures of polymers were determined with IR,1H-NMR,13C-NMR,and MS.The results of GPC showed that the molecular weight(Mn)of CTOL5%,CTOL10%and CTOL20%are 24609,25503,and 27413 g/mol successively.By integrating the test results of particle size,Zeta potential and SEM for above four kinds of polymers,those functionalized polymers are likely to form spherical nanoparticles,and displayed excellent dispersibility in PBS buffer.Especially,the polymer modified with lactobionic acid are easily to combine with DNA and generate approximate spherical particle with about 80 nm of diameter.The results indicated that those nanoparticles or nanocarriers/DNA complexes are readily to transport into cytomembrane.To CTO,CTOL5%,CTOL10%and CTOL20%,we systematically studied their biological properties by agarose gel electrophoresis,cytotoxicity test,GFP-pDNA transfection,FITC-DNA transfection and siRNA delivery in vitro.The results demonstrated that CTO,CTOL5%,CTOL10%and CTOL20%were able to combind with DNA well at ratio of N/P=0.3.Comparing with CTO,all of CTOL5%,CTOL10%and CTOL20%show lower cytotoxicities,and CTOL20%especially exhibited the lowest cytotoxicity(the survival rate of HepG2 cells is up to 90%at the concentration of 100?g/mL).Most importantly,CTOL displayed high efficiency of cell transfection and excellent ability of targeting HepG2 cells in GFP-pDNA transfection,FITC-DNA transfection and siRNA delivery.In conclusion,we have successfully prepared gene nanocarriers based on curdlan,which do possess lower cytotoxicity,cell-specificity of HepG2 cells and higher gene delivery efficiency.Those curdlan nanocarriers have potential value for overcoming the contradiction between the siRNA load capacity and the functionized modification,and provide us with the example on how to construct new targeted nanocarriers for RNAi.The gene vectors studied in this hesis has not been reported in the literature at present.