To Study The Preparation Of Chitosan Nanoparticle Loaded With PE-GFP-C1 Gene And Its Transfection

Objective : To prepare appropriate chitosan nanoparticles by optimizing the experimental condition. To load it with plasmid and studv its loading capability and protection ability to plasmid .To transfect it to HepG-2 cell and evaluate the possibility and efficiency of chitosan nanoparticle as a vector in gene transference compared to cationic liposomes.Methods:To prepare the chitosan nanoparticle with cocervation process;To measure its diamerter ,the diametric distribution range and its form by scanning electron microscope and laser atomic examining apparatus.The enhanced green fluorescent protein as the reporter gene is adsorbed to the chitosan nanoparticle by electrostatic forces; The potential of adsorbing DNA on nanoparticles was analyzed by agarose gel electrophoresis and spectrophotometer.To check its protection ability to DNA by DNase I digestive experiment. To transfect it to HepG-2 cell and evaluate the possibility and efficiency of chitosan nanoparticle as a vector in gene transference compared to cationic liposomes by fluorescence microscope.Results:1. Chitosan nanoparticles is showed to be spherical particles distributed evenly,the least nanoparticles is 36nm in diameter, d(0.5)is95nm,by scanning electron microscope and laser atomic examining apparatus.2.Agarose gel electrophoresis showed that the enhanced green fluorescent protein was adsorbed to the chitosan nanoparticle effectively.The encapsulation efficiency of nanoparticles -DNA mixed by different ratio(nanoparticle: plasmid) is 100% (50: 10), 100% (50: 25), 100% (50: 50), 92% (50: 75), 69% (50: 100) by the examining of fluorescence microscope.3. DNase I digestive experiment showed :there is no apparent DNA strip when we use no nanoparticles or nanoparticles -DNA accounting to the ratio of 5:50,while there is an apparent DNA strip when we use nanoparticles-DNA accounting to the ratio of 50:50,100:50. And it showed that chitosan nanoparticles can protect DNA well at such ratio.4. By fluorescence microscope,it was found that its transfection efficiency is higer than that of cationic liposomes when we use 30ul chitc san nanoparticle which has combined with pE-GFP-Clgene.The difference has statistical meaning.Conclusions : homogeneous and small in diameter chitosan nanoparticles has been prepared and loaded with plasmid effectively. It can protect the plasmid from been digested. HepG-2 cell is transfected successfully by the chitosan nanoparticles,and its transfection efficiency is higer than that of cationic liposomes.