Preparation Of FA-CS/rAAV Nanoparticles And Study Of Their Targeting Effect To Tumor Cell

Gene therapy is referred to put the purposed genes into target cells, to correct genetic defects or have the function of treatment. After decades of research, gene therapy shows its broad prospect of application. In terms of gene delivery system, Viral vector is convenient to carry medicine, also has obvious advantages of high efficiency expression for a long time. While Non viral vector has its unique advantages of low immunogenicity and high biocompatibility. Combined with the characteristics of viral vector and non viral vector, to develop more excellent gene carrier. Compared with other viral vectors, AAV vectors have the advantage of no pathogenic to human, host range, low pathogenicity and has good thermal stability, resistance to acid, alkali and organic solvent resistance and so on, which lead it to one of the most promising gene carrier at present. But lack of target to tumor cells, and the neutralizing antibodies in the blood and phagocytes have higher recognition and consuming ability to AAV. Chitosan has lower immunogenicity and good biocompatibility. Folic acid can be combined with folate receptor specificity, using folic acid receptors with high expression in most tumor cells and low expression in normal cells to target tumor cells. Chitosan parcel adeno-associated virus to improve its targeting property, reduce its immunogenicity and carrying capacity, providing a new thought and basic research for Gene therapy of tumor.This subject adopts the water-soluble Chitosan as a skeleton, Polyethylene glycol(PEG) and folic acid modified water soluble Chitosan, using Ionic crosslinking method to prepare the nanoparticles which package the virus, and regard egfp gene as report gene, research the expression in cells of carrying the gene carrier. This subject includes the following content:1. Purified Chitosan, synthesis different grafting rate of Chitosan- Folic, acid-Polyethylene glycol(PEG)-Chitosan in aqueous solution, testing its purity through GPC, Infrared spectrum and Nuclear magnetic to confirm the compound structure.2. Optimize the conditions of preparation of nanoparticles: in different buffer; different quality ratio of Chitosan and TPP; different rAAV amount3. Regard egfp gene as report gene, transfect cells, comparing the targeting function of nanoparticles with different folic acid content on A549 and KB cells.Conclusion: using G-25 to pure Chitosan to have the Chitosan with high deacelation degree and good water soluble, the molecular weight is between 3 kd-5 kd, synthetic FA-PEG-CS, FA-CS, PEG-CS, and identify the compound structure. Optimize the conditions of preparation of nanoparticles: 0.5 mg/mL chitosan,0.5 mg/mL Sodium phosphate polymer, quality ratio is 6:1, has uniform sizes and minimum radius, the average particle size is 220 nm. using Chitosan derivatives to package rAAV effectively, also to maintain the activity of the virus in partial acid environment. Virus nanoparticles can be highly expressed effectively in Folate receptor positive cells KB, also the express efficiency is higher than rAAV, while the express efficiency lower when in A549 cells. FA-CS/rAAV can target effectively to folate receptors high expression cell. In the folate receptor positive cells, compared with rAAV, FA-PEG-CS/rAAV can improve transfection efficiency of KB cells(folate receptor positive cells).