Study On DNA Vaccine Loaded Microspheres Drug Delivery System

DNA vaccines, also referred to as nucleic vaccines, are recombinant plasmid DNAencoding antigen which can express the antigen protein and elicit protective immuneresponse after inoculated in bodies. DNA vaccines are considered as the third generationvaccines which are based on the technology of gene therapy and transgene. However,DNA vaccines are new findings with many limitations which restrict its developmentspeed. Specifically, DNA vaccines tend to be relatively poorly immunogenic and havelow bioavailability , often requiring large doses to be effective in large animal tests andclinical trials. Besides the immunogenic characteristics of DNA vaccines, the major reasonof the poor immunogenicity is the low drug transport efficacy across the cell membrane.Because pDNA, composed by 2 to 10 kbp, with a molecular weight of millions, is veryhydrophilic and has a low o/w partition coefficient. Therefore, after intramusclarinnoculation with solution injection dosage of DNA vaccines, only very small amount ofthe pDNA can be transported into the professional antigen presenting cells such asphagocyte cells and dendritic cells. Most of the drug is degraded by DNA enzyme in thetissue liquid. Also, compared with oral delivery, injection delivery has the limitation ofinconvenience, higher cost, safety issue and tissue response. Oral immunization may elicitmucosal protective immune response which is very important for most infectious diseases.But DNA vaccine cannot be used orally without protection because it will be soondegraded in the gastric liquid and can not be absorbed across the intestinal mucus easily.How to increase the immunogenic effect and elicit mucosal immune response is theinternational focus of the DNA vaccines research. In order to improve the transport efficacy of DNA vaccines, increase the immuneresponse, reduce the dose, increase the choice of administration route and induce mucosalimmune response, this paper is designed to prepare DNA vaccine loaded microsphereswith the biodegradable material, sodium alginate and PLGA , as carriers andFoot-Mouth-Disease DNA vaccine as model drug. The formulation, preparationtechnology, in vitro release characteristics and its influence factors, physicochemicalcharacteristics, stability of DNA vaccine, the mechanism of immune response, immuneeffect in vivo, cytotoxicity and tissue irritation of the microspheres were evaluated. The emulsification-ion cross-linking method was used to prepare Foot-Mouth-Disease 4第二军医大学博士学位论文 DNA 疫苗微球给药系统的研究 英文摘要DNA vaccine loaded sodium alginate microspheres (DNA-SA-MS) to overcome thedifficulty in the traditional preparation method for sodium alginate microspheres. Thepreparation method is mild without organic solvent, so it is suitable for keeping theintegrity and biological stability of DNA vaccine. Uniform design is used to evaluate theeffects of the preparation condition and formulation on the mean diameter, drugentrapment efficacy. The statistical analysis showed that mean diameter was significantlyaffected by stirring rate and MC amount (P<0.01) .The drug entrapment efficacy wassignificantly affected by nominal drug content and stirring rate (P<0.01).The in vitrorelease test showed that DNA-SA-MS without chitosan cross-linking released fast andbecame slower after cross-linking with different extent of chitosan. The effects of the drugcontent and the kinds of release medium on the release rate were little. The electrophoresistest showed that integrity of DNA vaccine was well kept after the preparation andlyophilization process. For the purpose of industrialization, spray-drying method was used to prepareDNA-SA-MS. This method is simple, fast, without organic solvent residual and suitablefor industrial amplification. Orthogonal test was used to evaluate the effect of theformulation and process factors on the...