Investigation Of Biodegradable Materials As Controlled-release Gene Carriers

Biodegradable materials as controlled-released coatings of drugs and proteins or peptides have been widely reported. The biodegradable polymeric materials as gene carriers provided a controlled release of DNA. They could also protect the entrapped DNA from degradation due to nucleases and increase the efficiency of cellular DNA uptake. The prolonged availability could also lead to a prolong duration of gene expression. In this paper, we have described three kinds of synthesized biodegradable materials PLA, PELA and PLGAE including their synthesis, the factors influencing the loading efficiency, the release of microspheres in vitro, transfection and cytotoxicity. At the same time, we selected a constructed HCV core gene DNA vaccine by DNA recombination to induce immune response in the C57BL/6 mice immunized with the HCV core gene vaccine delivered by polymeric microspheres. According the currentinvestigation, the biodegradable polymeric microspheres showed their potential superiority. The investigation include two parts:The first part: To study the biodegradable polymers as gene controlled-released coatings for gene transfer. The poly-Dl-lactic (PLA), poly-Dl-lactic acid-polyethylene glycol) (PELA) and poly(lactic acid)-co-poly(ethylene glycol)-co-poly(glycolic acid) random copolymer (PLGAE) were synthesized in solvent evaporation-double emulsion method and prepared as the coatings of plasmid DNA in the transfection. All kinds of factors affecting the loading efficiency, cytotoxicity, transfection efficiency and the course of the degradation and release in vitro were discussed. The average diameters of microspheres of PLA and PELA were 1-3 u m and PLGAE microspheres was 0.72 u m . The loading efficiency of them was 32%, 62% and 70% respectively. The cytotoxicity of three kinds of delivery system for COS-1 cells was little. The transfection efficiency of PLA microspheres was higher than PLGAE and PELA microspheres and all of them could maintain gene expression for 96h. The PLGAE microspheres show more apparent controlled-release than others.The second part: The three kinds of biodegradable polymeric microspheres loading plasmid pCDNA3.1(+)HCV core as gene vaccines were used to vaccinate the C57BL/6 mice. 33 C57BL/6 male mice at the age of 6-8 weeks were divided into 11 groups and were vaccinated intramuscularly or orally respectively twice with PBS, plasmid pCDNA3.1(+), naked recombinant plasmid and the DNA/polymeric microspheres. Among them the DNA/PELA was designed vaccinated once with the above-mentioned ways. Serum samples were collected from mice at 3th, 5 th,7 ^,9th week after the first inoculation. The immune response of HCV core protein was detected by ELISA and MTT. The levels of antibody in mice vaccinated with all of the DNA/ microspheres were higher than the naked recombinant DNA and both the DNA/PLGAE and DNA/PELA showed similarapparent controlled-release in vivo. The oral experimental groups also showed a desirable result compared with the naked recombinant plasmid group. The stimulation index of spleencytes to ConA of mice vaccinated by plasmid pCDNA3.1(+)HCV core was significantly higher than that of mice vaccinated with plasmid pCDNA3.1(+) and PBS. These results suggested that the DNA vaccine encapsulated by biodegradable polymeric microspheres could induce spectific immune responses in C57BL/6 mice effectively.