| Oral vaccine delivery antigen through the gastrointestinal mucosa, produce immune response similar to that of the conventional injection through the digestive tract immune response pathways. Since the immunogenic substances, such as protein, nucleotide drugs, are unstable. After entering the digestive tract, they are vulnerable to badly enzymes and pH environment in the digestive tract, resulting in a large number of degradation. Secondly, the macromolecular drugs are difficult to break through the epithelial barrier of digestive tract into the submucosa, interact with immune response pathways. Thus the gastrointestinal mucous immune response to the antigen is weak and the quantity of antigen for oral immune is large, which limited the application of oral vaccine. With the development of medical and molecular biology technology, it has been concerned that the polymer nanocarriers can effectively protect the macromolecular drug from degradation, extend the residence time in the digestive tract mucosa, overcome the gastrointestinal epithelial barrier, improve the interaction between antigen and immune response pathways, and can effectively induce the body's immune response.In this research ovalbumin(OVA) with immunogenicity was used as a model drug. The penetrating peptide TAT based polymer drug carriers, i.e. TAT modified poly lactide glycolide-?-poly glutamic acid nanoparticles(TAT PLGA-?-PGA NPs), were designed and prepared. The vaccine delivery system using the cell penetration function of TAT to overcome the mucosal epithelial barrier, induced body's immune response, which povided the valuable reference for the design of oral vaccine delivery system.In the first chapter of this research, the polymeric nanoparticles were prepared and characterized. Firstly, the PLGA-COOH and ?-PGA were connected covalently by 2,2'-(Ethylenedioxy)bis(ethylamine)(EbE) to synthesize the copolymer PLGA-?-PGA. Characterized by 1H NMR spectra, the copolymer PLGA-?-PGA was synthesized successfully. Secondly, the copolymer PLGA-?-PGA was connected with TAT covalently to synthesize the TAT modified PLGA-?-PGA. The OVA loaded PLGA-?-PGA NPs and TAT PLGA-?-PGA NPs were prepared by W/O/W double emulsion-solvent evaporation method. The obtained OVA loaded PLGA-?-PGA NPs and TAT PLGA-?-PGA NPs showed particle size of( 279.4±14.4) nm and(262.1±9.6)nm respectively with uniform particle size distribution. The zeta potential were(-27.9±2.6) m V and( 20.0±1.8) m V. The drug loading were(6.43±0.26)% and(4.54±0.83)%. The Entrapping Efficiency were(68.7±3.0)% and(47.7±9.1)%. The SEM morphology of the polymer nanoparticles were both spheroidal with porous structure, and distributed uniformly. The drug release experiment results showed polymer nanoparticles significantly slow down the OVA release rate, and the drug release behavior of TAT PLGA-?-PGA NPs was more slowly than PLGA-?-PGA NPs.In the second chapter, Caco-2 cell used as the cell model, we studied the mechanism of cellular uptake and transport of the polymer nanoparticles via fluorescent probe. The result of cellular uptake showed the intracellular fluorescence intensity of TAT PLGA-?-PGA NPs was higher than PLGA-?-PGA NPs, increasing with the quantity of TAT modification. It indicated that TAT can significantly improve the cellular uptake ability of PLGA-?-PGA NPs. To study the cellular uptake mechanism of polymer nanoparticles, the cellular uptake inhibition study was carried out. The results showed that TAT PLGA-?-PGA NPs interacted with cell surface heparan sulfate receptors by TAT, taked into cell through the clathrin and caveolin/lipid rafts mediated endocytosi, not the micropinocytosis. The result of intracellular transport pathway study showed that the TAT PLGA-?-PGA NPs transport pass the endoplasmic reticulum and golgi apparatus after uptake into cell, not lysosomes pathway. The Caco-2 cell monolayer cultured for 21 days was used as the model to study the transmembrane transport capacity of polymer nanoparticles. After transmembrane transport for 2 hours, the cell monolayer was scanned for 3D image along the Z-axis under confocal microscope. The polymer nanoparticles all distributed in the basolateral of cell monolayer, and the transported TAT PLGA-?-PGA NPs were significantly more than PLGA-?-PGA NPs. At the same time, we found amounts of TAT PLGA-?-PGA NPs across the cell monolayer and accumulated in the transwell polycarbonate membrane channels, but a few of PLGA-?-PGA NPs. It demonstrated the epithelial barrier transport capacity of TAT PLGA-?-PGA NPs was better than PLGA-?-PGA NPs.In the third chapter, the female Balb/c mice were used as animal model to evaluate the capacity of polymer nanoparticles overcome the intestinal epithelial barrier. The DiO and DiI co-labeled polymer nanoparticles were gavaged to mice, after 2 hours the duodenum, jejunum and ileum were cut into slices to observe the uptake and distribution of polymer nanoparticles in the intestine under confocal microscope. The result showed that the PLGA-?-PGA NPs mainly distributed in the intestinal lumen side, few uptake into intestinal villi. However, amounts of TAT PLGA-?-PGA NPs attached on the intestinal surface, many absorbed into intestinal villi and distributed in lamina propria. The amount of TAT PLGA-?-PGA NPs absorbed into intestinal villi was 4-5 folds more than PLGA-?-PGA NPs. While we found polymer nanoparticles were absorbed into intestinal peyper's patch. The TAT PLGA-?-PGA NPs were absorbed 1.2 folds more than PLGA-?-PGA NPs in intestinal peyper's patch. The animal study result indicated that the TAT PLGA-?-PGA NPs can across the intestinal epithelial barrier efficiently and entry into the lamina propria and lymphatic system, enhance the association between polymer nanoparticles and gastrointestinal immune system.In the forth chapter, the female Balb/c mice were used as animal model to evaluate the immune response of ovalbumin(OVA) loaded carriers in digestive tract. The level of ovalbumin specific secretory IgA(OVA-s IgA) produced by mucosal immune in digestive tract and ovalbumin specificIgG(OVA-sIgG) in blood were measured. The results shown the polymer nanoparticles can deliver vaccines effectively via oral, induced systemic and mucosal immune response in mice. The quantity of OVA-sIgG and OVA-sIgA induced by OVA TAT PLGA-?-PGA NPs were higher than other oral groups. It indicated that TAT PLGA-?-PGA NPs we designed and builded that increased the oral vaccine delivery effectively, improved the ability of systemic and mucosal immune response in mice. It provided valuable reference for the design of oral vaccine carriers.Above all, OVA TAT PLGA-?-PGA NPs could efficiently across the intestinal epithelial barrier via the cell penetrating function of TAT, significantly improved the polymer nanoparticles interaction with immune cells in lamina propria and lymphatic system, and improved the systemic and mucosal immune response of OVA. |
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