| Polylactic acid-hydroxyacetic acid polymer（PLGA）is a copolymer of non-functional side groups polymerized by lactic acid and hydroxyacetic acid,because of its controlled release,degradability,biocompatibility and non-toxicity characteristics are often used as drug carriers,has been widely used in targeted drug delivery systems.Nanoparticles（NPs）are skeleton entities composed of polymer materials,and drugs can be modified on the surface by chemical coupling or physical adsorption,or the drugs are wrapped inside the skeleton by special preparation methods.Eucommia polysaccharide is a traditional Chinese medicine polysaccharide,which has good anti-tumor,hypoglycemic,immune enhancement and other functions.Dendritic cells（DCs）are a type of antigen-presenting cell that is responsible for receiving and processing antigens and passing them on to other immune cells.Mannose receptors are abundantly expressed as important pattern recognition receptors on the surface of dendritic cells.In this experiment,Eucommia polysaccharides and pattern antigen ovalbumin OVA were encapsulated in PLGA by taking advantage of the good biocompatibility and sustained release of PLGA nanoparticles,and the mannose ligands were modified on the surface of Eucommia polysaccharide PLGA nanoparticles（MN-EOPP/OVA）,and their immune activity in vitro was explored,laying the foundation for the application of PLGA nanoparticles to clinical immune adjuvants.The ethical methods involved and the results of the experiment are as follows:1.Preparation and optimization of Eucommia polysaccharide PLGA nanoparticles.Eucommia polysaccharide PLGA nanoparticles were prepared by reemulsion solvent volatilization method,and the PLGA nanoparticles encapsulating Eucommia polysaccharides and free Eucommia polysaccharides were separated by high-speed centrifugation,and the encapsulation rate and drug loading capacity of Eucommia polysaccharide PLGA nanoparticles were calculated.Based on the univariate results,the optimal preparation conditions of EOPP were determined by BBD response surface optimization method,the mass concentration of PLGA was 34%,the concentration of EOP was 50mg/m L,the volume ratio of the outer aqueous phase to colostrum was 11:1,the encapsulation rate of the final EOPP was 44.89±1.76%,and the drug load was7.49±0.21%,and the final models obtained under this condition had high statistical significance（P<0.001）,the correlation R2=0.9507 for encapsulation rate,RAdj2=0.8872for correction,R2=0.9752 for drug load,and RAdj2=0.9434 for the correction coefficient,indicated that the fit of the model was good.2.Preparation and characterization of mannose Eucommia polysaccharide PLGA nanoparticles.This chapter experiment couples mannose to EOPP by covalent modification and evaluates its characterization by ultraviolet spectrometer,laser particle size analyzer,scanning electron microscopy,transmission electron microscopy,and in vitro release assay.The results showed that the UV spectrogram of MN-EOPP had a characteristic absorption peak of mannose,and the coupling rate of mannose calculated by UV spectrometer could reach 24.33±1.78%,and MN-EOPP was uniformly spherical,with a particle size of 159.64±4.9 nm,an electric potential of-47.6±1.6 m V,and the absolute potential value was higher than that of EOPP’s-29.3±1.4 m V,which also showed that the nanoparticles had higher stability after coupling mannose.In vitro release tests have found that EOPP have good sustained-release effects,and the drug is mainly Fick diffusion.3.Effect of mannose Eucommia polysaccharide PLGA nanoparticles（MN-EOPP）on immune activity of splenocytes.In this chapter,mouse splenocytes were selected for in vitro experiments,the cytotoxicity of MN-EOPP on splenocytes was evaluated by CCK-8 method,the maximum safe concentration of EOPP and MN-EOPP and the following two concentrations were selected to co-culture with splenocytes,the effect of MN-EOPP on the proliferation of splenocytes and（B,T）lymphocytes alone was observed,and the effect of MN-EOPP on the secretion level of splenocytes IL-6 and IFN-γwas explored.The results showed that under the action of 135μg/m L,MN-EOPP was not significantly toxic to splenocytes,and MN-EOPP could significantly promote the secretion of IL-6 and IFN-γby splenocytes in vitro.4.Effect of mannose Eucommia polysaccharide PLGA nanoparticles on immune function of dendritic cells.In order to investigate the function of MN-EOPP on the presentation of mouse bone marrow-derived dendritic cell antigens,we first prepared MN-EOPP/OVA encapsulated with patterned antigen OVA,characterized MN-EOPP/OVA and determined the encapsulation rate of OVA in MN-EOPP/OVA,and then explored the effect of MN-EOPP/OVA on the cytoskeleton of dendritic cells and the effect of dendritic cells on MN-EOPP/OVA by laser confocal scanning microscopy.The uptake capacity of the OVA was then evaluated,the secretion level of MN-EOPP/OVA acting on dendritic postcellular cytokines（IL-6,IL-12,IFN-γ,TNF-α）was evaluated,and the effect of MN-EOPP/OVA on gene expression in dendritic cells was studied by high-throughput sequencing.The results showed that the MN-EOPP/OVA particle size was 190.24±3.4 nm,and the encapsulation rate of OVA was as high as84.17%.The fluorescence intensity of FITC-OVA in the MN-EOPP/OVA group was significantly greater than that in the OVA,EOP/OVA and EOPP/OVA groups,indicating that dendritic cells have a strong ability to uptake and phagocytote MN-EOPP/OVA,and can significantly upregulate the secretion levels of IL-6,IL-12,IFN-γand TNF-αin dendritic cells,and promote dendritic cell activation and maturation.Finally,high-throughput sequencing dendritic cell gene profiling can find that after treating cells with MN-EOPP/OVA,2114 related genes were differentially expressed,of which 1108 were significantly upregulated and 1006 were significantly downregulated,and multiple immune-related pathways were activated,such as iron death signaling pathway,NF-kapa B signaling pathway,complement and coagulation cascade signaling pathway,etc.According to the review,the optimal conditions for the preparation of Eucommia polysaccharide PLGA nanoparticles are 34%mass concentration of PLGA,50mg/m L concentration of Eucommia polysaccharide,volume ratio of external aqueous phase to colostrum is 11:1,encapsulation rate is 44.89±1.76%,and drug load is 7.49±0.21%.On this basis,by covalently modifying mannose on the surface of nanoparticles,MN-EOPP can enhance the proliferation ability of splenocytes and enhance the immune activity of splenocytes;MN-EOPP/OVA can affect the cytoskeleton of BMDCs,strengthen their uptake capacity,and promote the activation and maturation of BMDCs.Finally,according to the high-throughput sequencing results,it is found that MN-EOPP/OVA can activate multiple immune-related pathways,and this study provides some theoretical basis for the development of a new PLGA vaccine adjuvant. |
