Preparation Of PLGA Nanoparticles Targeting DEC-205 Eucommia Ulmoides Leaf Polysaccharide And Its Activation Of Dendritic Cells

Dendritic cells（DC）are specialized antigen-presenting cells with the unique ability to capture,process and present antigens to induce adaptive immune responses against viruses,pathogens and cancers.Plant polysaccharides have immunomodulatory and antiviral effects,and are good immune enhancers.Eucommia Ulmoides leaf polysaccharide（EULP）has the ability to promote dendritic cell maturation and enhance immune function.The efficiency of vaccination depends on the effective antigen presentation to T cells.By encapsulating the adjuvant and antigen in nanoparticles and then modifying the surface of nanoparticles,the antigen capture ability of antigen-presenting cells（APC）can be improved to activate T cells and enhance the immune function.DEC-205 receptor-mediated targeting of dendritic cells nanoparticles as a novel drug delivery system allows antigen and adjuvant to act simultaneously on dendritic cells,and the slow-release effect of nanoparticles can stimulate a strong and sustained immune response,providing a reference for the construction of novel vaccines with high efficiency and sustained efficacy.1.The preparation and optimization of EULP-PLGA nanoparticles were investigated.EULP-PLGA was prepared by the solvent volatilization method of double water-oil-water emulsion（W₁/O/W₂）.The optimal conditions were screened by orthogonal method analysis.Laser particle size analyzer was used for characterization.Study on drug release in vitro by dialysis.The results showed that the optimal preparation scheme was the internal aqueous phase to oil phase volume ratio of 1:7,PLGA concentration of 30 mg/m L,PVA concentration of 2%,and colostrum to external aqueous phase volume ratio of 1:10.Under these conditions,the encapsulation rate reached 65.08%drug loading 5.497%,The particle size was 269.8±11.6 nm,homogeneous dispersion and good stability.the PLGA followed a biphasic release behavior with a significant slow release effect through the combined effect of both drug diffusion caused by polymer surface hydrolysis and skeletal dissolution.2.The preparation and evaluation of anti-DEC-205m Ab-modified PLGA nanoparticles were explored.OVA was wrapped into PLGA nanoparticles with EULP by a two-water oil-water emulsion（W₁/O/W₂）solvent volatilization method.The anti-DEC-205m Ab was modified onto the nanoparticles by BS3.Infrared spectroscopy was used to determine the attachment of the bifunctional cross-linker.The modification of anti-DEC-205m Ab was qualitatively analyzed by fluorescence microscopy and quantitatively analyzed by BCA protein assay.Transmission electron microscopy and scanning electron microscopy were used to observe the nanoparticle morphology.The results showed that the encapsulation rate of nanoparticles OVA reached more than 80%and that of EULP was 64.66%.FTIR results showed that the cross-linker was inserted into the nanoparticle surface by non-covalent insertion.Fluorescence microscopy results showed that anti-DEC-205m Ab was successfully modified on the surface of the nanoparticles with an antibody attachment efficiency of 84.5±0.3%.The particle size was 291.0±12.8 nm.the nanoparticles were spherical with uniform size under transmission electron microscopy and scanning electron microscopy.3.The targeting and activation effects of EULP-OVA-PLGA-a DEC-205m Ab nanoparticles on dendritic cells were investigated.Dendritic cells were isolated from mouse bone marrow cells and cultured in vitro.The toxicity of nanoparticles on cells and the effect of dendritic cells on promoting lymphocyte value-added were examined by CCK-8 assay.The ability of nanoparticles to target dendritic cells was assessed by flow cytometry.Laser confocal microscopy to observe the antigen-presenting ability of nanoparticles.Flow cytometry to detect the effect of nanoparticles on the phenotypic expression of dendritic cells.The results showed that EULP-OVA-PLGA-a DEC-205m Ab was almost non-toxic to DCs at 150μg/m L and increased DCs-stimulated lymphocyte proliferation.Flow cytometry results showed that the nanoparticles modified by anti-DEC-205m Ab had higher affinity for DCs.EULP-OVA-PLGA-a DEC-205m Ab stimulated dendritic cells showed significant upregulation of CD80,CD86,MHC-II molecules and increased levels of IL-12,IL-6,TNF-αimmunomodulators in cell supernatants.EULP-OVA-PLGA-a DEC-205m Ab effectively induced dendritic cell maturation and activation.In summary,PLGA nanoparticles accurately targeting to the DEC-205 receptor of dendritic cells and loaded with antigen（OVA）and adjuvant（EULP）were successfully prepared as a novel drug delivery system in this experiment.The optimally prepared EULP-OVA-PLGA-a DEC-205m Ab nanoparticles showed a high encapsulation rate for both EULP and the model antigen OVA.anti-DEC-205m Ab was successfully modified on the nanoparticles.The nanoparticles were effectively taken up by dendritic cells,increasing the delivery of antigen（OVA）and adjuvant（EULP）to DCs,upregulating the expression of stimulatory molecules on the surface of DCs,promoting the secretion of cytokines,and facilitating the maturation and activation of DCs.In this study,a carrier was designed to simultaneously deliver antigen and adjuvant to dendritic cells,which provides a basis for the development of a novel drug delivery system.