吸入式核壳微粒结核疫苗的制备及其免疫应答水平初探OA

OBJECTIVE To prepare an inhalable core shell microparticle tuberculosis vaccine and evaluate its immunogenicity.METHODS ① Preparation and characterization:The core-shell microparticle vaccine ANBM-MP encapsulating the ANBM antigen was constructed via double emul-sion-solvent evaporation,self-healing,and layer-by-layer self-assembly techniques.Microparticles were morphologically characterized by scanning electron microscopy,particle size was measured by laser diffraction changes in porosity were determined by mercury intrusion porosimetry,antigen distribu-tion was examined by confocal laser scanning microscopy,encapsulation efficiency and loading capacity were quantified by ultraviolet spectrophotometry,and aerodynamic performance was evaluated using a next-generation cascade impactor(NGI).② The in vivo release study:ANBM was labeled with indocya-nine green(ICG-ANBM).Male C57 mice were assigned to either an ICG-ANBM group or an ICG-ANBM-MP group,each receiving intratracheal administration of 10 μg ANBM antigen.Tissue distribution in the heart,liver,spleen,lung,and kidney as well as the alterations in pulmonary fluorescence intensity were monitored using a small animal imaging system on days 0,1,4,7,14,21,and 28.Pulmonary fluores-cence distribution was examined by photoacoustic imaging on day 4.③ Cellular uptake:Murine alveolar macrophage MH-S cells and dendritic cell DC2.4 cells were used for uptake assays.Cells were incu-bated with FITC-ANBM or FITC-ANBM-MP(6 μg antigen,4 h).In parallel,the PLGA skeleton was labeled with Nile Red,cells were incubated with 0.5 μL of Nile Red-microparticles for 4 h.Uptake was visualized by confocal microscopy and quantified by flow cytometry.④ Immune response evaluation:Male C57 mice were divided into a negative control group(saline),a blank microparticle group(unloaded microparticles,intratracheal),and an ANBM-MP group(10 μg ANBM,intratracheal).On days 5 and 14,expressions of CD45,CD64,CD24,MHC Ⅱ,CD11b,CD11c,CD86,and CD206 in lung tissues were analyzed by flow cytometry.At weeks 2,4,and 6,serum samples were collected and antigen-specific IgG,IgG1,and IgG2a titers were measured by ELISA.RESULTS ① The inhalable core-shell microparticle vaccine ANBM-MP was constructed,with a median particle size of 5.954 μm.Pores were distributed in the 1-10 μm range and markedly reduced after self-healing,with antigens predominantly localized with-in the pores.Encapsulation efficiency and loading capacity were(63.04±2.56)%and(0.50±0.064)%,respectively.Aerodynamic performance was favorable,with a mass median aerodynamic diameter(MMAD)of(6.843±0.127)μm,a fine particle fraction(FPF)of(29.182±1.209)%,and a geometric standard deviation(GSD)of 1.836±0.019.② ANBM-MP was rapidly released from day 4 to day 14,followed by sustained release through day 28,with good lung targeting.③ ANBM-MP significantly enhanced antigen uptake by APCs,and intact microparticles were also visibly internal-ized by cells.④ Compared with the negative control,the ANBM-MP group showed increased macro-phage recruitment,a significantly higher proportion of M1-polarized macrophages,and sustained high IgG titers over 6 weeks,with IgG2a as the predominant subtype.CONCLUSION An inhalable core-shell microparticle tuberculosis vaccine with favorable aerodynamic properties is developed,which can promote APC-mediated antigen uptake and elicits robust humoral and cellular immune responses.