| Background:Autoantigen-activated effector T cells,especially T helper 1(Th1)and T helper 17(Th17),are involved in the pathogenesis of autoimmune uveitis and other autoimmune diseases.In an overactivated immune microenvironment,programmed cell death protein 1(PD-1)could express on these effector T cells accompanied by deficiency of specific elimination signaling.Therefore,PD-1 continues to be highly expressed.Meanwhile,due to the significantly increased energy metabolism demand of hyperactive effector T cells,the energy metabolism manner will be transformed from conventional oxidative phosphorylation or fatty acid oxidation to pyruvate kinase M2(PKM2)mediated aerobic glycolysis pathway.Therefore,the present study proposed the hypothesis that the synergy of metabolic inhibition and PD-1 receptor guidance might target and down-regulate these hyperactive effector T cells to achieve anti-immune effects.Methods:Using polyethylene glycol(PEG)modified poly(lactic-co-glycolic acid)(PLGA)as nanoplatform matrix,TPP nanoparticles were prepared by double-emulsification method to encapsulate TEPP-46 and carbodiimide method to conjugate PD-1 antibody.The surface morphology and structure of TPP nanoparticles were observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The particle size distribution,polydispersion index and zeta potential were measured by Malvern laser particle size analyzer.According to Brunauer,Emmett&Teller(BET)theory,nitrogen adsorption and desorption test was carried out to analyze the surface pore structure.Fluorescence microscopy was used to observe the binding of PD-1 antibody to PLGA-PEG,and flow cytometry was used to quantify the binding rate.The absorption spectra of TEPP-46,PLGA-PEG and supernatant were detected by Nanophotometer 80.Histological analysis of paraffin sections was performed by hematoxylin eosin staining.Laser scanning confocal microscopy(LSCM)and fluorescence microscopy were used to observe the binding of cells to nanoparticles,and flow cytometry was used to detect the binding rate.Cell counting kit-8(CCK-8)assay and hemolysis test were used to study its biosafety in vitro,and blood cell analysis,blood biochemical analysis and histological analysis were used to evaluate its biosafety in vivo.Finally,the proportion of cells producing interleukin-2(IL-2)/interleukin-17A(IL-17A)/interferon-γ(IFN-γ)was determined by flow cytometry.Experimental autoimmune uveitis model was established using female C57BL/6J mice aged 4 to 6 weeks by interphotoreceptor retinoid-binding protein(IRBP)immunization.Results:TPP nanoparticles presented a spherical and solid structure with porous surface and a particle size of 218.7±13.9 nm,a zeta potential of-17.4±0.7m V,narrow polydispersion index and uniform distribution.The connection rate between PD-1 antibody and PLGA-PEG was 80.5%.TPP nanoparticles showed good stability in vitro and high level of biosafety in vitro and in vivo.Encapsulated TEPP-46 showed a sustained release profile with burst,steady and slow release periods.TPP nanoparticles showed significant targeting effect on activated PD-1~+lymphocytes in vitro and had no targeting effect on PD-1~-cells.TPP nanoparticles can effectively inhibit the early activation and proliferation of activated lymphocytes in vitro.TPP nanoparticles inhibited Th1 and Th17cell proportion in vivo and in vitro,and could significantly alleviate autoimmune responses in vivo.Conclusion:The prepared novel nanoplatform TPP had satisfied characteristics,high level of biosafety,effective targeting ability and sustained drug release profile.It could significantly inhibit the active PD-1~+lymphocytes in association with suppressed early activation and proliferation in vitro.And the differentiation or proliferation of Th1 and Th17 cells was significantly reduced in vitro and in vivo,which provided a significant anti-inflammatory effect on experimental autoimmune uveitis mouse model.TPP provided a novel approach to ameliorate autoimmune diseases mediated by these cells. |
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