Nano-drug Delivery System Enhanced The Therapeutic Effects Of Experimental Autoimmune Encephalomyelitis By Promote The Expansion Of Antigen Specific Regulatory T Cells

Background and objective:Currently,immunosuppressive medications are always used for autoimmune diseases therapy.However,besides the cost,patients have suffered from serious side effects,such as infectious malignancies,complications,and metabolic disorders.The main cause of autoimmune diseases is the disrupted immune balance,resulting in activating the effector T cells that recognizing autoantigens.The therapeutic strategy that expansion Treg in vitro and infusion them to increase the Treg level in vivo has become a research hotspot for autoimmune diseases therapy.However,there are some limitations for Treg-based therapy,such as long culture period,low proliferation efficiency,and reduced cell stability and function.Therefore,successful expansion Treg cells,especially antigen-specific Treg cells,has become the main problem to improve the therapeutic effect of autoimmune diseases.Tolerant dendritic cells(DCs)present antigens to the TCR of T cells through major histocompatibility complex(MHC)molecules on the surface.In addition,low expression of costimulatory molecules on DC’s surface cannot effectively bind to receptors on the surface of T cells,resulting in imbalance between Th1 and Th2 cells,inhibiting Th17 cell differentiation,and promoting Treg cell proliferation.Recent studies show that 1α,25-hydroxyvitamin D3(VD3)can effectively induce tolerogenic DCs in vitro.However,as a hydrophobic small molecular drug,it is difficult to achieve the optimal concentration for VD3 to induce tolerogenic DCs and Treg cell proliferation in vivo.In addition,whether combination of VD3 and autoantigens can promote the expansion of antigen-specific Treg cells is still unclear.However,in order to effectively expand antigen-specific Treg cells,it is necessary to simultaneously deliver VD3 and antigen into DCs in vivo.The nano-drug delivery system(NDDS)can improve the solubility,prolong the circulation time,enhance targeted delivery,and reduce side effects of drugs.Hence,the VD3 and autoantigens co-loaded NDDS may provide a useful strategy to expand antigen-specific Treg in vivo.Methods:In this study,a double emulsion method was introduced to prepare a PEG-PLGA nano-drug delivery system carrying both VD3 and MOG peptides(named as NP/VD3/MOG).The particle size and zeta potential of the NP/VD3/MOG were analyzed by a dynamic light scattering.The mouse bone marrow-derived dendritic cells(BMDCs)were cultured with blank NP,NP/VD3,and NP/VD3/MOG for 24 h,and stimulated with 15μg/m L OVA for another 24 h.The expression levels of MHC-Ⅱ and other costimulatory molecules were assessed using flow cytometry.C57BL/6 mice received intradermal injection of NP/Di D were sacrificed,the draining lymph nodes were isolated.The distribution of Di D in the lymph nodes was evaluated by flow cytometry and confocal microscopy.B10.A5R-gfp-Foxp3 or C57BL/6 mice were intradermal injected with PBS,NP/VD3,NP/MOG,or NP/VD3/MOG.The MOGspecific Treg cells in draining lymph nodes were measured by flow cytometry.The C57BL/6 mice,which was induced experimental autoimmune encephalomyelitis,were intradermal injected with PBS,free VD3,free MOG,NP/VD3,or NP/VD3/MOG.The clinical scoring was done daily.At 17 th day after the first treatment,the spinal cords from mice were harvested and level of Treg were detected by flow cytometry.Results:1.NP/VD3/MOG was prepared by the double emulsion method,and the particle size was about 343 nm and the zeta potential was about-27.3 m V.2.NP/VD3 and NP/VD3/MOG can induce the tolerogenic DCs in vitro.3.Intradermal injected NP/DID accumulated in the draining lymph nodes,and mainly distributed in the dendritic cells.4.NP/VD3 and NP/VD3/MOG significantly increased the level of Treg cells in draining lymph nodes.5.NP/VD3/MOG significantly delayed the EAE development and inhibited the clinical scores in mice.Conclusion:In this study,a VD3 and MOG peptides co-loaded nano-drug delivery system was successfully prepared.NP/VD3/MOG significantly induce the tolerogenic DCs in vitro.NP/VD3/MOG obviously delivered VD3 and MOG peptide into DCs in draining lymph node after intradermal injection,resulting in the expansion of tolerogenic DCs and antigen-specific Treg cells.Importantly,NP/VD3/MOG significantly delayed the EAE development and inhibited the clinical scores in mice.The NP/VD3/MOG offers a potentially effective system for autoimmune diseases therapy.