Mechanism And Application Of Regulating Of CD40 And Lkb1 SiRNA By Using Biodegradable Drug Delivery System To Promote Immune Tolerance

Background and Objective: Autoimmune disease is a chronic inflammatory disease with high morbidity and disability rate.The current treatment for autoimmune disease involves the administration of broad-spectrum,nonspecific anti-inflammatory,or immunosuppressive drugs,which interfere with the normal immune environment of the body and increase the risk of severe infection and malignant tumor.Cellular immunomodulatory therapy has obvious advantages such as precise targeting and small side effects.Dendritic cells(DCs)are core entities between mediating central and peripheral immune tolerance mechanisms,therefore they are ideal targets for immunotherapy.At present,DCs can be regulated by a variety of regulatory means to induce immune tolerance.T cell immune response initiated by DCs is the basis of peripheral immune tolerance.Interference with the costimulatory signaling pathway of DCs and T cells interaction can induce immune tolerance.In addition to interfering with DCs costimulatory signaling pathways,regulating DCs metabolism can also control the direction of DCs differentiation.It is well known that CD40/CD40 ligand(CD154)pathway is a key costimulatory signaling pathway on DCs surface.Disturbing CD40 expression on DCs could inhibit the activation of DCs and T cells and induce the differentiation of T cells toward Treg,which has been shown to play a key role in inhibiting autoimmune diseases and allograft rejection.This immune process is also regulated by the level of metabolic and metabolites in DCs.DCs are developed towards immune tolerance when catabolic activity is dominant in DCs.On the contrary,when the DCs metabolism level elevated,DCs become activated to enhanced cellular immune activity,thus anti-tumor,anti-inflammatory and other abilities are improved.Therefore,interfering with the level of nutrient metabolism in cells will greatly affect the immune state of cells.Lkb1 is a key regulator for cell metabolism,growth and development.When Lkb1 gene is knocked out in DCs,glycolysis level is increased,cell lipid metabolism is vigorous,and extracellular acidification rate is increased.At the same time,DCs regulate the differentiation of T cells in the inhibitory direction,inducing the body to produce immune tolerant environment for the treatment of autoimmune diseases and asthma.Regulating the expression of CD40 and Lkb1 in DCs to regulate its immune function in vivo,has become an urgent problem to be solved.One possible solution is RNA interfere(RNAi),which plays a regulatory role by specifically silencing m RNA expression in host cells.Although RNAi therapy is highly targeted and accurate,it is difficult for a siRNA to be internalized to the target cells directly because of its large molecule weight,electronegativity and vulnerability to enzymes in the body.Thus,it must be assisted by external carriers to enter cells.With the development of material science,nano drug delivery system has become an ideal carrier,which can carry immunomodulatory substances targeting antigen presenting cells and induce immune tolerance.They have good prospect for immunotherapy toward autoimmune diseases,allergies and transplant rejection.Therefore,this work intends to develop a biodegradable nano drug delivery system for the co-delivery of siCD40 and siLkb1,and explore the synergic effect of the co-delivery of CD40 and Lkb1 expression on the two RNA-silenced DCs on the induction of immune tolerance.Methods:(1)Nanoparticles loaded with siRNA were fabricated using the w/o/w double emulsion solvent evaporation method.(2)The size and surface ?-potential of particles(NPs)were obtained by using dynamic light scattering(DLS),and the morphology of nanoparticles were observed by TEM.(3)Cellular uptake of NPs in vitro and in vivo,and the silencing capability of NPs encapsulated with siCD40 and siLkb1 were investigated.(4)The effects of co-delivery of siCD40 and siLkb1 to immune system were detected.(5)EAE mouse model was established.(6)Detect the effects of siCD40 and siLkb1 delivered by drug delivery system in EAE curing,and explore the possible mechanisms.Results: 1.The nanocarriers were prepared by w/o/w double emulsion method,and size was about 112 nm and the zeta potential was about 29.5 m V,the encapsulation efficiency was above 80%.2.The nanocarriers can deliver siCD40 and siLkb1 to DCs in vitro and silence the m RNA expression.3.The nanoparticles can deliver siCD40 and siLkb1 to splenocyte phagocytes in vivo.4.The nanocarriers were administered to healthy mice through the tail vein.The results showed that codelivery of siCD40 and siLkb1 could reduce the expression of MHC-II molecules on DCs,decrease the percentage of CD4+T cells and improve the number of Tregs.5.NP/siCD40+siLkb1 could delay the onset of EAE and reduce the severity of the disease.The data showed that NP/siCD40+siLkb1 inhibited the differentiation of CD4+ Th1 and Th17 cells by reducing the expression of MHC-II molecule on DCs.Conclusion: In this study,siCD40 and siLkb1 encapsulated nanoparticles were prepared.Such delivery systems could co-deliver siCD40 and siLkb1 to DCs in vivo and in vitro,and silence the expression CD40 and Lkb1.Codelivery of siCD40 and siLkb1 can induce immune tolerance,delay the onset of EAE,and reduce the severity of disease,inflammatory cell infiltration and tissue damage in spinal cord.Furthermore,NP/siCD40+Lkb1 interfered with the naive T cells differentiating into inflammatory Th1 and Th17 cells by decreasing the expression of MHC-II molecules on DCs and induced immune tolerance to autoimmune reactions.