The Preparation And Analysis Of Nanoparticle-Encapsulated Small-Molecule TNF-α Inhibitor C87 Using In Vitro And In Vivo Models

Background:TNF-α is a 233 amino acid protein cytokine produced by activated macrophages,monocytes.It exerts a important roles in innate immunity and inflammation,It plays a key role in responding to infection and/or inflammation,and initiating immunity to pathogens.TNF-α is a major pro-inflammatory cytokine.Excessive production of TNF-α can cause rheumatoid arthritis,compulsive spondylitis,inflammatory bowel disease and other diseases.When TNF-α is produced,it is transported to the cell membrane and expressed as a membrane-associated trimer.Membrane TNF-α can then be cleaved by TNF-αconverting enzyme(TACE)and enter the circulation as a trimer.Both the membranebound form(mTNF-α)or the soluble form(sTNF-α)are active.There are two types of TNF-α receptors,TNF receptor 1(TNFR1)is a 55 kDa protein,and TNFR2 is a 75 kDa protein.TNFR1 is ubiquitous,and TNFR2 is restricted to certain cells(especially immune cells).TNFR1 can be activated by membrane form and soluble form of TNF-α.Most reports indicate that TNFR2 is only effectively activated by membrane form.TNF-α can regulate a variety of inflammatory and autoimmune processes.It is a pleiotropic cytokine involved in cell apoptosis,differentiation and cell recruitment.The combination of TNF-α and TNFR-1 plays an important role in activating and recruiting immune cells to cause inflammation.TNF-α activates the transcription pathway,induces oxidative stress,and then oxidative stress and inflammation influence each other to promote cell degradation.In most autoimmune inflammatory diseases,the overproduction of TNF-α may lead to the occurrence of the disease.When treated with TNF-α biological inhibitors,most patients have chronic inflammation and RA,ulcerative colitis,Crohn’s disease,psoriasis,psoriatic arthritis,ankylosing spondylitis,and juvenile idiopathic arthritis(JIA)Most of the disease symptoms have been improved.In the past few decades,various clinical trials have been conducted for these compounds,and these trials have been shown to improve the clinical conditions of RA patients.These TNFa antagonists,including infliximab,certolizumab,etanercept,golimumab,and adalimumab,have completely changed autoimmune diseases,especially rheumatic inflammatory diseases.Treatment programs.These drugs bind to TNFa dimers to form a complex,thereby blocking the binding of TNFα receptors,thereby preventing the activation of downstream pathways that induce inflammation and other signaling pathways.The drugs reported so far are proteins or antibodies with high molecular weights and are associated with various side effects,such as tuberculosis,congestive heart failure,lupus,demyelinating diseases,injection site reactions,autoantibody production,and systemic Side effects,as well as its high production cost,greatly reduce the clinical therapeutic effect of biological agents.Obviously,small molecule drugs with the efficacy of anti-TNF biologics can benefit more patients.Objective:1.To prepare and characterize nanoparticles loaded with small molecule TNF-αinhibitor C87,2.To evaluate their effects on inhibiting TNF-α activity in L929 cells3.To evaluate their effects on inhibiting TNF-α activity in Concanavalin A(Con A)induced autoimmune hepatitis mouse models.4.To evaluate the therapeutic effect of C87 on mice with rheumatoid arthritis.Methods:1.C87-loaded nanoparticles(C87 NP)with the feature of slow release and long work time,were prepared by nanoprecipitation method.The physicochemical properties and in vitro drug release profile of C87 NP were evaluated.2.The anti-TNF-α activity of C87 NP in L929 cells at different doses was implemented by MTT assay.3.C87 NP was administrated intravenously beforehand in Con A induced immunemediated hepatitis mouse model and the survival rate of mice was assessed.4.The cytokine le vels in the serum of mice were detected by LEGENDplexTM.5.The distribution,proportion and number of T cells and its subsets,and NK cells in liver and spleen were measured by flow cytometry.6.The mouse model of collagen-induced rheumatoid arthritis was established to observe the swelling degree of mouse paw,mouse arthritis severity score,Micro-CT analysis of mouse paw,pathological HE staining of mouse paw.Results:1.C87 NP had good stability with a relatively high loading content(LC%)of 34.4%and encapsulation efficiency(EE%)of 48.1%.The average particle size of C87 NP was 82.57 nm,and the polydispersity index was 0.115.C87 NP was electrically neutral and displayed spherical structure in transmission electron microscopy image.C87 NP slowly released C87 in a sustained manner with the duration for no less than 8 hours.2.In vitro study demonstrated that C87 NP could antagonize TNF-α in a concentration-dependent manner(IC50=9.13 μmol/L)in L929 cells.3.Animal experiment results showed that C87 NP increased the survival rate to 66.7%.Further biochemical and pathological analysis demonstrated that prophylactic C87 NP significantly reduced the level of serum ALT,AST and several cytokines(P<0.01)12 hours after ConA administration.Liver injury and infiltrated cells(CD4+T,CD8+T,NK cells,etc)in mouse liver and spleen(P<0.01)were reduced while the proportion of Treg cells in spleen was increased(P<0.05).4.C87 treatment significantly reduced the arthritis scores of collagen-induced arthritis in mice(P<0.05),and alleviated joint bone erosion and inflammatory cell infiltration in mice.arthritis disease models.Conclusion:1.Successfully prepared nano-drug C87 NP,which has the characteristics of sustained release and long-term effect.2.C87 NP has a good inhibitory effect on TNF-α cytotoxicity in vivo and in vitro,which lays the foundation for its clinical application in the future.3.C87 can effectively alleviate the severity of mouse arthritis disease models.