Application Of Dextran-methotrexate Conjugates In Rheumatoid Arthritis Treatment

Rheumatoid arthritis(RA) is a kind of chronic autoimmune inflammatory disease,which is characterized by synovium hypertrophy and articular cartilage destruction. It leads to the decrease of joint function, or even dysfunction in the affected joints, and possesses high incidence of disability and deformity in the late stage of disease. Now,the therapeutic strategy of RA is mainly composed of surgical and drug treatment.Surgical therapy including arthroscopy and arthroplasty can alleviate the symptoms and increase the life quality through removal of abnormal synovium and remodeling the joint. However, RA is a systemic disease along with symmetrical polyarticular involvement, which limits the application of surgical treatment. The therapeutic agents for RA mainly divide into four categories, including disease-modifying antirheumatic drugs(DMARDs), glucocorticoids(GCs), nonsteroidal anti-inflammatory drugs(NSAIDs) and biological agents. In spite of providing remarkable inhibition of inflammation and remission of symptoms, side effects and tolerance by the long-time administration of drugs impose restrictions on the clinic application.With the development of nanocarriers, the limitation of some drugs in clinic is overcome. Nanocarriers possesses several advantages, such as the passive and active targeting delivery properties, controlled release of the drugs, prolonged pharmacokinetics, decrease of drug concentration in unwanted areas, and reduction of side effects in off-targeted sites. Therefore, exploration of drug delivery system becomes hotspot research area of RA targeted therapy, which can increase the concentration of drugs in the lesion sites, prolong the pharmacokinetics and reduce the side effects.Activated macrophages play an important role in pathogenesis of RA. It is widely shared that the activated macrophages, as the main source of various inflammatory mediators, are the key effectors in the development of RA. There is a definitely obvious correlation between the recruitment of multiple potent mediators,including pro-inflammatory cytokines(e.g., tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β), and interleukin-6(IL-6)), chemokines, and metalloproteinases,and so on, and the progress level of disease. Moreover, metabolism of cartilage and bone is involved by upregulation of many pro-inflammatory cytokines and proteinase.On the activated macrophages membrane, many receptors are overexpressed, such as scavenger receptor(SR), folate receptor, and integrin. The expression of these receptors is a little or none on the macrophages in normal tissues, but abundant under the inflammatory conditions. Consequently, the activated macrophages become ideal targets in RA targeted therapy.The objective of this work is discussion of inhibition effect of inflammation,protection mechanism of articular cartilage and evaluation of the potential and advantage of activated macrophages-targeted conjugates in RA treatment.Two activated macrophages-targeted conjugates Dex-g-MTX/FA and DS-g-MTX was prepared, in which FA could specifically recognized the FR and DS could bind to SR with high affinity, and non-targeted conjugate Dex-g-MTX as control. In this work, the three conjugates were prepared by a one-step condensation reaction, and the chemical construction was determined by proton nuclear magnetic(1H NMR) and Fourier-transform infrared(FT-IR). In aqueous environment, the three conjugates self-assembled into micelle, and the hydrodynamic diameters(Dhs) tested by dynamic laser scattering(DLS) were around 100 nm, which was suitable for passive target strategy. The critical micelle concentrations(CMCs) were evaluated,and the results illustrated the CMCs of the three conjugates were far low than the therapeutic concentrations. In the drug release assay, the controlled release of MTX from the three conjugates was demonstrated. Then, the cellular uptakes of the three conjugates were demonstrated by confocal laser scanning microscope(CLSM) and flow cytometry(FCM). The results indicated that the cellular uptakes of Dex-g-MTX/FA and DS-g-MTX towards activated macrophages were significantly increased by high affinity of FA and DS to FR and SR, respectively. In MTT assays,the cytotoxicity of Dex-g-MTX/FA and DS-g-MTX towards the activated macrophages was higher than that of Dex-g-MTX, the results was further indicatedthat the cellular uptakes of Dex-g-MTX/FA and DS-g-MTX towards activated macrophages was increased by FR and SR, respectively, and accordingly enhance the cytotoxicity. At last, the therapeutic efficacy of Dex-g-MTX/FA and DS-g-MTX was evaluated in CIA model, respectively. The results were suggested that more significant remission of arthritic symptoms, inhibition of inflammation and synovium hypertrophy were respectively demonstrated in Dex-g-MTX/FA and DS-g-MTX group compared with non-targeted conjugate Dex-g-MTX. In addition, the more remarkable protection of joints and cartilage against destruction and inhibition of systemic inflammatory level were respectively demonstrated by more powerful expression suppression of pro-inflammatory cytokines in the in Dex-g-MTX/FA and DS-g-MTX group.In this research, two active targeted conjugates Dex-g-MTX/FA and DS-g-MTX were synthesized by chemical conjugation. The two conjugates possessed suitable size, favorable biocompatibility, and controlled release of MTX. However, it was confirmed that the targetablility and therapeutic efficacy of DS-g-MTX was superior to that of Dex-g-MTX/FA. Most importantly, the significant enhancement of anti-inflammation efficacy and protection of articular cartilage were gained by active targeted strategy to activated macrophages, which might possess a great potential in targeted therapy RA or other chronic inflammatory diseases.