The Effective Delivery Of Methotrexate In Autoimmune Diseases And Its Mechanism Of Precise Treatment

Autoimmune diseases（AIDs）are result from a dysfunction of the acquired immune system and caused by the immune response of the body to its antigens,resulting in cell and tissue damage.About 80 to 100 diseases have been identified as autoimmune in nature,among these,rheumatoid arthritis and psoriasis are two of the most common and well-known AIDs.The factors that cause abnormal autoimmunity are complex and unknown etiologies.Most AIDs cannot be cured by effective drugs,and the main treatment strategy is to relieve the symptoms of the disease and balance the body’s autoimmune function.Currently treatments for AIDs include conventional immunosuppressive drugs（ISDs）,disease-modifying anti-rheumatic drugs（DMARDs）,and emerging biologic therapies.Nonetheless,there are still unmet medical needs as a lack of patient and/or disease responsiveness,the resistance to longterm treatment,concerns regarding high costs,and increased infection risks of these biologic agents in the treatment of AIDs.Methotrexate（MTX）is a folate analogue that can competitively inhibit dihydrofolate reductase,blocking the conversion of dihydrofolate to tetrahydrofolate,thus inhibiting DNA synthesis and mitosis.MTX has been in clinical use as anti-tumor agent since 1947,and its anti-inflammatory effects make it use for psoriasis（since 1960s）and rheumatoid arthritis（since 1980s）.The aim of using methotrexate for the treatment of autoimmune inflammatory diseases is to dampen the overactive immune cells without killing normal cells.Therefore,in order to avoid toxicity on normal cells,the doses used are much lower than the ones used for the treatment of neoplastic diseases.As a traditional first-line drug for rheumatoid arthritis and psoriasis,MTX can effectively inhibit the activation of immune cells and the abnormal proliferation of synovial fibroblasts or keratinocytes,but it still faces many challenges in clinical application,such as poor water solubility,more and serious adverse reactions,insufficient concentration at focal sites after systemic administration and poor percutaneous permeability.Therefore,to improve the local drugs concentration at the lesion site and reduce or avoid systemic adverse reactions,two of the most common AIDs（rheumatoid arthritis and psoriasis）were selected as examples to carry out the research work in this paper,and realized the precise and effective delivery of MTX and achieved good therapeutic effects.Experiment 1:In the study of rheumatoid arthritis,we synthesized a well water-soluble Au₂₅nanocluster using GSH as a template.In vitro,Au₂₅ nanoclusters can significantly modulate phenotypic polarization of a pro-inflammatory M1 phenotype to an anti-inflammatory phenotype M2 and reduce the expression of inflammatory cytokines in macrophages by restraining phosphorylated p65.In TNF-?-activated synovial fibroblasts model,Au₂₅ nanoclusters can effectively inhibit thioredoxin reductase（Trx R）and the phosphorylation of STAT3 similarly to monovalent gold compounds which result in the accumulation of reactive oxygen species（ROS）and the apoptosis of synovial fibroblasts.In vivo experiments,MTX was conjugated with Au₂₅ nanocluster by the hydrophobic interaction that can improve the water solubility.In the arthritis rat AA model induced by adjuvant,Au₂₅+MTX can effectively relieve the paw swelling and cartilage erosion,and there is no obvious systemic toxicity.Experiment 2:Topical administration is the preferred method for the treatment of skin diseases.Therefore,in the study of psoriasis,a sprayed NIR-responsive composite hydrogel with good photothermal effect and sol-gel-sol reversible phase transition characteristics was developed for MTX local delivery in the psoriatic epidermis.MTX can be encapsulated with formation of spherical micelles with hydrophilic outer layer and hydrophobic inner layer which self-assembled by the synthesized PDLLA-PEG-PDLLA triblock copolymer,resulting in not only improving the water solubility of MTX,but also increasing the uptake capacity of keratinocytes.In vitro experiments,the keratinocytes apoptosis induced by the PLEL@GNR+MTX hybrids hydrogel was due to the synergistic effect of MTX release and photothermal therapy.In vivo experiments,the drug delivery system based on PLEL@GNR+MTX hydrogel was particularly appealing for psoriasis with thickened stratum corneum,given that their double phase can be reversibly switch to improve skin penetration and retention time.In addition,via establishing Imiquimod（IMQ）-induced psoriatic dermatitis in mice,we demonstrated that PLEL@GNR+MTX hydrogel was effective in the prevention and treatment of IMQ-induced psoriatic dermatitis.What’s more,PLEL@GNR+MTX hydrogel achieved similar efficacy compared with the commercial betamethasone/carpotriol ointment,without obvious skin irritation and biological toxicity.