Macrophage-Based Secondary Delivery System For Ferroptosis And Photothermal Synergistic Therapy Of Rheumatoid Arthritis

Rheumatoid arthritis（RA）is an autoimmune disease associated with chronic joint inflammation and cartilage destruction,which is characterized by infiltration of immune cells that trigger inflammatory process and proliferation of fibroblast-like synoviocytes（FLSs）that destruct the joints,ultimately leading to bone destruction,joint deformities,and eventually disability.Currently,the common treatment for RA is the combination of nonsteroidal anti-inflammatory drugs,cytotoxic drugs and immunosuppressants.However,due to the nonspecific targeting of these drugs,there is always the risk of severe side effects,such as osteoporosis,muscular atrophy,and impaired immune function,thus greatly limiting their therapeutic efficacy.Therefore,it is necessary to develop alternative methods to specifically kill inflammatory cells and proliferating FLSs at the site of inflammation in order to improve efficacy and reduce toxicity.It is reported that chemokines and cytokines secreted by the inflammatory site can recruit macrophages in large numbers,thus causing recruitment of macrophages to inflammatory sites,indicating that macrophages can be used as drug carriers to improve the targeting efficiency of drugs to RA sites.In view of this,we constructed sulfasalazine（SSZ）,ferroptosis inducers,loaded iron oxide nanoparticles（IONPs）,after being encapsulated by macrophages,utilizing the active chemotaxis of macrophages,IONPs loaded with the therapeutic drug SSZ were accurately delivered to the inflammatory site and directly targeted at inflammatory cells and proliferating FLSs,which aims to achieve specific ferroptosis and photothermal synergistic therapy.The photothermal effect generated by IONPs could kill macrophages to trigger the fast release of therapeutic drugs.The released IONPs were degraded in RA microenvironment to generate Fe²⁺,catalyzing the fenton reaction of cellular hydrogen peroxide（H₂O₂）to produce reactive oxygen species（ROS）,and the released SSZ could inhibit the cystine/glutamate transporter（X_c^-）system,reducing the production of glutathione（GSH）,and downregulating the activity of glutathione peroxidase 4（GPX4）expression,reducing the antioxidant capacity of cells,further leading to accumulation of lipid peroxidation（LPO）,and synergizing with Fe²⁺to exert highly effective ferroptosis effect to cure inflammatory arthritis.The specific research is as follows.1.Particle size optimization of IONPs for ferroptosis research.IONPs with different sizes（10,40 and 70 nm）were synthesized by solvothermal method.The effects of different sized IONPs on the generation of Fe²⁺,·OH and the loading capacity of macrophages were studied.The results showed that the small-sized IONPs（10 nm）were unstable in acidic medium and could produce higher Fe²⁺by acid degradation,thereby catalyzing the fenton reaction to generate higher·OH.While the large-sized IONPs（70 nm）were stable,generated lower Fe²⁺and·OH,and had the highest cellular uptake.Therefore,intermediate-sized IONPs（40 nm）could better exert therapeutic effect as primary delivery system due to the comprehensive ferroptosis effect and loading capacity.2.Construction and in vitro evaluation of nano-targeted drug delivery system based on macrophages and IONPs.Firstly,SSZ-loaded IONPs（IONP-SSZ）were fabricated.In vitro photothermal experiments and·OH generation indicated the good photothermal performance and free radical generation ability of IONP-SSZ.Then,IONP-SSZ were co-incubated with macrophages to construct macrophage-based drug delivery system（Mφ-IONP-SSZ）.Cell migration and cytokines detection assays showed that IONP-SSZ scarcely affected the activity and function of macrophages.In vitro photothermal cytotoxicity assay confirmed the photothermal property of Mφ-IONP-SSZ to damage cells.Cellular ferroptosis inhibition assay,intracellular GSH,GPX4 and malondialdehyde（MDA）contents,and intracellular ROS and LPO levels all proved that IONPs could exert synergistic ferroptosis effect in combination with the ferroptosis inducer SSZ.FLSs were isolated,cultured and identified in vitro,and cytotoxicity assays showed that IONP-SSZ could produce enough cytotoxicity through photothermal therapy combined with ferroptosis to kill FLSs.3.Macrophages and IONPs-based nano-targeted drug delivery systems for in vivo therapeutic research of RA.Collagen induced arthritis（CIA）model mice were initially established.In vivo imaging assays showed that drug-loaded macrophages could actively target at inflammatory sites to achieve high and durable drug accumulation.In vivo photothermal experiments proved that intravenous injection of Mφ-IONP-SSZ could produce excellent photothermal effects.Mφ-IONP-SSZ combined with near-infrared（NIR）laser could effectively inhibits the occurrence of RA,which was as follows:the thickness of the paw of the treated CIA mice was significantly decreased and close to that of normal mice;the content of pro-inflammatory cytokines in serum returned to normal level;and pathological changes such as synovial hyperplasia,cartilage damage,and inflammatory cell infiltration were remarkably reduced.