Mechanism Of Fe₃O₄@PDA Magnetic Targeting Mesenchymal Stem Cells For The Treatment Of Rheumatoid Arthritis And Its Toxicological Evaluation

Rheumatoid arthritis（RA）is a chronic systemic autoimmune disease that occurs symmetrically in the small joints of the human body.Early clinical manifestations include joint swelling,pain,and limited range of motion.Late stages may result in decreased joint mobility and even complete loss of mobility.Its pathological feature is the abnormal changes of inflammatory factors throughout the body,causing the body to produce autoimmunity.It also promotes the formation of neovascular condyles in the local joint synovium,leading to inflammation and proliferation of the joint synovium,and eventually the osteochondral damage at the damaged joints.Mesenchymal stem cells（MSCs）,as a kind of low-immunogenic cells with high differentiation ability,have also shown great advantages in the treatment of RA in recent years.It can effectively control inflammation throughout RA and repair osteochondral damage in RA.However,due to its poor targeting,this therapy also has certain shortcomings.Fe₃O₄ nanoparticles are the most commonly used superparamagnetic nanoparticles.Using the endocytosis of MSCs to magnetically label them to form magnetized cells,and to place magnetic fields on the damaged joints to magnetically guide them can make more MSCs locally targeted for enrichment.At the same time,the potential nanotoxicity of nanoparticle applications is also a problem that cannot be ignored,and has attracted more scholars’attention in the toxicological evaluation of nanoparticles and in reducing the coating of toxic materials.Studies have shown that,although poly dopamine（PDA）-coated Fe3O4nanoparticles can effectively reduce its toxicity,and cell membrane bio-functionalized nanoparticles have also become one of the most popular nanomaterials attenuated coating methods,the functional comparison of the two coating methods needs to be further explored,which can provide a theoretical basis for finding better biocompatible nanomaterials.Therefore,this research is mainly divided into the following two parts:First,Fe₃O₄@PDA magnetic targeting human umbilical cord mesenchymal stem cells for the treatment of rabbit rheumatoid arthritis osteochondral defect modelIn this study,Fe₃O₄@PDA was magnetically labeled to form magnetized cells by endocytosis of HUMSCs,and rabbit RA models were treated by injection of rabbit ear veins into rabbits and local magnetic guidance of damaged joints.The rabbits were divided into the following 8 groups:（1）blank control group（no sensitization,no treatment）;（2）sham operation group（no sensitization,no treatment,skin cut and then suture the skin）;（3）saline treatment group;（4）Fe₃O₄@PDA treatment group;（5）Fe₃O₄@PDA+Magnet treatment group;（6）HUMSCs treatment group;（7）Fe₃O₄@PDA+HUMSCs treatment group;（8）Fe₃O₄@PDA+HUMSCs+Magnet treatment group.Then application of macrophase observation,macroscopic score of ICRS cartilage repair,hematoxylin eosin staining（H&E）staining,ICRS visual tissue score,toluidine blue（TBO）staining,safranin O staining,horse Masson’s trichrome staining,MicroCT in rabbit femoral condyles and other methods were used to detect the repair of osteochondral bone.The results showed that at 4 weeks,12 weeks and 24weeks,the Fe₃O₄@PDA+HUMSCs+Magnet treatment group obtained the best osteochondral structure formation and the highest score,and the repair effect was more significant with time.By using ELISA,RT-PCR and other methods for the inflammatory factors IL-1β,IL-6 and TNF-α,osteogenic factors OCN and OPN,chondrogenic factors SOX9,COLII and GAGs.The results showed that at 4 weeks,12 weeks and 24 weeks,the Fe₃O₄@PDA+HUMSCs+Magnet treatment group had the least expression of inflammatory factors and the best osteochondral repair effect,and the treatment effect was more significant with time.It is shown that magnetically targeted HUMSCs can provide a new feasible solution for the treatment of osteochondral defect RA model.Second,Effects of three kinds of superparamagnetic iron oxide nanoparticles based on apoptosis and autophagy in kidney of mice based on PI3K/AKT/mTOR signaling pathwayIn this study,we mixed HUMSCs membrane vesicles with Fe₃O₄@PDA core with a weight ratio of polymer to membrane protein of 2:1 and obtained stem cell membrane-camouflaged Fe₃O₄@PDA,namely MSC（Fe₃O₄@PDA）by extrusion.Then a suspension of Fe₃O₄,Fe₃O₄@PDA and MSC（Fe₃O₄@PDA）with Fe³⁺concentration of 10 mg/mL was prepared.ICR mice weighing（20±2）g were randomly divided into four groups,namely（1）saline control group,（2）Fe₃O₄ exposure group,（3）Fe₃O₄@PDA exposure group and（4）MSC（Fe₃O₄@PDA）exposure group.The Fe₃O₄,Fe₃O₄@PDA and MSC（Fe₃O₄@PDA）suspensions were injected into mice for 4weeks according to the Fe³⁺concentration dose(1/10 LD₅₀)of 45mg/kg.kw/d.After the mice were sacrificed,serum and kidney tissues were collected.Tunel staining was used to analyze renal apoptosis,and transmission electron microscopy was used to analyze renal autophagy.Detection of mRNA and protein expression levels of apoptosis-related factors Bax,Blc-2,Caspase-3,Caspase-3 and Caspase-9 and autophagy-related factors LC3,P62,Beclin-1 and ATG5 by RT-PCR and Western blot.Finally,RT-PCR and Western blot were used to detect the mRNA and protein expression levels of PI3K/AKT/mTOR signaling pathway-related factors upstream of apoptosis and autophagy.The study found that,in contrast,the MSC（Fe₃O₄@PDA）exposure group caused the smallest amount of renal cell autophagy and apoptosis,and was related to its upstream PI3K/AKT/mTOR signaling pathway.It shows that Fe₃O₄@PDA camouflaged with human umbilical cord mesenchymal stem cell membrane has less cytotoxicity,and provides a new feasible solution for the better application of nanomaterials in the medical field in the future.