PEG-PLGA Nanoparticles Loaded With Rapamycin For Targeted Treatment Of Mice Rheumatoid Arthritis

Background and objective: Rheumatoid Arthritis(RA)is a progressive autoimmune disease with invasive synovitis of small joints.The bone and cartilage structures are deformity as the disease progresses.At present,there are several disadvantages in clinical drugs of RA,such as low utilization rate,systemic side effects and high price.Rapamycin(RAPA),as a macrolide immunosuppressor,can regulate the polarization of macrophages in the synovial joint,and inhibit the local inflammatory environment.However,how to improve the efficacy of RAPA and reduce the ectopic effect need to be solved urgently.To improve the utilization efficiency of RAPA drugs,this study intends to use phacoemulsification to build PEG-PLGA nanoparticles loaded with RAPA,with the increase of vascular permeability in the synovial site of RA to realize the enrichment of nanoparticles in the joint synovial membrane,which provides a new idea for the treatment of RA.Methods: PEG-PLGA nanoparticles(PEG-PLGA-RAPA)loaded with RAPA were synthesized by phacoemulsification.The size,stability and loading rate of PEG-PLGA-RAPA nanoparticles were detected by transmission electron microscopy,granularity potentiometer,HPLC,etc.In macrophage cell line(RAW264.7),the effect of PEG-PLGA-RAPA on LPS-induced polarization of macrophage cell lines was assessed by flow cytometry.Effect of PEG-PLGA-RAPA on expression of inflammatory cytokines after LPS treatment was evaluated by q PCR and ELISA.The regulation of RAPA on the downstream signal of LPS was measured by Western blot.The RA model of male DBA/1 mice was constructed by injecting PEG-PLGA-RAPA into the tail vein.The targeting effect of PEG-PLGA-RAPA at RA site was detected by small animal imaging.Flow cytometry and ELISA were used to evaluate the polarization of macrophages and expression of inflammatory factors in synovial tissue.H&E staining was used to observe the infiltration of ankle synovial inflammatory cells.Body weight and hind paw thickness were measured,and arthritis index scores were calculated.The effects of PEG-PLGA-RAPA on blood routine and biochemistry of mice were testedResults: PEG-PLGA-RAPA nanoparticles were successfully synthesized by phacoemulsification,with uniform particle size(200～230 nm)and negative electricity on the particle surface.The results showed that the PEG-PLGA-RAPA nanoparticles obtained after ultrasonic mixing have high drug loading efficiency and could be long-range slow-release RAPA in physiological solution.PEG-PLGA-RAPA inhibited M1 polarization and decreased secretion of proinflammatory factors.q PCR results suggested that PEG-PLGA-RAPA inhibited m RNA expression of Il-1β,Tnfa and other pro-inflammatory factor IL-4 m RNA.Western blot showed that RAPA inhibited m TOR signaling induced by LPS,and RAPA decreased activation of p-m TORC1,p-S6 K and p-S6.Live imaging of small animals passively enriched PEG-PLGA-RAPA in RA facets.After PEG-PLGA-RAPA treatment,IL-4 increased in joint tissues,decreased IL-1β,TNF-α,and differentiated macrophages to M2 type.PEG-PLGA-RAPA inhibited synovial hyperplasia,reduced joint thickness and reduced the inflammatory response in mice.PEG-PLGA-RAPA also showed good biocompatibility.Conclusion: PEG-PLGA with RAPA can form a stable and consistent PEG-PLGA-RAPA loading system,which can effectively inhibit inflammation,induce m TOR signal activation and suppress macrophage M1 polarization,promote macrophage M2 polarization and reduce secretion of pro-inflammatory cytokines.PEG-PLGA-RAPA can target the RA synovial tissue in mice,inhibit synovial hyperplasia,reduce joint thickness,and improve the symptoms of RA.