Effects And Mechanism Of Reactive Oxygen Species-responsive Dexamethasone-loaded Nanoparticles For Targeted Treatment Of Rheumatoid Arthritis

Background:Rheumatoid arthritis（RA）is an immune-mediated inflammatory disease that results in synovitis,cartilage destruction,and even loss of joint function.Although the precise pathogenesis of RA has not been fully elucidated,it has been demonstrated that macrophages and fibroblast-like synoviocytes（FLS）in the joint synovium and lead to the severe destruction of articular cartilage.In particular,activated macrophages are the most prominent cells in the inflamed joints of RA and it can produce a plethora of cytokines such as tumor necrosis factor alpha（TNF-α）,interleukin-1β（IL-1β）and B-cell activation factor（BAFF）,which derive the vicious cycle of inflammation and tissue damage.It is the inactive rhomboid protein 2（i Rhom2）in macrophages that contributes to the secretion of TNF-α,which can simultaneously induce the expression of BAFF.Clinical trials have proved that inhibitors for i Rhom2 and its mediated production of TNF-αand BAFF can alleviate the inflammatory process of RA,thereby delaying the progression of the disease.Profit from its strong anti-inflammatory,anti-rheumatic and immunosuppressive effects,Dexamethasone（Dex）can inhibit the expression of TNF-αand BAFF in RA macrophages,thereby reducing bone erosion.In the treatment of RA,Dex can quickly relieve the joint swelling and pain symptoms of RA patients,but long-term and high-dose Dex treatment can cause systemic side effects,such as avascular necrosis of the femoral head,osteoporosis,and fractures.Therefore,it is necessary to consider how to effectively target Dex into RA macrophages,reduce the number of administrations and doses,and reduce its side effects,thus improving its application in RA.Nanoparticles（NPs）came in and proved to be a reliable,effective,and clinically applicable strategy,which can effectively deliver drugs to the lesion.In this process,the high expression of folate receptor（FR）in the activated macrophages and the significant increase of reactive oxygen species（ROS）provide beneficial conditions for the targeted delivery of NPs.Based on the above,a folic acid-modified ROS responsive material（Oxi-αCD）was prepared in this project by the method of nano-precipitation self-assembly to package Dex and assembled into nanoparticles to explore whether it can target activated macrophages and its roles in therapeutic effect for RA.Objective:To investigate the therapeutic effect of ROS-responsive Dex-loaded nanoparticles on rheumatoid arthritis and its underlying mechanisms.Methods:1.Synthesize ROS-responsive materials Oxi-αCD and Cy5-labeled Oxi-αCD through chemical reactions,and perform proton NMR characterization of the materials.2.Prepare ROS-responsive NPs by the nanoprecipitation method,and perform transmission electron microscopy,nanometer size,polymer dispersion index,Zeta potential characterization,and freeze-drying method to detect the drug loading rate of the prepared NPs.3.The in vitro drug release and hydrolysis mechanisms of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs were tested by semipermeable membrane dialysis experiments.4.The hydrogen peroxide detection kit was used to determine the H₂O₂ content in activated mouse macrophages cell line Raw264.7 after NPs treatment.5.Confocal laser scanning microscopy was used to detect the uptake of Cy5-labeled NPs by mouse macrophages cell line Raw264.7 and human fibroblast synovial cell line MH7A.6.The m RNAs levels of the three FRs in MH7A cells were detected by q PCR experiments,and the protein levels of the three FRs in Raw264.7 and MH7A cells were detected by WB experiments.7.The protein levels of i Rhom2,TNF-αand BAFF and the m RNA levels of i Rhom2 in activated Raw264.7 cells were detected by WB and q PCR,respectively.8.The effect of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs on the viability of Raw264.7 cells was determined using the cell counting kit-8.9.The in vivo biological safety of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs was evaluated by observing the body weight,blood routine test and H&E staining of main internal organs of CIA mice after administration treatment.10.The in vivo biodistribution and arthritis-targeting ability of Cy5-labeled Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs were detected by living imaging system in the CIA animal model.11.The anti-inflammatory effects of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs in CIA mice were evaluated by observing the paw thickness,AI index and direct photograph observation after administration of CIA mice.12.The micro-CT,H&E and Safranin O staining of the metacarpal joints of mice after administration of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs were used to observe the joint synovial damage and cartilage erosion of CIA mice.13.The pro-inflammatory cytokine levels of TNF-αand BAFF in serum of CIA mice after administration were measured using ELISA-specific quantification kits.14.WB was used to detect the expression levels of pro-inflammatory genes i Rhom2,TNF-αand BAFF in joint tissue proteins of CIA mice after administration treatment.Results:1.Successfully prepared spherical NPs with regular morphology,uniform size and about 200nm,including Blank/Oxi-αCD NPs,Dex/Oxi-αCD NPs,Dex/FA-Oxi-αCD NPs,Cy5-labeled Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs.2.Semipermeable membrane dialysis experiments show that Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs accelerate degradation in the presence of H₂O₂,and they can significantly reduce the intracellular H₂O₂ content of activated Raw264.7 cells.3.The expression of FRs in Raw264.7 cells was significantly higher than that in MH7A cells.Laser confocal detection confirmed that Raw264.7 cells effectively uptake Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs in a time-dependent manner,which was significantly higher than that in MH7A cells,and the uptake of Dex/FA-Oxi-αCD NPs is stronger.4.Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs significantly inhibited i Rhom2 RNA expression and i Rhom2,TNF-αand BAFF protein expression in activated Raw264.7 cells in a dose-dependent manner.But different concentrations of NPs had no significant effect on the viability of Raw264.7 cells.5.Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs had good biosafety in CIA mice,and Dex/FA-Oxi-αCD NPs had the strongest and lasting ability to accumulate in the joint joints of CIA mice.6.After treatment with Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs,the synovial inflammation and cartilage erosion in the joints of CIA mice were significantly reduced,and the treatment effect of Dex/FA-Oxi-αCD NPs was better.7.Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs treated CIA mice significantly reduced serum TNF-αand BAFF levels,and inhibited the expression of i Rhom2,TNF-αand BAFF proteins in the joints of CIA mice.The inhibitory effect of Dex/FA-Oxi-αCD NPs is more significant.Conclusion1.We finally developed a Dex-loaded ROS-responsive nanoparticles（named Dex/Oxi-αCD NPs）and an FA modified Dex-loaded ROS-responsive NPs（named Dex/FA-Oxi-αCD NPs）that can specifically and efficiently bound with the activated macrophages.2.Dex/FA-Oxi-αCD NPs can be actively targeted and taken up by FRs,especially FRβ,in activated Raw264.7 cells,and can effectively eliminate the elevated H₂O₂ in activated Raw264.7 cells,causing the spherical structure of NPs to collapse to release Dex.The released Dex exhibited anti-inflammatory activity by inhibiting the expression of i Rhom2,TNF-αand BAFF.3.In CIA mice,we confirmed that Dex/FA-Oxi-αCD NPs has good safety and active targeting.It can effectively delivered and accumulated to the inflamed joints,strongly reduced joint inflammation and alleviated cartilage destruction of CIA mice through the i Rhom2/TNF-α/BAFF signaling pathway,and play an anti-inflammatory effect.It is suggested that Dex/FA-Oxi-αCD NPs may be used as effective drugs to treat RA.