Preparation Of Methotrexate-gold Nano-Targeted Formulation And Its Treatment Of Rheumatoid Arthritis

Objective:This project uses functionalized gold nanoparticles（GNPs）as the carrier,methotrexate（MTX）as the model drug,and folic acid（FA）as the target molecule to synthesize a GNPs/MTX-Cys-FA drug delivery system with joint targeting performance,reducing the toxic and side effects of MTX,and to study its therapeutic effect on RA.Methods:1.Preparation and characterization of nanomaterials and nanodrug delivery systems.MTX-Cys-FA was synthesized by solid-phase organic synthesis and purified,and GNPs/MTX-Cys-FA nano-drug delivery system was prepared by self-assembly of GNPs and thiol groups in cysteine（Cys）.It was characterized by particle size and Zeta potentiometer,TEM,SEM,FT-IR,DSC,X-ray and TG analysis.Hemolysis behavior and storage stability in different environments were investigated.HPLC method was established for in vitro analysis of MTX.The release behavior of the nanoconjugates in different p H environments（7.4 and 5.5）was investigated by dialysis method,and drug loading and encapsulation rate were determined.2.In vitro and in vivo targeting studies.Confocal laser microscopy and flow cytometry were used to study the uptake of the nanoconjugates.A multimode small animal imager was used to observe the imaging of nano-conjugated compounds at different time points in rats.3.Cellular behavioral study of HFLS-RA.CCK-8 assay was used to detect cytotoxicity.Cell proliferation,cell scratches and transwell assay were used to observe the effects of the nanoconjugates on HFLS-RA cells.4.In vivo pharmacokinetics and tissue distribution.To establish the quantitative analysis method of MTX content in rat plasma and tissue homogenates by LC-MS/MS,and to compare the pharmacokinetics and tissue distribution of GNPs/MTX-Cys-FA and MTX.5.Study on pharmacodynamics in rats.The successful modeling of rats was judged by the score of toe joint.Arthritis index and toe volume were used to record the treatment of CIA rats.X-ray small animal imaging was used to evaluate the degree of bone destruction.Micro-CT was used to evaluate bone erosion,joint space and bone destruction.The tissue sections of rat joints were evaluated by HE staining and safranin fast green staining.In addition,enzyme-linked immunosorbent assay（ELISA）was used to detect the changes of pro-inflammatory cytokines TNF-α,IL-1β,IL-6 and IL-17 and plasma iron reduction capacity（TRAP）.Results:1.TEM and SEM showed that GNPs and GNPs/MTX-Cys-FA were spherical.After drug loading,the average particle size of GNPs/MTX-Cys-FA increased to 103.06±3.88 nm,and the Zeta potential reached-33.68±1.02 m V.FT-IR results show that GNPs/MTX-Cys-FA is successfully constructed.In vitro release results showed that the nanoconjugates were p H sensitive with drug loading of 11.04±0.28%and encapsulation rate of 73.61±1.91%.Hemolysis test showed that the nanospheres had good biocompatibility.2.The results of CCK-8 showed that the cell line survival rates of GNPs were all greater than 80%,but the cytotoxicity of the nanoconjugates increased significantly with the increase of concentration.The results of cellular uptake indicated that the nanoconjugates with FA could be efficiently taken up by RAW 264.74 cells.In vivo imaging results showed that drug fluorescence could still be observed after 24 h in GNPs/MTX-Cys-FA.3.The CCK-8 results screened GNPs/MTX-Cys-FA dose concentration of 1.25μg/m L,2.5μg/m L,and 5μg/m L.Cell proliferation was the lowest at high concentration（5μg/m L）,and cell proliferation was significantly inhibited（P<0.001）.The results of cell scratch and transwell experiments confirmed that the nanoconjugates inhibited the migration of HFLS-RA cells in a time-and dose-dependent manner after TNF-αstimulation.4.The methodological investigation of the established LC-MS/MS quantitative analysis method conforms to the provisions of pharmacopoeia.The pharmacokinetics results showed that the half-life(t_1/2)of the nanoconjugates was prolonged by 2.1 times,and the mean retention time(MRT_0-t)was increased by 1.74 times（P<0.01）.The targeting efficiency of GNPs/MTX-Cys-FA was 6.9 times higher than that of free MTX（P<0.01）.5.Pharmacodynamics results showed that compared with the free MTX group,the toe volume of CIA rats was significantly reduced after intravenous GNPs/MTX-Cys-FA（P<0.01）,and toe swelling and bone injury were improved.Histopathology showed that the surface of articular cartilage was smooth and intact without injury,and the articular cavity was obvious.Conclusion:The GNPs/MTX-Cys-FA nano drug delivery system prepared in this topic has high encapsulation rate and drug loading capacity for MTX,small particle size and uniform particle size distribution,which can give full play to the synergistic effect of FA and MTX in the combined application.The targeted delivery of inflammatory sites can also reduce the damage of drugs to normal tissue cells,improve the therapeutic effect of drugs on RA,improve the therapeutic effect of drugs and reduce their toxic and side effects.In summary,GNPs/MTX-Cys-FA nanodrug delivery system has great potential in RA targeted therapy.