Study On The Effect And Mechanism Of Peptide-Anchored Neutrophil Membrane-Coated Biomimetic Nanodrug For Targeted Treatment Of Rheumatoid Arthritis

Background Macrophage polarization determines the production of cytokines that further promote the development of rheumatoid arthritis(RA).However,coordinated modulation of macrophages in the synovium and synovial fluid has not been achieved thus far.Therefore,the regulation of macrophage polarization may be a potential therapeutic strategy for RA.Objective In this study,we intend to construct a biomimetic Apo A-I mimetic peptide R4F-modified neutrophil membrane(NM)-wrapped F127 polymerbionic with the targeting function of Scavenger Receptor class Btype1(SR-B1)receptor(R4F-NM@F127),so as to achieve the specific targeting of arthritis in the direction of inflammation.To study the effect and mechanism of R4F-NM@F127-Cel targeted therapy for RA by reprogramming synovium and synovial fluid macrophages with core-loaded celastrol(Cel).Methods(1)Neutrophils were isolated to obtain neutrophil membranes.Pluronic F127polymer loaded with hydrophobic drugs was prepared by thin-film hydration,further mixed with NMs with a liposome extruder to prepare NM@F127 nanoparticles.NM@F127 was functionalized with R4F peptide to prepare R4F-NM@F127.The basic properties of R4F-NM@F127 were determined with a Malvern Particle Size Analyzer.The morphology of R4F-NM@F127 was examined using transmission electron microscopy(TEM).The encapsulation characteristic and absorption wavelength of DiR-BOA-loaded R4F-NM@F127was measured with a high-performance liquid chromatography(HPLC)and UV?visvisible spectrophotometer.(2)The targeting ability of R4F-NM@F127 to macrophages was determined by confocal laser scanning microscopy and flow cytometry.(3)The targeting ability of R4F-NM@F127 was detected by a live animal imaging system;The co-localization of macrophages from R4F-NM@F127 to synovium and synovial fluid was determined by flow cytometry and confocal imaging.(4)In vitro,LPS(100 ng/mL)induced RAW264.7 cells inflammation model was established,after incubation with varying concentrations of Cel,NM@F127-Cel and R4F-NM@F127-Cel for 6 h or 12 h to detect the efficacy of cell therapy.The m RNA expression of macrophage markers in M1 type macrophages(TNF-α,IL-1β,i NOS)and M2 type macrophages(IL-10,Arg-1)was detected by real-time quantitative PCR.The polarization markers of M1/M2 macrophages and the expressions of proteins related to NF-κB and MAPK signaling pathways were detected by Western blot.The mean fluorescence intensity of M1/M2 macrophage markers CD86 and CD206 was measured by flow cytometry.(5)The collagen-induced arthritis(CIA)animal model was established in vivo.The therapeutic effect of R4F-NM@F127-Cel was evaluated by mouse body weight,joint score,left and right paw thickness,joint pathological tissue staining,Micro CT analysis,synovial and synovial fluid macrophage polarization.Results(1)The average size of R4F-NM@F127 was 48.09±5.82 nm,the zeta potential was -2.76±0.54 mV,and the PDI was 0.08±0.005.The white light image showed a distinct color change of R4F-NM@F127 from colorless to black?blue after encapsulation of DiR-BOA,and the UV–visible absorption spectrum also showed that both free DiR-BOA and DiR-BOA-labeled R4F-NM@F127 had obvious enhancement peaks at 730 nm.(2)Targeting results showed that R4F-NM@F127 has the ability to selectively target SR-B1~+cells and effectively target SR-B1~+macrophages,while in vivo fluorescence imaging showed that R4F-NM@F127 preferentially accumulates in inflamed joints and is phagocytic by joint synovium and synovium fluid macrophages.(4)In vitro cell experiments showed that R4F-NM@F127-Cel significantly inhibited the expression of M1 macrophage markers and increased the expression of M2 macrophage markers in a concentration-dependent manner.At the same time,R4F-NM@F127-Cel can significantly reprogram RAW264.7 cell polarization by inhibiting the phosphorylation of important proteins in NF-κB and MAPK signaling pathways in a concentration-dependent manner in vitro.(5)In vivo animal experiments have shown that R4F-NM@F127-Cel can significantly reduce joint scores,improve paw swelling,and reduce synovial inflammation and cartilage destruction in CIA mice.More importantly,R4F-NM@F127-Cel effectively inhibits synovial inflammation and improves joint damage by reprogramming macrophage polarization.Conclusion R4F polypeptide modified neutrophil membrane coated biomimetic nanomaterials(R4F-NM@F127-Cel)have obvious targeting ability of SR-B1~+cells,and can effectively inhibit synovial inflammation and alleviate joint injury by targeting synovial fluid macrophages through SR-B1 receptors,thus providing a promising strategy for clinical treatment of RA.