Inhibiting synovial inflammation and promoting cartilage repair in rheumatoid arthritis using a matrix metalloproteinase-binding hydrogel

Abstract

Originating from synovial tissue, matrix metalloproteinase-9 (MMP-9) is a key inflammatory factor that promotes the formation and invasion of synovial pannus, leading to cartilage matrix destruction in rheumatoid arthritis (RA). However, clinical trials of systemic use of MMP-9 inhibitors are not successful due to severe side effects. Thus, locally inhibiting MMP-9 may be an alternative in the treatment of RA. Herein, we developed MMP-9 binding peptide-functionalized copper sulfide nanoparticles (CuS-T NPs) and delivered them with light crosslinking chondroitin sulfate methacrylate (ChSMA) hydrogel. We found that the CuS NP-doped hydrogels could inhibit synovial inflammation. Specifically, the CuS-T/ChSMA hydrogel could rapidly bind to MMP-9, thereby inhibiting not only the invasion of RA fibroblast-like synoviocytes but also the polarization of inflammatory M1-type macrophages. The underlying mechanism involved the inhibition of the MAPK pathway. Moreover, ChSMA hydrogel provided a cartilage matrix-mimic microenvironment and synergistically promoted the generation of collagen-2 and aggrecan with CuS NPs. In an adjuvant-induced arthritis mouse model, the intra-articular injection of ChSMA/CuS-T hydrogel significantly alleviated synovial inflammation and accelerated cartilage repair without causing any side effects, killing two birds with one stone in RA therapy.

Keywords: Chondroitin sulfate methacrylate; Copper sulfide nanoparticles; Matrix metalloproteinase-9; Rheumatoid arthritis.

Graphical abstract

Scheme 1

Illustration of the application of ChSMA/CuS-T hydrogel in RA therapy. The inflammatory M1 type macrophages and synoviocytes release MMP-9 and cytokines to erose articular cartilage in RA. The ChSMA/CuS-T hydrogel can not only rapidly bind MMP-9 but also inhibit the invasion of synoviocytes as well as polarization of M1 macrophages. The hydrogel also exhibits strong chondroinductivity due to the presence of CuS NPs and chondroitin sulfate even under inflammatory microenvironment. Hence, intra-articular injection of ChSMA/CuS-T hydrogel alleviates joint inflammation and promotes cartilage regeneration, achieving an efficient RA therapy.

Fig. 1

Preparation and characterization of the NPs and hydrogels. (a) Illustration of synthesis of CuS-T NPs and ChSMA/CuS-T hydrogel. CuS NPs were modified with PAA to obtain carboxy groups, and then CuS-PAA NPs were conjugated with NH₂-rich MMP-9-binding peptides (cTTHWGFTLc, termed T) via an EDC/NHS reaction. A mixture of ChSMA/LAP and CuS-T NPs was crosslinked under 405 nm light irradiation for 30 s to form the ChSMA/CuS-T hydrogel. (b) TEM image of CuS and CuS-T NPs. Insert: photo of CuS (40 mg/mL) and CuS-T (1 mg/mL) NP solution. Scale bar = 50 nm. (c) Zeta potentials of CuS, CuS-PAA, and CuS-T NPs. The changes of zeta potentials from negative charge to positive charge suggested the successful conjugation of peptides on the NPs. (d) Absorption spectra of the NPs. A new peak was observed in ChSMA/CuS-T NPs in the range of 230–290 nm after peptide conjugation. (e) Photos of the hydrogels. (f) SEM images showing the porous structure of the hydrogels after freeze-drying. Scale bar = 50 μm. (g) Rheology test of ChSMA/CuS-T hydrogel. (h) ELISA of MMP-9 content in the supernatant. This result indicated that ChSMA/CuS-T hydrogel could bind MMP-9. (i) Fluorescence imaging of mice after intraarticular injection of Cy5.5-labeled ChSMA/CuS-T hydrogel.

Fig. 2

Cytotoxicity, inhibition of RA-FLS migration and invasion. (a) Cell viability of BMSCs cultured with different hydrogel extracts. (b) Live/dead staining of BMSCs. Scale bar = 100 μm. (c) Hemolysis ratio of the hydrogels. Inset: photo of blood samples after incubated with (I) PBS (negative control), (II) double distilled water (positive control), (III) ChSMA hydrogel, (IV) ChSMA/CuS hydrogel, and (V) ChSMA/CuS-T hydrogel, respectively. (d) Wound healing of RA-FLS treated with different hydrogels. (e) Transwell assay of RA-FLS cultured with the hydrogels. Scale bar = 100 μm. Quantitative analysis of wound healing rate (f) and transwell assay (g). ∗∗P < 0.01, ∗∗∗∗P < 0.0001, compared with the control group.

Fig. 3

Anti-inflammatory effect of the hydrogels. (a) Relative gene expression levels of MMP-9 and TNF-α of macrophages after different treatments. RAW264.7 cells without LPS stimulation were served as the blank control. In other groups, RAW264.7 cells were treated with 250 ng/mL LPS to maintain the inflammatory M1 type. (b) Statistical analysis of immunofluorescence intensity derived from panel (c). (c) Fluorescence images of the expression of M1 macrophage markers TNF-α and MMP-9. Scale bar = 25 μm. These results indicated that ChSMA/CuS-T hydrogels could inhibit MMP-9 and TNF-α expressions in M1 type macrophages. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared with the control group. ^#P < 0.05, ^##P < 0.01, ^####P < 0.0001 compared with the blank group.

Fig. 4

Mechanism of the anti-inflammatory effect. (a) Volcano plots revealing that there were 54 up-regulated genes and 139 down-regulated genes in the comparison of the ChSMA/CuS-T hydrogel group with the PBS group. (b) KEGG pathway enrichment analysis showing that the MAPK, Rap1, and Ras pathways are highly related to the anti-inflammatory effect of ChSMA/CuS-T hydrogel. (c) GO analysis showing that GTPase activity is enriched within biological process and molecular functions. (d) GSEA confirming that the MAPK, Rap1, and Ras pathways are inhibited in the ChSMA/CuS-T hydrogel groups.

Fig. 5

Chondroinductivity of the hydrogels. (a) Relative gene expression levels of Col-2, ACAN and SOX9 of BMSCs in different groups. (b) Immunohistochemistry staining of Col-2 and ACAN expressions in cell pellets. Scale bar = 100 μm. (c) Statistical analysis of Col-2 and ACAN expressions derived from the immunohistochemistry staining of cell pellets. (d) Illustration of the chondroninduction study of BMSCs under inflammatory microenvironment. The culture supernatant of M1 type macrophages were mixed with different hydrogels extracts to obtain the conditional media. (e) Relative gene expression levels of Col-2, ACAN and SOX9 of BMSCs after cocultured with different conditional media. These results indicated that ChSMA/CuS-T hydrogel could promote the chondrogenic differentiation of BMSCs even under inflammatory microenvironment. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, compared with the control group. ^####P < 0.0001, compared with the blank group.

Fig. 6

Therapeutic effect of the hydrogels on AIA mice. (a) Illustration of establishment of AIA mice and treatment design. (b, c) Representative H&E staining of the knee joints of AIA mice after different treatments. Synovial invasion was highlight by yellow arrows. Scale bar = 200 μm. (d, e) Safranine O-fast green staining of the knee joints. Cartilage destruction was highlight by yellow arrows. Scale bar = 200 μm. (f) Histological evaluation of knee joint sections using Synovitis score and Mankin score. (g) Statistical analysis of gait analysis of AIA mice after treatment. (h) Representative gait diagram of mice in different groups. RH = right hind paw, LH = left hind paw, RF = right front paw, LF = left front paw. These results indicated that ChSMA/CuS-T hydrogel exhibited the best therapeutic effects on AIA mice among all groups. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared with the PBS group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Immunofluorescence staining of knee joints. (a) MMP-9 expression in synovial tissue, TNF-α synovium in synovial tissue and cartilage, and Col-2 and ACAN expressions in cartilage. (b) Statistical analysis of immunofluorescence intensity derived from panel (a). The yellow arrows show the areas of synovium invasion and the dotted white lines showed the cartilage area in knee joint. These results indicated that ChSMA/CuS-T hydrogel could inhibit MMP-9 and TNF-α expressions while promoting cartilage repair under inflammatory microenvironment. Scale bar = 100 μm ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, compared with the PBS group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

In vivo safety of hydrogels. (a) H&E staining of the main organs after treatments, including heart, liver, spleen, and lung. All organs presented normal morphology. Scale bar = 100 μm. (b) Serum ALT, AST, Cr, and UA levels of mice were within the normal range, indicating that ChSMA/CuS-T hydrogel did not cause any undesired effects on liver and kidney functions.