Injectable bioadhesive and lubricating hydrogel with polyphenol mediated single atom nanozyme for rheumatoid arthritis therapy

Abstract

Rheumatoid arthritis (RA) is a common chronic autoimmune condition accompanied by lubrication dysfunction, inflammatory infiltration, and cartilage wear. Long-term improvements in joint lubrication, inflammation elimination, and worn cartilage repair are crucial for effective RA treatment. Herein, we present an injectable bioadhesive and lubricating hydrogel containing a dopamine-modified hyaluronic acid (DA-HA) network, sulfonated hyaluronic acid (SO₃^--HA) network, and kartogenin (KGN)-grafted dopamine-hybridized graphene quantum dot-supported Cu single-atom nanozyme (DAGQD@Cu@KGN SAN) designed to restore cartilage lubrication and repair worn cartilage in RA. DA within the hydrogel networks provides bioadhesion, allowing it to persist in the joint cavity for extended periods. The hydrogel with SO₃^- group offer lubricity, reducing friction coefficient and alleviating cartilage wear. The DAGQD@Cu@KGN SAN exhibits excellent superoxide dismutase, catalase, and •OH scavenging activities, effectively inhibiting inflammation. KGN is sustainably released from the hydrogel, recruiting bone marrow mesenchymal stem cells to repair damaged cartilage by promoting their differentiation into chondrocytes. In vivo experimental results demonstrate that this injectable bioadhesive and lubricating hydrogel not only prevents cartilage wear and tear, providing long-term anti-oxidation and anti-inflammatory effects in early RA, but also repaired damaged cartilage in late-stage RA. This bio-adhesive and lubricating hydrogel presents a potential full-cycle strategy for RA therapy.

Fig. 1. Schematic diagram illustrating the synthesis of DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel and its application in RA treatment.

A Synthesis of DAGQD@Cu SAN and DAGQD@Cu@KGN SAN. B Synthesis of DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel. C DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel for RA therapy. (I) Hydrogel self-cures in situ within the joint cavity; (II, III) DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel displays lubrication and adhesion properties; (IV) Regulation of inflammatory microenvironment facilitated by SAN with remarkable ROS-scavenging capabilities; (V) KGN released from the hydrogel promotes the recruitment of BMSCs and induces their differentiation into chondrocytes, facilitating the repair of cartilage damaged by RA.

Fig. 2. Characterization of SAN.

TEM images of (A) DAGQD@Cu SAN and (B) DAGQD@Cu@KGN SAN. C High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) image of DAGQD@Cu SAN. D The UV-Vis spectrum of DAGQD, DAGQD@Cu SAN, and DAGQD@Cu@KGN SAN. E The C1s spectrum and (F) Cu2p spectrum of DAGQD@Cu SAN. G Hydroxyl radical scavenging ability, (H) SOD activity, and (I) CAT activity of GQD, DAGQD, DAGQD+Cu²⁺, DAGQD@Cu SAN, and DAGQD@Cu@KGN SAN (n = 3 samples). J The mechanism of ROS elimination of DAGQD@Cu SAN. (Data are presented as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *, **, and **** indicate significance at p < 0. 05, p < 0.01, and p < 0.0001, respectively).

Fig. 3. The self-healing, adhesion, lubricity properties of the injectable hydrogel.

A Photographs depicting the self-curing process of SO₃⁻/DA-HA hydrogel precursor initiated by DAGQD@Cu@KGN SAN. B The injectable property of DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel. C Rheological properties of DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel. D Images illustrating the self-curing process of DAGQD@Cu@KGN-SO₃⁻/DA-HA hydrogel in the cartilage defect of a rabbit knee joint. E Hydrogel self-healing ability. F Schematic depiction of the self-healing mechanism. G Coefficient (COF) comparison between Cu²⁺-SH-HA hydrogel, SAN-SH-HA hydrogel, and SAN-SO₃⁻-HA hydrogel (n = 3 samples). H COF variation of SAN-SO₃⁻/DA-HA hydrogel with different ratios of SO₃⁻-HA and DA-HA (n = 3 samples). I Adhesion strength of SAN-SO₃⁻/DA-HA hydrogel with different ratios of SO₃⁻-HA and DA-HA on cartilage samples (n = 3 samples). J Adhesion strength of the hydrogel with different concentrations of SAN on cartilage samples (n = 3 samples). K Anatomic picture of hydrogel adhesion in joint. L The FESEM and (M) metallographic microscopy image of hydrogel-cartilage cross-section (n = 3 independent experiments). N Illustration of the bioadhesion mechanism of DAGQD@Cu@KGN SAN hydrogel on cartilage tissue, involving (1) hydrogen bonds, (2) cation-π interaction, and (3) covalent linking. O Compressive strength and (P) compression modulus analysis of the hydrogel with varying concentrations of DAGQD@Cu@KGN SAN (n = 3 samples). (Data are presented as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test, with a value of *P < 0.05, **P < 0.01, and ****P < 0.001).

Fig. 4. In vitro biological performance of the hydrogel.

A Intracellular oxygen level of RAW264.7 cells subjected to different treatments was tested using RDPP (n = 3 independent experiments). Scale bar = 100 μm (B) Flow cytometry analysis of RDPP-labeled cells for different groups. C Intracellular ROS staining images of RAW264.7 cells subjected to different treatments (n = 3 independent experiments). Scale bar = 100 μm. D Flow cytometry analysis of ROS-labeled cells for different groups. E–G ELIZA results showed extracellular expression of (E) TNF-α, (F) IL−1β, and (G) IL−10 (n = 3 samples). H BMSCs proliferation on various hydrogels was evaluated using CCK8 after 3 or 7 days of cultivation (n = 3 samples). I Spreading morphology of BMSCs on hydrogel after being cultured for 12 h (n = 3 independent experiments). Scale bar = 200 μm. J–M The expression of cartilage-related markers Sox9, aggregatan (Acan), type II collagen (Col II), and collagen type X (Col X) (n = 3 samples). (Data are presented as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test, with a value of *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001).

Fig. 5. RNA sequencing analysis.

A The different changes of the expression of genes (Group 2 v.s. Group 1). B The different changes of the expression of genes (Group 3 v.s. Group 1) (C) Venn diagram of differentially expressed genes among the three groups. D GO enrichment of sole differentially expressed genes in Group 3. BP, biological processes. CC, cellular component. MF, molecular functions. E GSEA analysis of Foxo signaling pathway, Lysosome, osteoclast differentiation, TNF signaling pathway of DAGQD@Cu-SO₃⁻/DA-HA hydrogel. F Heatmap analysis of differentially expressed genes involved in anti-oxidative stress, autophagy, repairment, inflammation. G Illustrating the potential mechanism by which DAGQD@Cu-SO₃⁻/DA-HA hydrogel enhances cartilage repair and suppresses inflammation.

Fig. 6. Hydrogel prevents cartilage wear and tear in early RA.

A Schematic illustration of the experimental procedure. B Micro-CT images of rat knee joints after 4 weeks of treatment. The yellow dotted line represents the location of the histological section of the rat. C, H, E and (D) Safranine-O stain images of rat knee joints after 4 weeks of treatment (n = 3 independent experiments). E–G Immunohistochemical staining of cartilage markers (Sox9、Col II and Acan). The black symbol represents the positive expression. Scale bar = 200 μm. H–J The quantitative analysis of cartilage markers (Sox9、Col II and Acan) (n = 3 samples). K–M Levels of IL-6, IL−1β, and IFN- γ in rat serum after 4 weeks of treatment (n = 3 samples). (Data are presented as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test, with a value of *P < 0.05, **P < 0.01, and ***P < 0.005).

Fig. 7. Hydrogel promotes cartilage reconstruction in late RA.

A Schematic illustration of the experimental procedure. B Macroscopic observation of repaired cartilages at 8 weeks (black arrow: defect cartilage site). C Micro-CT images of rabbit’s knee joints after 8 weeks of treatment. The blue dotted line represents the location of the histological section of the rabbit. D, H, E Safranine-O stain images of the rat knee joints after 8 weeks of treatment (n = 3 independent experiments). Scale bar = 2 mm. F Quantitative analysis of bone volume over total volume ratio (BV/TV) and (G) trabecular number (Tb.N) of CIA rats in different treated groups (n = 3 samples). H Histological synovitis scoring (HSS) and (I) modified Osteoarthritis Research Society International (OARSI) score of rabbit knee joints after 8 weeks of treatment (n = 5 samples). J Indentation test results and (K) corresponding reduced modulus of the newly formed tissues in different groups (n = 6 samples). (Data are presented as the mean ± SD. Ordinal data such as HSS score and OARSI score was analyzed by Kruskal-Wallis test and continuous data was analyzed by one-way ANOVA followed by Tukey’s post-hoc test, with a value of **P < 0.01, ***P < 0.005, and ****P < 0.001).