An injectable hydrogel to disrupt neutrophil extracellular traps for treating rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function. The therapeutic efficacy of RA treatments with the severely affected side remains unsatisfactory despite current treatment methods that primarily focus on anti-inflammatory activity, largely because of the complicatedly pathological mechanisms. A recently identified mechanism for RA development involves the interaction of RA autoantibodies with various proinflammatory cytokines to facilitate the formation of neutrophil extracellular traps (NETs), which increased inflammatory responses to express inflammatory cytokines and chemokines. Therefore, NETs architecture digestion may inhibit the positive-feedback inflammatory signal pathway and lessen joint damage in RA. In this work, deoxyribonuclease I (DNase) is connected to oxidized hyaluronic acid (OHA) via Schiff base reaction to extend the half-life of DNase. The modification does not influence the DNase activity for plasmid deoxyribonucleic acid hydrolysis and NETs' architecture disruption. Carboxymethyl chitosan is crosslinked with DNase-functionalised OHA (DHA) to form an injectable, degradable, and biocompatible hydrogel (DHY) to further strengthen the adhesive capability of DHA. Importantly, the collagen-induced arthritis model demonstrates that intra-articular injection of DHY can significantly reduce inflammatory cytokine expression and alleviate RA symptoms, which can be significantly improved by combining methotrexate. Here, a DNase-functionalised hydrogel has been developed for RA treatment by constantly degrading the novel drug target of NETs to decrease inflammatory response in RA.

Keywords: Rheumatoid arthritis; anti-inflammation; deoxyribonuclease; hydrogel; neutrophil extracellular traps.

Scheme 1.

An illustration of DNase-functionalized hydrogel with MTX loading for rheumatoid arthritis treatment. (A) The synthesis of DNase-coupled oxidized hyaluronic acid. (B) The prepared process of carboxymethyl chitosan. (C) Intra-articular injection of DNase-functionalized hydrogel for the treatment of RA.

Figure 1.

Characterization of DNase-functionalized hydrogel. (A) Color change for the indication of Schiff base reaction between OHA and DNase for different time. (B) Coomassie blue staining of free DNase, DNase-modified OHA (DHA) and free OHA. (C) FT-IR spectra of DHA, CMCS and DHY. (D) Tube inversion experiments and SEM images of DHY with different mass ratio of DHA and CMCS. (E) The cumulative release of MTX from DHY under different pH conditions.

Figure 2.

In vitro NETs digestion mediated by DHA. (A) Cytotoxicity of CMCS, DHA, and DHY on RAW264.7 cells determined by MTT assay. Cell viability measurements were performed after incubating the RAW264.7 cells with different concentrations of the formulations for 48 h. (B) Endonuclease activity of DHA. Plasmid DNA was incubated with 1 mg/mL of free DNase or DHA at 37 °C for 30 min. Control DNA was incubated in parallel without enzyme addition. The digestion product was run on 1% agarose gel. (C) Neutrophils were stimulated with PMA (100 nM) for 2 h followed by incubation with free DNase and DHA for 30 min. Neutrophils/NETs were washed and stained with 4′,6-diamidino-2-phenylindole and antibodies for elastase detection (green).

Figure 3.

DNase-functionalised hydrogel inhibited NETs-promoted M1 macrophage polarization. Neutrophils separated from mice with collagen-induced arthritis were stimulated with PMA (100 nM) for 2 h, followed by incubation with RAW264.7 cells in the transwell system. (A-B) RAW264.7 cells were cultured in the lower chamber and incubated with NETs upon different treatments (upper chamber) for 48 h. The CD86 of M1 macrophage marker was determined by flow cytometry. (C) Fluorescence detection for nitric oxide in RAW264.7 cells with different treatments. (D-E) Enzyme-linked immunosorbent assay for inflammatory factors in the RAW264.7 cell supernatants.

Figure 4.

In vivo controlled release behaviors of DHY. (A-B) IVIS images during different time intervals after the intra-articular injection of free Cy5.5 or DHY-encapsulated Cy5.5 (A) and determination of the fluorescence intensity (B). (C-D) IVIS images for different periods after the intra-articular injection of Cy5.5-labelled DNase with different formulations in CIA mice (C) and relative fluorescence intensity determined at each time interval (D).

Figure 5.

In vivo therapeutic effect of DHY in a collagen-induced arthritis mouse model. (A) Overall experimental timeline for in vivo therapeutic effect evaluation. (B) Representative images of arthritic paws from different groups at the end of the experiments. (C) Hind paw thickness and (D) arthritis scores.

Figure 6.

The combined anti-inflammatory effects of DHY and MTX on the RA model. (A-B) H&E (A) and safranin O/fast green (B) staining of the knee joint tissue. (C-D) IHC staining of tumor necrosis factor-α (C) and interleukin-6 (D). Scale bar, 100 μm.

Figure 7.

H&E staining of major organs tissues from different groups, scale bar, 200 μm.