Injectable Hydrogels for VHH Delivery to Restore Joint Homeostasis

Osteoarthritis (OA) is a chronic joint disease characterized by cartilage degradation and inflammation driven by pro-inflammatory cytokines like Tumor Necrosis Factor-alpha (TNF-α). Neutralizing TNF-α using antibody fragments is a promising therapeutic approach, yet rapid clearance limits the effectiveness of intra-articular treatments. Variable domains of single-chain heavy-chain antibodies (VHHs) are highly specific and stable TNF-α inhibitors, but their small size necessitates a delivery system for sustained presence in the joint. This study aims to develop an injectable hydrogel enabling dual-triggered VHH release, using enzymatic degradation by hyaluronidase (HAase) and matrix metalloproteinases (MMPs) for disease-responsive, long-lasting OA therapy. Hydrogels were synthesized from hyaluronic acid-tyramine (HA-TA) and dextran-tyramine (Dex-TA), functionalized with maleimide (Mal) groups for VHH conjugation via thiol-maleimide chemistry. The VHH was engineered with a GS10-linker or an MMP-cleavable site for dual enzymatic release and a cysteine group in its C-terminus. In-situ gelation was achieved through horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ). Polymer ratios were optimized for balanced degradation and release. Degradation and VHH stability were evaluated using HAase and MMP-13 enzymes. Functional assays, including ELISA and SPRi, assessed VHH release and TNF-α neutralization. In-vitro studies employed HEK293 NF-kB reporter cell line and a cartilage-on-chip (CoC) platform with mechanically stimulated chondrocytes. Ex-vivo cartilage explants were evaluated for matrix degradation by TNFα in the presence or absence of VHH. Hydrogels demonstrated successful in-situ gelation within 60 seconds. High VHH-polymer coupling efficiencies were achieved: 95% for HA-TA-Mal and 75% for Dex-TA-Mal. A 50:50 HA:Dex ratio provided sustained VHH release over four weeks, with initial burst release from uncoupled VHH followed by controlled enzymatic degradation. The dual-release system allowed degradation-responsive delivery while maintaining VHH stability and TNF-α binding. In-vitro assays showed complete TNF-α inhibition with VHH at 100× molar excess, outperforming Adalimumab, which achieved only 30% inhibition. The CoC platform was employed to assess the potency of the VHH in neutralizing TNF-α. This model showed that mechanical stimulation, combined with TNF-α exposure, increased cellular sensitivity to TNF-α evidenced by increased cell death. However, blocking TNF-α activity with VHH successfully mitigated this adverse effect. Finally, explant studies confirmed the therapeutic efficacy of VHH, demonstrating reduced TNF-α levels and protection against cartilage degradation. This dual-triggered hydrogel offers a novel platform for sustained, targeted intra-articular delivery of TNF-α blocking VHH. By tailoring release to enzymatic activity linked to OA progression, this approach enables precision therapy and long-lasting, disease-modifying treatment for joint diseases.