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 (H2O2). 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.