Tunable hydrogel-based VHH delivery for intra-articular osteoararthritis therapy

Purpose (the aim of the study): Current osteoarthritis (OA) treatments based on intra-articular injections provide only temporary relief due to fast clearance from the joint. This calls for innovative therapeutic strategies for the long-term sustained release of therapeutics inside the joint space. This research focuses on developing an injectable hydrogel system as a tuneable localized drug delivery platform. The study explores TNFα-neutralizing variable domain of single chain heavy chain only antibody (VHH) delivery to validate this concept, emphasizing tuneable hydrogel degradation and diverse release kinetics profiles.

Methods: Hyaluronic acid (HA) and Dextran (Dex) were functionalized with tyramine (TA) and maleimide (Mal) groups and analyzed with 1H-NMR. Injectable hydrogels were created using various HA:Dex ratios and polymer percentages. Crosslinking was initiated by mixing the polymer conjugates with the enzyme horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) during injection, allowing rapid in situ hydrogel formation. Hydrogel degradation was assessed by exposing the gels to hyaluronidase in order to mimic in vivo conditions of the joint, and the degradation profiles were monitored over time. A TNFα-targeting VHH was conjugated to the polymers using thiol-maleimide chemistry after engineering a free cysteine in the C-terminal tail of the VHH. Varying hydrogel compositions and VHH conjugation sites (table 1) were examined to determine their impact on VHH release kinetics. Release profiles were quantified using ELISA at different time points. Finally, Surface Plasmon Resonance imaging (SPRi) and ELISA assays were used to confirm the maintained biological activity of released VHH in inhibiting TNFα.

Results: 1H-NMR spectra of the polymers showed that the number of substituted TA and Mal moieties per 100 disaccharide units in HA-TA-Mal were 7.5 and 12, and per 100 saccharide units in Dex-TA-Mal 9.5 and 2.5, respectively. The hydrogels displayed tunable degradation based on HA:Dex ratios. Dex-only hydrogels exhibited no degradation, while HA-only hydrogels showed the fastest degradation profile. In situ gelation was modulated by HRP concentration, maintaining a constant amount of crosslinks by having a constant concentration of H2O2.

Directed coupling of the VHH via the engineered free cysteine to the maleimide group in the polymers presented a coupling efficiency of 95% for HA-TA-Mal and 75% for Dex-TA-Mal. As mentioned before, the hydrogel degradation was influenced by polymer composition, and release profiles of VHH were modulated by both hydrogel composition and conjugation sites. Over a period of four weeks, it was seen that the hydrogels containing a 50:50 HA:Dex mix showed a sustained and stable release of VHH over time. Furthermore, coupling the VHH to HA-TA-Mal demonstrated a significant increase in the sustained VHH release performance compared to the VHH coupled to Dex. When the hydrogels were incubated in PBS, the release profile only showed a burst release of the VHH during the first days due to the uncoupled VHH diffusion out of the gels. Finally, bioactivity assays confirmed the maintained functionality of the released VHH.

Conclusions: This research pioneers tuneable hydrogel systems for controlled VHH delivery upon intra-articular injection, showcasing the potential for precision osteoarthritis therapy. Successful VHH conjugation and controlled release underscore the system's viability. By tailoring hydrogel composition and conjugation strategies, this study introduces a novel therapeutic avenue for targeted and sustained osteoarthritis treatment, with implications for future clinical applications.