(T1330-01-02) Optimizing Gel Network Architecture for Oxanorbornadiene-Based Degradable Hydrogels

Purpose: Injectable hydrogels are a promising platform for delivering therapeutic molecules, offering enhanced stability and reduced dosing frequency. This study presents a degradable hydrogel system utilizing oxanorbornadiene (OND) linkages, which undergo retro-Diels–Alder (rDA) fragmentation upon reaction with thiol nucleophiles. The rDA reaction of OND-thiol adducts exhibits tunable half-lives ranging from minutes to months, providing precise control over hydrogel degradation rates. While PEG-based hydrogels have previously demonstrated high encapsulation efficiencies ( >95%) and diverse release kinetics for monoclonal antibodies (mAbs) and nanoparticle vaccines, challenges such as protein aggregation ( >10 mg/mL) and potential immunogenicity limit their broader application. To address these limitations, PEG was replaced with short thiol-containing linkers, such as ethylene glycol di-thiol derivatives, to optimize gel network properties. This study provides insights into engineering advanced hydrogels for therapeutic delivery.

Methods: OND linkages with various fragmentation half-lives were synthesized via [4+2] cycloaddition of furans with alkynes. These ONDs were attached to dextran sidechains through esterification or copper-catalyzed azide-alkyne cycloaddition (CUAAC) click reactions. The degree of substitution (DS) of ONDs on dextran was analyzed via 1H NMR spectroscopy. A standard Ellman’s assay quantified the free SH% of multi-valent PEG-thiols and ethylene glycol di-thiol linkers. The IgG cargo was dye-labeled, and the protein solution was used to dissolve OND and thiol-containing components separately. These were then mixed in predefined ratios to fabricate hydrogels containing 10–20 wt% of materials in 100 μL volumes. Additional buffer solution was added as the medium for the gel-entrapped IgG cargo, and release kinetics were determined by measuring fluorescence in the supernatant. All gels were incubated at 37 °C and tested every 12 or 24 hours.

Results: OND linkers with differing rDA rates were attached to dextran at DS values ranging from 7% to 31%. Ellman’s assay reported the SH% of both 4-armed PEG-SH and ethylene glycol di-thiol linkers to be >90%. The gelation speed of each hydrogel was engineered to occur within 1–3 minutes. The injectability of the gel materials was validated using a clinically approved syringe-to-syringe transfer technique followed by injection through a needle. Once formed, the hydrogels degraded spontaneously in a programmable manner by applying different OND linkers and tuning their topology parameters, allowing the entrapped IgGs to release according to pre-set kinetics. Preliminary results demonstrate sustained IgG release for over one month, with hydrogels formulated using short thiol-containing linkers exhibiting non-first-order release kinetics over 23 days.

Conclusion: Hydrogels encapsulating IgG were successfully formulated using dextran-OND and different thiol-containing components, including both traditional PEG and short ethylene glycol linkers. Hydrogels formulated with short linkers exhibited tighter mesh sizes, successfully sustaining IgG release for over three weeks while maintaining structural integrity. These findings underscore the potential of modifying gel network architecture with small thiol linkers to overcome the limitations of PEG-based hydrogels.

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Figure 1. In vitro release of mAb from hydrogels at 37°C, with repeat trials (n = 3). The red curve represents the preliminary results for hydrogels synthesized using ethylene glycol di-thiol short linkers.