Coupled Environmental Effects in Competitive Inhibition of Dynamic Hydrazone Hydrogels

Dynamic benzyl-hydrazone crosslinks are widely used in covalent adaptable hydrogels due to their hydrolytic stability and tunable exchange kinetics. This reversibility enables competitive inhibition strategies in which small-molecule competitors modulate network dynamics by replacing crosslinking sites. Here, we demonstrate that adding molecules to compete with crosslinks can unintentionally perturb hydrogel mechanics through environmental effects, including changes in pH and ion concentration, in addition to direct bond disruption. In benzyl-hydrazone hydrogels, methyl hydrazine competitors unexpectedly increased the average relaxation time rather than accelerating network exchange. To decouple environmental effects from competitive inhibition, we systematically investigate how pH, ion concentration, and buffer composition influence hydrogel mechanics. We find that increasing ion concentration enhances the storage modulus, consistent with salt-mediated stabilization. Comparison across biologically relevant media further reveals that relaxation dynamics vary significantly with buffer identity, even when elastic moduli remain similar. Notably, more basic environments lead to markedly slower network relaxation, even in the presence of competitors to the crosslink. These findings demonstrate that, in competitively inhibited benzyl-hydrazone hydrogels, pH, ion concentration, and buffer identity can influence network mechanics in ways that differ from the expected effects of competitor addition alone, highlighting the need for careful interpretation of competitive inhibition experiments.