Stiffening and Toughening Protein Hydrogels by Tuning Electrostatic Interactions

Electrostatic interactions play a central role in governing the swelling and mechanical properties of protein-based hydrogels, yet their influence near the isoelectric point (pI) remains poorly understood in chemically cross-linked protein hydrogels. Herein, we investigated the pH-dependent swelling and mechanics of a chemically cross-linked recombinant protein hydrogel constructed from GRG5RG4R (pI ≈ 4.6), an engineered elastomeric protein consisting of folded globular domains GB1 interspersed with unstructured resilin sequences. We found that at pI, the hydrogel equilibrated in NaCl solution not only showed the smallest swelling ratio due to minimized electrostatic repulsion and Donnan osmotic pressure at charge neutrality but also exhibited the highest Young's modulus and toughness. These results demonstrated the feasibility of tuning the electrostatic interactions to engineer stiff and tough protein hydrogels, a long-standing challenge in material engineering. Moreover, in citrate and sulfate buffers, while the hydrogel displayed the smallest swelling ratio at the pI, the maximum Young's modulus was observed at pH 3, which is below pI, despite increased swelling. This result demonstrated the decoupling of hydrogel's swelling and stiffness. We propose that bivalent anions interact with clustered cationic surface patches of GB1 domains, acting as a secondary ionic cross-linking to enhance the network stiffness. These findings highlight the feasibility of using pH and citrate as mechanisms to tune hydrogel mechanics without altering network chemistry.