Spectroscopic Determination of Ion-Binding Environment in Alkali-Driven Ionically Cross-Linked Hyaluronic Acid Hydrogels

Hyaluronic acid’s (HA) interaction with metal cations impacts its phase behavior in an ion-specific, and pH and ion concentration-dependent manner. Under alkaline conditions, bivalent metal cations can cross-link HA into hydrogels. However, the underlying mechanism remains unclear. Herein, we used CMP-NMR and FTIR-ATR to identify interactions that contribute to alkali-driven ionic cross-linking by Mg(II) and Cu(II). We show that the interactions and resulting polymer rigidity differed based on the ion used and correlated with each ion’s propensity for cross-link formation. Interaction with Cu(II) limited the segmental mobility across the entire polymer, while Mg(II) decreased the segmental mobility specifically around the carboxylate group. Further, we found that ionic cross-linking is driven by unidentate coordination with the carboxylate as well as interaction with the deprotonated acetamide nitrogen present only at high pH. Our findings offer a molecular perspective into ionic cross-linking of HA and its dependence on ion identity.