Tuning the Structure of Galacturonate Hydrogels: External Gelation by Ca, Zn, or Fe Cationic Cross-Linkers

We show here how the nature of various divalent cations M²⁺ (Ca²⁺, Zn²⁺, or Fe²⁺) influences the structure and mechanical properties of ionotropic polygalacturonate (polyGal) hydrogels designed by the diffusion of cations along one direction (external gelation). All hydrogels exhibit strong gradients of polyGal and cation concentrations, which are similar for all studied cations with a constant ratio R = [M²⁺]/[Gal] equal to 0.25, showing that every M²⁺ cation interacts with four galacturonate (Gal) units all along the gels. The regions of the hydrogels formed in the early stages of the gelation process are also similar for all cations and are homogeneous, with the same characteristic mesh size (75 ± 5 Å, as measured by small angle neutron scattering (SANS)) and the same storage modulus G' (∼5 × 10⁴ Pa). Conversely, in the regions of the gels formed in later stages of the process there exist differences in mechanical properties, turbidity, and local structure from one cation to another. Zn(II)-polyGal and Fe(II)-polyGal hydrogels display mesoscopic heterogeneities, more marked in case of Fe than for Zn, that are not present in Ca(II)-polyGal hydrogels. This comes from the mode and the strength of association between the cation and the Gal unit (bidentate for Ca²⁺ and monodentate "egg-box" for Zn²⁺ and Fe²⁺). Cross-links formed by Zn²⁺ and Fe²⁺ have a higher stability (lower ability to untie and reform) that induces the formation of local heterogeneities in the early stages of the gelation process whose size progressively increases during the gel growth, a mechanism that does not occur for cross-links made by Ca²⁺ that are less stable and enable possible reorganizations between polyGal chains.