Rheological Properties of Hemoglobin-Based Physical and Chemical Gels, and Their Hybrid

Gel viscoelasticity governs the mechanical identity of functional biomaterials. This paper presents the design and rheological characterization of hemoglobin (Hb)-based hydrogels, in which the network is maintained either by native Hb or by intramolecularly ββ-cross-linked Hb (XLHb). Native Hb has a α₂β₂ structure constructed via reversible, noncovalent interactions between αβ subunits, whereas in XLHb the tetrameric α₂β₂ structure is stabilized covalently. Conjugation of the β subunits of native Hb with four-armed 10 kDa polyethylene glycol (PEG) produces a physically cross-linked supramolecular gel exhibiting liquid-like viscoelasticity and self-healing behavior. By contrast, XLHb yields a chemically cross-linked gel exhibiting solid-like mechanical properties attributed to a permanent covalent network among PEG termini. Additionally, to explore intermediate states, we synthesized a hybrid gel by reacting a combination of Hb and XLHb with four-armed PEG. Rheological analysis revealed a critical transition from liquid-like to solid-like viscoelastic behavior between 80% and 90% XLHb ratios. These findings provide a mechanistic rationale for designing hybrid gel systems with precisely tunable viscoelastic properties through control of cross-linking modalities and through application of unified network backbone strategies.