Engineering Multilayer Poly(ethylene glycol) Hydrogels via Off-Stoichiometry Thiol-Ene Chemistry

Multilayer hydrogels with spatially controllable mechanical and functional properties are increasingly valuable, yet their utility is often constrained by fabrication challenges such as weak interfacial adhesion as well as reliance on cytotoxic photoinitiators and UV exposure. This study presents a simple, robust, and photoinitiator-free method to engineer multilayer poly-(ethylene glycol) (PEG) hydrogels using off-stoichiometry thiol-ene (OSTE) Michael addition chemistry. By deliberately utilizing an alternating thiol-rich and vinyl sulfone-rich PEG formulation, residual functional groups are preserved at the layer interfaces and subsequently form covalent bonds between the adjacent layers, enabling strong interfacial adhesion without UV irradiation, photoinitiators, or adhesives. The resulting hydrogels exhibited rapid and tunable gelation (1-5 min), with mechanical properties spanning ∼0.5-6 kPa through varying polymer concentration (5-10 wt %) and thiol-ene ratio (0.6-1.6). Ellman's assay confirmed the presence of stoichiometry-dependent unreacted thiol groups, validating the proposed interfacial bonding mechanism. Peel tests revealed strong interfacial adhesion, with failure occurring within the bulk material rather than at the interface. Nanoindentation further revealed spatially defined stiffness profiles across the multilayer construct, confirming precise mechanical compartmentalization. The hydrogels displayed tunable swelling behavior and degradation profiles depending on the formulations. Additionally, all single- and multilayer hydrogels maintained high cytocompatibility (>90% NIH/3T3 mice fibroblast viability) independent of polymer concentration, thiol-ene ratios, or layering. Incorporation of alginate microparticles provided an additional strategy for modulating hydrogel properties, significantly reducing swelling and enhancing stiffness while preserving structural integrity. Overall, the findings established a platform for engineering heterogeneous hydrogel constructs with spatially controlled properties, biofunctionality, and compatibility features that make them relevant for biomedical applications.