Multiple approaches to generate microstructured hydrogels have emerged in order to control microscale properties for applications ranging from mechanical reinforcement to regenerative medicine. Here, we report new heterogeneous hybrid hydrogels comprising emerging resilin-like polypeptides (RLPs); the hydrogels can be engineered with controlled microstructure and distinct micromechanical properties via the liquid-liquid phase separation (LLPS) of aqueous solutions of the RLPs and poly(ethylene glycol) (PEG). The microstructure in the hydrogels was captured by cross-linking a phase-separated RLP and PEG solution via a Mannich-type reaction with the cross-linker tris(hydroxymethyl phosphine) (THP). Phase diagrams of the RLP/PEG system were generated in order to define solution parameters that would yield micron-scale domains in the hydrogels with diameters on the order of 20-90 μm; the production of RLP- and PEG-rich domains with these dimensions was confirmed via confocal microscopy. The hydrogel mechanical properties were assessed via oscillatory rheology and atomic force microscopy (AFM), with the hydrogels exhibiting a moderate bulk shear storage modulus (ca. 600 Pa) and micromechanical properties of the domains (Young's modulus ca. 13 kPa) that were distinct from those of the matrix (ca. 6 kPa). These results demonstrate that tuning the parameters of the aqueous-aqueous phase-separated RLP/PEG solutions provides a simple, straightforward methodology for fabricating microstructured protein-containing hydrogels, without extensive material processing or purification. Given the unusual mechanical properties of the resilins, these methods potentially could be useful for engineering the micromechanical properties and cellular behavior in phase-separated protein-polymer hydrogels.
