Aqueous Liquid–Liquid Phase Separation of Resilin-Like Polypeptide/Polyethylene Glycol Solutions for the Formation of Microstructured Hydrogels

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 mechanica...