In Situ Phase Transition of Elastin-Like Polypeptide Chains Regulates Thermoresponsive Properties of Elastomeric Protein-Based Hydrogels

Engineering protein-based hydrogels that can change their physical and mechanical properties in response to environmental stimuli have attracted considerable interest due to their promising applications in biomedical engineering. Among environmental stimuli, temperature is of particular interest. Most thermally responsive protein hydrogels are constructed from thermally responsive elastin-like polypeptides (ELPs), which exhibit a lower critical solution temperature (LCST) transition, or nonstructured elastomeric proteins fused with ELPs. Here we report the engineering of thermally responsive elastomeric protein-based hydrogels by fusing ELPs to elastomeric proteins made of tandemly arranged folded globular proteins. By fusing ELP sequence (VPGVG)n to an elastomeric protein (GR)4, which is made of small globular protein GB1 (G) and random coil sequence resilin (R), we engineered a series of protein block copolymers, Vn-(GR)4. The fusion proteins Vn-(GR)4 exhibit temperature-responsive behaviors in aqueous solution that are different from that of Vn-ELPs, as they did not exhibit the macroscopic phase transitions in the turbidity test. Instead, V48-(GR)4 and V72-(GR)4 form micelles at temperatures higher than the transition temperature of V48 and V72 at the same concentration. Using the well-developed ruthenium-mediated photochemical cross-linking method, Vn-(GR)4 polymers can be cross-linked into hydrogels, in which Vn-ELP serve as side chains of the hydrogel network. These hydrogels exhibited thermoresponsive properties due to the temperature dependent phase transition behaviors of the incorporated Vn-ELPs blocks. At elevated temperatures, the Vn-ELPs side chains in the hydrogel network underwent aggregation, leading to secondary physical cross-linking. The aggregation of the Vn-ELPs resulted in higher Young's modulus and reduced swelling ratio. Furthermore, the amplitude of such property changes can be tuned by side chain length and composition. These results demonstrate that in situ phase behaviors of ELP side chains can regulate thermoresponsiveness of protein-based hydrogels. We anticipate that this method can be applied to other elastomeric proteins for potential biomedical applications.