Imparting new stimuli-responsive behaviors in protein–polymers via self-immolative linker conjugation

The development of “smart” polymers capable of responding to physiologically relevant stimuli is essential for engineering dynamic sensing and actuation systems that leverage biological signals under specific (patho)physiological conditions. In this study, we present a general and versatile strategy to engineer novel stimuli-responsive behaviors in temperature-responsive protein-based polymers (PBPs) via site-specific conjugation with self-immolative molecules. Specifically, we developed hydrogen peroxide (H₂O₂)- and β-galactosidase (β-gal)-responsive elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs). Using a library of ELPs with varying numbers of conjugation sites, we demonstrate that this approach enables precise modulation of stimulus-responsive phase transitions, providing a tunable temperature window of up to 50 °C for stimuli-controlled phase transition. We further show that incorporation of these responsive ELPs into collagen hydrogels allows for controlled, dose- and time-dependent release of the ELPs, accompanied by stimulus-induced changes in the hydrogel's transparency, and storage and loss moduli. Additionally, we engineered diblock copolymer nanostructures comprising ELP–ELP or RLP–ELP segments for encapsulation and stimulus-triggered release of a hydrophobic model payload (Nile red) with varying release profiles. Together, these results establish a robust platform for imparting environmentally responsive functionalities to PBPs by integrating recombinant synthesis with chemically triggered actuation, thereby enabling the rational design of adaptive biomaterials with tunable physicochemical and biological properties for a wide range of biomedical and biotechnological applications.