Proteolysis-Programmed Reopening of Bilayer Hydrogel Actuators

Soft hydrogel actuators that interface with living tissues must combine reliable attachment with programmed disengagement after completing their function. Here, we introduce a programmed-reopening bilayer hydrogel actuator with an intrinsic material off-switch. Using multilayer digital light processing (DLP), we fabricate poly(N-isopropylacrylamide)/bovine serum albumin-poly(ethylene glycol) diacrylate(Trypsin) (PNIPAM/BSA-PEGDA(Trypsin)) bilayers in which the PNIPAM layer drives closure at physiological temperature (37°C), while the trypsin-loaded BSA-PEGDA layer undergoes proteolysis-programmed softening. The progressive loss of stiffness relaxes curvature and reopens the device. We quantify print-parameter-dependent actuation, trypsin encapsulation and release, retained proteolytic activity, and time-dependent softening of the enzyme-programmed layer. Lap-shear testing and microscopy characterize the bonded bilayer interface. Mechanistic anchoring is visualized on a mucin-mimicking interface by microscopy and time-lapse imaging, and ex vivo tests on porcine small intestine demonstrate robust retention under controlled dynamic motion with millinewton-scale gripping forces. These findings establish a materials strategy for programming the functional lifetime and reopening of tissue-interfacing hydrogel actuators. STATEMENT OF SIGNIFICANCE: Tissue-interfacing hydrogel actuators are promising for gastrointestinal retention and local therapy, but most existing systems deform in one direction and lack an intrinsic mechanism for disengagement. We introduce a programmed-reopening bilayer actuator in which proteolysis-programmed mechanical relaxation acts as an internal off-switch. Multilayer digital light processing (DLP) produces poly(N-isopropylacrylamide)/bovine serum albumin-poly (ethylene glycol) diacrylate (Trypsin) (PNIPAM/BSA-PEGDA(Trypsin)) bilayers that close at physiological temperature, release active trypsin, and progressively reopen as the protein-polymer layer softens. The study integrates release, mechanical relaxation, preliminary cytocompatibility, mucin-interface anchoring, and ex vivo retention on porcine intestine. It establishes a materials design principle for programming actuator lifetime and termination, advancing autonomous and transient soft biomedical devices.