Previously, we developed the "protein activation and release from cage by external light" (PARCEL) method for controlling the function of proteins by encapsulating them in a photodegradable hydrogel and subsequently releasing them by ultraviolet (UV) irradiation of the gel. However, controlling small proteins is difficult because small proteins can leak from the gap (ca. 12.4 nm) of the mesh structure of the hydrogel without irradiation. Here, we developed a photodegradable gel with a smaller mesh size (~3.6 nm) and used the new gel to control the function of three small enzymes (trypsin, chymotrypsin, and elastase) and several small nonprotein molecules. The new gel showed reduced leakage of the proteins without irradiation, and tryptic activity increased approximately 78-fold upon irradiation of gel-encapsulated trypsin. The new gel also permitted encapsulation and release of 4',6-diamidino-2-phenylindole (DAPI, molecular weight 277), a small DNA-specific fluorescent probe. After irradiation to the gel-encapsulated DAPI and subsequent addition of DNA, strong fluorescence of the DAPI-DNA complex was observed. Our results indicate that reducing the gel mesh size from 12.4 to 3.6 nm should allow the encapsulation of various proteins and small molecules in an inactive state and their subsequent light-induced release. We expect this method to be useful in preparation of photoactivated biosensors, drug delivery systems, and catalysis.

Herein we report an approach to encapsulate enzymes within polymeric nanocapsules dispersed in an organic solvent via inverse miniemulsion periphery RAFT polymerization (IMEPP). Glucose oxidase (GOx), which has various applications but is unstable at elevated temperature and in organic solvents, was chosen as a model enzyme. In this study, we have explored the use of photoinitiation under visible (blue) light instead of thermal initiation to avoid enzyme denaturation by heating. GOx was successfully encapsulated within polymeric nanocapsules (∼200 nm) and showed high activity (71−100% relative to free GOx in PBS) dispersed in toluene/t-BuOH. The nanocapsules were thus able to protect GOx and enable it to function in an organic solvent mixture where native GOx would otherwise undergo denaturation. This approach of enzyme encapsulation is significant as it may lead to increased industrial applications of enzyme biocatalysis, expanding the use of enzymes as nontoxic and environmentally friendly biocompatible catalysts.

Positively charged photodegradable nanoparticles that simultaneously encapsulated various compounds including small and large molecules were prepared. The nanoparticles were internalized to the cell by endocytosis and were stable within the cells for at least 4 days. The encapsulated molecules were released into the cytosol using light stimuli.