Small Mesh Size Hydrogel for Functional Photocontrol of Encapsulated Enzymes and Small Probe Molecules

Abstract

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.

Reduction of Molecular Leaching from a Gel Matrix for the Precisely Controlled Release of Encapsulated Molecules by Light Stimulus

“…We believe that the optimized gels, which exhibit minimal molecular leaching, developed in this study are excellent candidates for controlling the activity of encapsulated large proteins. In our previous study, 9) we prepared a photodegradable gel with a small mesh size by reducing the size of the branched monomer. We successfully used this gel to reduce molecular leaching from the gel before irradiation.…”

“…8) Because the gel can be used as a photocontrol method for the activity of various molecules, we have employed a method, which we refer to as PARCEL (Protein Activation and Release from Cage by External Light) methods, [9][10][11][12] for the spatiotemporal activation of molecules such as proteins, small molecules, and small interfering RNA (siRNA) within cells. 13) The reduction or disappearance of molecular leaching prior to the application of a stimulus is one of the critical remaining issues to be overcome in this area because molecules that are leached prior to the application of a stimulus show activity after irradiation, making the precise control of molecular functions difficult; many studies have been carried out on the reduction of leaching from the immobilizing matrix.…”

“…We have succeeded in reducing the leaching of small molecules by reducing the mesh size of the gel through reduction of the monomer size used in the preparation of the gel. 9) In this study, we performed detailed investigations into the effect of the mesh structure on molecular leaching from the gel. Two different types of monomer were used for the preparation of the photodegradable gel: a linear monomer, such as acrylamide (AA), that forms linear polymers after polymerization, and a branched monomer containing four-armed polyethylene 11) or polyamine moieties 10) that form branched structures within the gel.…”

Rapid and mild purification method for nanoparticles from a dispersed solution using a monolithic silica disk

“…The preparation scheme of 4-armed PEG is shown in our previous papers [21,22]. We mixed a solution of 800 L of 100 mg/mL PEG-Ac, 250 L of 1 mg/mL doxorubicin, 950 L of water, 250 L of 20 mg/mL APS, and 250 L of 0.1 M TEMED in 1 M Tris/HCl buffer in a tube and then stirring the mixture for 20 min using a vortex mixing device (VORTEX-GENIE 2, Scientific Industries, Inc., Bohemia, NY, USA).…”

Preparation and Release Properties of Sol-Gel Encapsulated Proteins

“…Hydrogels are excellent protein containers [8,9]. However, silica gel encapsulation of living cells and other biological components is not yet understood, due to the complexity of sol-gel chemistry and the variety of materials that may be considered for encapsulation [10].…”