Nanogels, also called hydrogels, are molecules coated in a gel layer that protects enzymes but allows them to be active. This layer can be thought of like a cross between a gel and a sponge, which allows water and other small molecules to diffuse in and out. The ability to embed and stabilize functional enzymes has enormous potential. In 2006, researchers from China’s Tsinghua University demonstrated the production of single-enzyme nanogels (SENs), which consist of a single enzyme molecule encased in this spongy, gel-like material. This area of research has since produced nanogels incorporating a wide variety of enzymes and other proteins that show promise for use as drug delivery systems, biocatalysis, biocidal agents, biosensors, pollution degradation agents, and other applications.

Researchers have thus far used a two-step fabrication technique to make these nanogels, says Guillaume Delaittre, a researcher at Karlsruhe Institute of Technology in Germany. This process involves first modifying the lysine residues of enzymes to introduce vinyl groups. During the second step, the vinyl groups serve to anchor the sponge layer formed by polymerization of acrylamide, leading to the nanogel structure. It would be advantageous to produce this product using a one-step technique, which could potentially save time and money.

Scientists have experimented with a variety of charged monomers to create nanogels in a single-step process, Delaittre says. But this process risks exposing the enzyme or proteins to charged moieties that can lead to a loss of enzyme stability or total inactivation, he says.

In an article published in a recent issue of Chemical Science, Delaittre, Ana Beloqui, and colleagues have now demonstrated a one-step process for the creation of SENs, incorporating a wide range of enzymes, by exposing them to a very simple substance: sucrose. They analyzed the SENs molecular structures and activity levels.

The work “provides a new strategy to [use a simple method to] encapsulate enzymes into nanocapsules,” says Zhen Gu, associate professor with the joint biomedical engineering department at North Carolina State University and University of North Carolina at Chapel Hill, who was not involved in this study.

To create the nanogels, the researchers mixed the enzyme (or protein) at a maximum concentration of 60 micromoles per liter with precursors of the hydrogel, acrylamide and bisacrylamide. To this milieu they added sucrose, and then a redox radical initiating system to start the polymerization process. “We find that in optimized conditions [polymerization] principally occurs at the surface of the enzyme and that few or no by-products are produced [such as] residual non-bound polymer, or non-encapsulated protein,” Delaittre says.

Although the team has not uncovered the exact mechanism by which sucrose promotes nanogel formation, they hypothesize it works by steering  hydrophilic and hydrophobic interactions between the enzyme surface and the acrylamide precursors. This allows the gel to more stably interact with and coat the enzyme. “Small carbohydrates, particularly disaccharides, may strengthen the monomer/protein interaction through reduction of hydrophobic/hydrophilic repulsion forces and, therefore, provide a surface microenvironment in which monomers accumulate,” the researchers write. “This may result in thicker polymeric shells and eventually lead to higher enzyme stabilization.”

The process worked for creating nanogels incorporating “a range of proteins with widely different structures, catalytic activities, and sizes, including redox enzymes (horseradish peroxidase, glucose oxidase, catalase, laccase, and alcohol oxidase), hydrolases (esterase, β-glucosidase, and lipase), as well as a non-catalytic protein (bovine serum albumin),” the researchers write in the study.

“The most important step forward is that this can now be performed in principle with any enzyme, even more fragile ones that normally cannot be chemically modified,” says Jan van Hest, a researcher with Eindhoven University of Technology in the Netherlands. “The robust and facile procedure looks really attractive.”

The team also investigated the structure of the nanogel by incorporating fluorescent molecules selectively at the surface of the enzyme and within the shell, and employed high-resolution fluorescence microscopy. Their findings suggest that the method produces SENs with an enzyme core surrounded by a gel “shell” and that the thickness of the shell can be varied by adjusting protein concentrations in the initial polymerization reaction.

The research group also measured the activity of various SENs under different pH conditions. Thicker shells, as expected, provide higher stability, as inferred from the widening of the pH range under which various enzymes performed optimally. However, the researchers noted an inverse relationship between enzyme activity and stability, as enzymes in thicker shells were more stable but less accessible for chemical reactions. This tradeoff must be appropriately tailored to the individual application, the researchers say.

How could this technique be used in the future? Delaittre says that the method is probably still too expensive for producing nanogels on a large scale, though it could potentially be already feasible for high-end applications, such as for use in biosensors. “What would be necessary for further applications on possibly larger amounts is to optimize the synthesis on larger volumes, [possibly] using flow chemistry, and to automate it,” he says.

Read the abstract in Chemical Science.