Combining Active Carbonic Anhydrase with Nanogels: Enzyme Protection and Zinc Sensing

Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization process, both the biological activity and the structural integrity of the enzymes. Here we report, for the first time, a proof-of-concept study for embedding active carbonic anhydrase (CA) into polyacrylamide (PAAm) nanogels. By immobilizing CA in these nanogels, we hope to provide important advantages, such as matrix protection of the CA as well as its targeted delivery, and also for potentially using these nanogels as zinc nano-biosensors, both in-vitro and in-vivo.

Two methods are reported here for CA immobilization: encapsulation and surface conjugation. In the encapsulation method, the common process was improved, so as to best preserve the CA, by 1) using a novel biofriendly nonionic surfactant system (Span 80/Tween 80/Brij 30) and 2) using an Al₂O₃ adsorptive filtration purification procedure. In the surface conjugation method, blank PAAm nanogels were activated by N-hydroxysuccinimide and the CA was cross-linked to the nanogels. The amount of active CA immobilized in the nanoparticles was quantified for both methods. Per 1 g nanogels, the CA encapsulated nanogels contain 11.3 mg active CA, while the CA conjugated nanogels contain 22.5 mg active CA. Also, the CA conjugated nanoparticles successfully measured free Zn²⁺ levels in solution, with the Zn²⁺ dissociation constant determined to be 9 pM.

This work demonstrates universal methods for immobilizing highly fragile bio-macromolecules inside nanoparticle carriers, while preserving their structural integrity and biological activity. The advantages and limitations are discussed, as well as the potential biomedical applications.