Nanographene Oxide Enables Activity-Preserving Immobilization and Enhanced Stability of Galactose Oxidase

Abstract

Enzyme-mediated generation of reactive oxygen species (ROS) has attracted considerable interest for cancer-related applications. However, the biomedical use of oxidase enzymes is often limited by their intrinsic instability and rapid loss of catalytic activity under physiological conditions. Here, we report a nanographene oxide (nGO)-based platform that enables activity-preserving immobilization and stabilization of galactose oxidase (GaOX), a copper-containing oxidase that catalyzes hydrogen peroxide (H2O2) generation using D-galactose as a substrate. GaOX was physically immobilized onto nGO through non-denaturing interactions with high loading efficiency, followed by surface coating with Pluronic F127 to form a stable nanocomplex (F127@nGO-GaOX). Structural analysis confirmed that immobilized GaOX retained its secondary structure and catalytic parameters, including substrate affinity and catalytic efficiency. Notably, the immobilized enzyme exhibited markedly enhanced stability under serum-containing physiological conditions compared with the native enzyme. In addition, the F127@nGO-GaOX nanocomplex facilitated efficient cellular internalization, enabling intracellular ROS generation in the presence of galactose and resulting in enhanced cytotoxicity in cancer cells in vitro. These findings establish nGO as a nanocarrier-based formulation platform that preserves oxidase activity while improving enzyme stability and intracellular delivery. This work introduces GaOX as a ROS-generating therapeutic enzyme candidate and provides a simple strategy for developing enzyme-based formulations for biomedical applications.