Engineering Glutathione Peroxidase-Loaded Polymeric Nanogels Through a Grafting-To Route for Enhanced Enzyme Stability and Activity

Nanogels provide unique opportunities for stabilizing fragile enzymes through soft, hydrated polymer networks. Here, we report the development of a glutathione peroxidase (GPx)-loaded nanogel (GPxNG) engineered via a mild "grafting-to" epoxy-amine coupling strategy to enhance enzyme stability and antioxidant function. An amphiphilic copolymer composed of methacrylated 2,2,6,6-tetramethyl-4-piperidyl (PMA) and glycidyl methacrylate (GMA) was synthesized by controlled reversible addition-fragmentation chain-transfer (RAFT) polymerization using a poly(ethylene glycol) (PEG) macro-chain transfer agent (macro-CTA), yielding well-defined polymer chains with reactive epoxy groups. Covalent conjugation between polymer epoxides and GPx enzyme surface amines generated soft, PEGylated nanogels with high coupling efficiency, uniform particle sizes, and excellent colloidal stability. The engineered nanogels exhibited shear-thinning injectability, robust storage stability, and non-cytotoxic behavior in RAW 264.7 macrophages. Compared with native GPx enzyme, GPxNGs demonstrated significantly enhanced reactive oxygen species (ROS) scavenging activity, including strong inhibition of lipid peroxidation and copper-induced low-density lipoprotein (LDL) oxidation. Importantly, the nanogels preserved GPx enzyme activity after extended storage, freeze-thaw cycles, and repeated catalytic use, whereas the free enzyme rapidly lost function. This protective effect arises from the nanoscale confinement of the GPx enzyme within the flexible PEG-based network, which limits unfolding and aggregation. Overall, this work introduces a simple and biocompatible "grafting-to" nanogel platform capable of stabilizing redox-active enzymes without harsh conditions. The GPx nanogels combine high enzymatic preservation, potent antioxidant activity, and excellent handling properties, highlighting their potential as a therapeutic nanoplatform for mitigating oxidative stress-associated disorders such as atherosclerosis.