Enzymes are biological catalysts that have many potential industrial and biomedical applications. However, the widespread use of enzymes in the industry has been limited by their instability and poor recovery. In biomedical applications, systemic administration of enzymes has faced two main challenges: limited bioactivity mostly due to rapid degradation by proteases and immunogenic activity, since most enzymes are from nonhuman sources. Herein, we propose a robust enzyme-encapsulation strategy to mitigate these limitations. Catalase (CAT) was encapsulated in nanoporous silica nanoparticles (CAT-SiNPs) by first chemically modifying the enzyme surface with a silica precursor, followed by silica growth and finally poly(ethylene glycol) (PEG) conjugation. The formulation was carried out in mild aqueous conditions and yielded nanoparticles (NPs) with a mean diameter of 230 ± 10 nm and a concentration of 1.3 ± 0.8 × 10¹² NPs/mL. CAT-SiNPs demonstrated high enzyme activity, optimal protection from proteolysis by proteinase K and trypsin, and excellent stability over time. In addition, a new electrochemical assay was developed to measure CAT activity in a rapid, simple, and accurate manner without interference from chromophore usually present in biological samples. Concentrations of 2.5 × 10¹⁰ to 80 × 10¹⁰ CAT-SiNPs/mL not only proved to be nontoxic in cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay but also conferred cell protection when cells were exposed to 1 mM hydrogen peroxide (H₂O₂). Finally, the ability of CAT-SiNPs to release oxygen (O₂) when exposed to H₂O₂ was demonstrated in vivo using a rat model. Following the direct injection of CAT-SiNPs in the left kidney, partial pressure of oxygen (pO₂) increased by more than 30 mmHg compared to the contralateral control kidney during the systemic infusion of safe levels of H₂O₂. This pilot study highlights the potential of CAT-SiNPs to generate O₂ to relieve hypoxia in tissues and potentially sensitize tumors against radiation therapy.

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