Nitroxide-modified protein-incorporated nanoflowers with dual enzyme-like activities

Purpose: Combined superoxide dismutase (SOD)/catalase mimetics have attracted much attention because of their efficacy against reactive oxygen species-associated diseases; however, their application is often limited owing to their poor stability and the absence of favorable grafting sites. To address this, we developed a new class of SOD/catalase mimetics based on hybrid nanoflowers, which exhibit superior stability and possess the desired grafting sites for drugs and endogenous molecules. Methods: In this work, for the first time, we used polynitroxylated human serum albumin (PNA) to mediate the formation of hybrid copper-based nanoflowers. H₂O₂ depletion and O₂ evolution assays were first performed to determine the catalase-like activity of the hybrid nanoflowers. Next, the xanthine oxidase/cytochrome c method was used to assay the SOD-like activity of the nanoflowers. Further characteristics of the nanoflowers were evaluated using scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), and Fourier-transform infrared spectroscopy (FTIR). Operational stability was assessed via the reusability assay. Results: The H₂O₂ depletion and O₂ evolution assays indicated that PNA-incorporated nanoflowers have genuine catalase-like activity. Kinetic analysis revealed that the reactions of the incorporated nanoflowers with H₂O₂ not only obey Michaelis–Menton kinetics, but that the nanoflowers also possess a higher affinity for H₂O₂ than that of native catalase. The FTIR spectra corroborated the presence of PNA in the hybrid nanoflowers, while the EPR spectra confirmed the intermolecular interaction of nitroxides bound to the human serum albumin incorporated into the nanoflowers. The remarkable operational reproducibility of the hybrid nanoflowers in catalase-like and SOD-like reactions was verified across successive batches. Conclusion: Herein, a comparison of Michaelis constants showed that the hybrid nanoflower, a catalase mimetics, outperforms the native catalase. Acting as a “better-than-nature” enzyme mimetics, the hybrid nanoflower with superior stability and desired ligand grafting sites will find widespread utilization in the medical sciences.