<p>Nitroxide-Modified Protein-Incorporated Nanoflowers with Dual Enzyme-Like Activities</p>

Abstract

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. H 2 O 2 depletion and O 2 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 H 2 O 2 depletion and O 2 evolution assays indicated that PNA-incorporated nanoflowers have genuine catalase-like activity. Kinetic analysis revealed that the reactions of the incorporated nanoflowers with H 2 O 2 not only obey Michaelis-Menton kinetics, but that the nanoflowers also possess a higher affinity for H 2 O 2 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.

Hybrid Nanoflowers Bloom From Disintegrated Food Proteins Towards Natural Pigments Stabilization

“…20−23 However, the preparation of protein-based HNFs currently mostly employs enzyme proteins for immobilization of enzymes to improve their stability and activity. 11,13,24 Food proteins have been rarely found to effectively produce HNFs and are limited to BSA, 25,26 concanavalin A, 27 immunoglobulin G, 28 human serum albumin, 29 hemoglobin, 30 and streptavidin 31 using the primitive method as introduced by Ge et al 9 Moreover, in most cases, these proteins are used because of their special enzyme mimicking activities or specific affinity with some substrates (especially antibodies), and the prepared HNFs are mainly applied as biomimetic catalysts or biosensors. We hypothesize that the limited use of food proteins for HNF fabrication is caused by the high-molecular weight and less flexible structures of general food proteins (particularly for plant proteins) compared to enzymes, where both factors have shown crucial roles in the formation of hierarchical structures by protein and inorganic species.…”

“…Among organic materials, only polysaccharides and polyphenols have been explored to carry some small biomolecules (e.g., 5-flurouracil and curcumin) for now. Compared to those two types of organic materials, protein is one of the most used biomaterials in the design of delivery systems because it not only can be easily obtained from numerous food resources but also has abundant functional groups that can combine with other biomolecules through hydrophobic interactions, hydrogen bonds, and electrostatic interactions. − Once incorporated into high surface-to-weight ratio HNFs, proteins might have a better stabilization of loaded cargos due to the synergistic effects of immobilized proteins as shown in enzyme protein-based HNFs. − However, the preparation of protein-based HNFs currently mostly employs enzyme proteins for immobilization of enzymes to improve their stability and activity. ,, Food proteins have been rarely found to effectively produce HNFs and are limited to BSA, , concanavalin A, immunoglobulin G, human serum albumin, hemoglobin, and streptavidin using the primitive method as introduced by Ge et al Moreover, in most cases, these proteins are used because of their special enzyme mimicking activities or specific affinity with some substrates (especially antibodies), and the prepared HNFs are mainly applied as biomimetic catalysts or biosensors. We hypothesize that the limited use of food proteins for HNF fabrication is caused by the high-molecular weight and less flexible structures of general food proteins (particularly for plant proteins) compared to enzymes, where both factors have shown crucial roles in the formation of hierarchical structures by protein and inorganic species .…”

Immobilization of Phospholipase A1 Using a Protein-Inorganic Hybrid System

“…Since the first report of protein–inorganic hybrid nanostructures in 2012 [ 24 , 25 , 26 ], this method had been successfully employed to immobilize many types of enzymes (i.e., laccase, lipase, carbohydrase, and cholesterol oxidase) [ 27 , 28 , 29 , 30 , 31 ]. For developing hybrid nanostructures, Cu 2+ , Ca 2+ , Mn 2+ , Mg 2+ , Zn 2+ , Co 2+ , and Fe 2+ are the mainly inorganic components that have been reported [ 32 , 33 , 34 , 35 ]. In general, depending on the high stability, durability, reusability, and biocompatibility, these protein–inorganic hybrid nanostructures show an extensive utilization in many fields such as biosensor manufacturing, food processing, and pharmaceuticals producing.…”