Inflammation, a crucial defense mechanism against infection, injury, and toxins, can become detrimental when excessive and uncontrolled. This uncontrolled inflammation is linked to numerous diseases, including cardiovascular disease, hepatitis, nephritis, and impaired wound healing. Oxidative stress, characterized by an imbalance between ROS production and antioxidant defense, plays a significant role in exacerbating inflammation. Excessive ROS in inflammatory responses intensifies tissue damage and leads to chronic inflammation. Current therapeutic strategies focus on scavenging ROS using antioxidants like N-acetyl cysteine and acetyl-L-carnitine. However, limitations such as poor bioavailability, low stability, and efficacy hinder their clinical application. Nanomedicine offers innovative approaches for ROS clearance, employing functional nanomaterials such as carbon, ceria, platinum, redox polymers, and polyphenol nanoparticles. Nanozymes, which mimic natural antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), represent a promising strategy to restore redox balance. Ideal nanozymes should possess high ROS scavenging capabilities, broad-spectrum activity, stability in disease environments, and rapid clearance for biocompatibility. Ultrasmall nanoparticles (hydrodynamic diameter <5.5 nm) are particularly attractive due to their high surface-to-volume ratio, leading to enhanced catalytic activity, and their capacity for rapid renal clearance through glomerular filtration. While some ultrasmall ROS scavenging nanomaterials have been developed, limitations such as low catalytic activity and high cost remain. This research aimed to synthesize a simple, cost-effective, and highly efficient ultrasmall copper-based nanozyme to address the challenges of existing ROS-related disease treatments.

The literature extensively documents the role of oxidative stress in inflammation and various diseases. Many studies highlight the benefits of ROS scavenging as a therapeutic strategy. Existing small molecule antioxidants such as N-acetyl cysteine and acetyl-L-carnitine have shown promise, but limitations in bioavailability and efficacy restrict their widespread use. The field of nanomedicine has emerged as a promising area for developing novel ROS scavengers, with research focusing on different nanomaterials with antioxidant properties. Existing literature demonstrates the successful development of various nanozymes, which mimic the function of natural antioxidant enzymes. These include nanozymes based on different materials such as carbon, ceria, and platinum. However, many of these nanozymes suffer from limitations regarding catalytic efficiency, cost-effectiveness, or biocompatibility. This study builds upon previous work exploring the use of copper-based nanoparticles for ROS scavenging, leveraging the known role of copper in various human enzymes. The researchers aimed to improve upon existing copper-based nanomaterials by developing ultrasmall nanoparticles with enhanced ROS scavenging capabilities and improved biocompatibility.

The researchers synthesized ultrasmall Cu₅₄₀ nanoparticles (Cu₅₄₀ USNPs) using a green, rapid, and cost-effective one-step method. They optimized the synthesis parameters, including the ratio of Cu²⁺ to L-ascorbic acid (AA), reaction temperature, and time, to achieve uniform nanoparticles with optimal catalytic activity. Transmission electron microscopy (TEM) was used to characterize the size and morphology of the synthesized USNPs. X-ray diffraction (XRD) and X-ray Auger electron spectroscopy (XAES) were employed to determine the composition and oxidation states of copper in the nanoparticles. The ROS scavenging activity of the Cu₅₄₀ USNPs was assessed using various methods: hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻), and hydroxyl radical (•OH) scavenging assays. The ABTS radical scavenging assay was also performed to evaluate the overall free radical scavenging capacity. Enzyme-mimicking activities (catalase-like, superoxide dismutase-like, and glutathione peroxidase-like) were investigated using appropriate assays. The in vitro biocompatibility of the USNPs was evaluated using a cell viability assay (CCK-8) and a hemolysis assay. In vivo biocompatibility studies were conducted using mice, assessing the effects of the USNPs on blood chemistry, inflammatory cytokine levels, and major organ histopathology. The pharmacokinetics and biodistribution of the USNPs in mice were investigated using inductively coupled plasma-atomic emission spectrometry (ICP-AES) and TEM. The therapeutic efficacy of Cu₅₄₀ USNPs was evaluated using established animal models of acute kidney injury (AKI), induced by glycerol or cisplatin, acute liver injury (ALI), and diabetic wound healing. For AKI, survival rates, serum creatinine (CRE) and blood urea nitrogen (BUN) levels, and histological analysis of kidney tissues were assessed. For ALI, serum alanine aminotransferase (ALT) and aspartate transaminase (AST) levels and histological analysis of liver tissues were evaluated. For wound healing, wound closure rates and histological analysis of wound tissues were performed. Finally, transcriptomics analysis was performed on kidney tissues from AKI mice to elucidate the molecular mechanisms underlying the therapeutic effects of the Cu₅₄₀ USNPs. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were also performed to validate the transcriptomic findings.

The synthesized Cu₅₄₀ USNPs exhibited an average diameter of 3.5-4.0 nm (dry state) and approximately 4.5 nm (hydrodynamic diameter), meeting the renal filtration threshold. They demonstrated remarkable ROS scavenging activity across a broad spectrum, with significantly lower working concentrations compared to other reported nano-antioxidants (25 ng mL⁻¹ in vitro and 2 µg kg⁻¹ in vivo for AKI). Cu₅₄₀ USNPs showed significant catalase-like, superoxide dismutase-like, and glutathione peroxidase-like activities. In vitro studies using HEK293 cells showed that Cu₅₄₀ USNPs effectively reduced ROS levels and protected cells from H₂O₂-induced damage. In vivo studies demonstrated excellent biocompatibility with minimal toxicity, even after repeated administration. Pharmacokinetic analysis revealed a relatively short blood circulation half-life and primarily renal clearance. Cu₅₄₀ USNPs showed significant therapeutic efficacy in AKI models (glycerol- and cisplatin-induced), significantly improving survival rates and kidney function indicators (BUN and CRE). The therapeutic effect extended to ALI models, where Cu₅₄₀ USNPs reduced liver damage markers (AST and ALT). Additionally, Cu₅₄₀ USNPs accelerated diabetic wound healing. Transciptomic analysis of AKI mice revealed that Cu₅₄₀ USNPs treatment modulated gene expression related to glutathione metabolism, MAPK signaling, and TNF signaling pathways, suggesting a role in suppressing inflammation and promoting tissue repair. Specifically, Cu₅₄₀ USNPs downregulated the MAPK and TNF signaling pathways and upregulated the expression of various antioxidant genes and genes associated with tissue repair.

The findings of this study demonstrate that ultrasmall Cu₅₄₀ USNPs are a promising therapeutic agent for ROS-related diseases. The exceptionally low working concentration and broad-spectrum ROS scavenging activity of Cu₅₄₀ USNPs are significant advancements over previously reported nano-antioxidants. The multi-enzyme mimicking capabilities of Cu₅₄₀ USNPs allow for a comprehensive approach to ROS detoxification and cytoprotection. The ultrasmall size and rapid renal clearance ensure excellent biocompatibility and minimize potential long-term toxicity. The effectiveness across multiple disease models (AKI, ALI, and diabetic wound healing) highlights the versatility of this approach. The transcriptomic analysis provided mechanistic insights, suggesting that the therapeutic effect is not solely attributable to ROS scavenging but also involves the modulation of crucial signaling pathways associated with inflammation and tissue repair. These results provide a strong basis for further development and clinical translation of Cu₅₄₀ USNPs.

This research successfully synthesized and characterized ultrasmall, biocompatible Cu₅₄₀ USNPs with remarkable ROS scavenging and multi-enzyme mimicking capabilities. These nanoparticles demonstrated significant therapeutic efficacy in preclinical models of AKI, ALI, and diabetic wound healing. The study's findings suggest a promising avenue for developing next-generation nanozymes for the treatment of various ROS-related diseases. Future research should focus on further optimizing the synthesis and formulation of Cu₅₄₀ USNPs, exploring their long-term effects, and conducting clinical trials to evaluate their efficacy and safety in humans.

While the study demonstrates significant promise, certain limitations should be acknowledged. The in vivo studies were conducted on animal models, and the results might not be directly translatable to humans. Long-term toxicity studies are needed to fully assess the safety profile of these nanoparticles. The precise mechanisms by which the nanoparticles interact with cells and modulate gene expression require further investigation. The study focused on a limited set of disease models, and further studies are necessary to determine the effectiveness of Cu₅₄₀ USNPs across a broader range of ROS-related conditions.