Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy

Single-atom catalysts (SACs) have emerged as a promising area of nanocatalysis, offering precise control over active sites and high catalytic efficiency. Their application in chemodynamic therapy, leveraging the generation of highly reactive hydroxyl radicals (·OH), is particularly intriguing. Traditional approaches often rely on endogenous H₂O₂ in the tumor microenvironment, but its concentration is often insufficient to achieve significant therapeutic effect. This research aims to address this limitation by developing a novel SAC-based nanomedicine that utilizes ambient oxygen as the primary source of ROS for enhanced tumor therapy. The use of oxygen as a readily available source offers a significant advantage over relying on the limited endogenous H₂O₂ levels found in tumors. The research question focuses on whether the strategic incorporation of atomically dispersed gold onto degradable zero-valent copper nanocubes can significantly enhance the production of ·OH radicals from oxygen, leading to improved tumor therapy. The context is the need for effective and biocompatible cancer treatments with minimal side effects. The purpose is to design, synthesize, and evaluate the efficacy of a novel nanomaterial for targeted cancer therapy. The importance lies in potentially overcoming the limitations of current chemodynamic therapies by providing a readily accessible source of ROS.

Previous studies employing single-atom catalysts (SACs) in tumor catalytic therapy predominantly focused on utilizing endogenous H₂O₂ for ·OH generation. However, the low intratumoral H₂O₂ levels often limit therapeutic efficacy. To address this, various strategies have been explored, including co-catalysis (e.g., using MoS₂ to accelerate Fenton reactions) and integrated cascade reactions to concurrently generate ·OH and O₂⁻. Recent efforts also explored the fabrication of nanozymes capable of producing H₂O₂ through glutathione-oxidase activity. This current work diverges from these approaches by focusing on utilizing ambient oxygen as a self-supplying source of H₂O₂ for the subsequent ·OH generation, circumventing the reliance on limited endogenous H₂O₂.

The researchers employed a galvanic replacement approach to synthesize atomically dispersed Au on degradable zero-valent Cu nanocubes. This method involved controlling the addition of HAuCl₄ precursor to achieve varying AuₓCuᵧ compositions. The resulting nanocubes were characterized using various techniques, including UV-Vis spectroscopy, TEM, XRD, and XAS. XAS, specifically XANES and EXAFS, provided crucial information on the oxidation states and local structures of Au and Cu atoms. The ability of the nanocubes to generate H₂O₂ and ·OH was assessed using colorimetric analysis (KMnO₄), fluorescence detection (hydrogen peroxide assay kit and terephthalic acid probe), and electron spin resonance (ESR) spectroscopy using DMPO. The stability and degradation properties of the nanocubes were evaluated under different conditions (water, PBS at pH 7 and 5.5). Density Functional Theory (DFT) calculations were performed to understand the reaction mechanisms involved in O₂ reduction to H₂O₂, and subsequent ·OH generation. In vitro cytotoxicity studies on HepG2-Red-FLuc liver cancer cells were conducted using MTT assays, live/dead cell staining, and flow cytometry to evaluate the anticancer potential. Finally, in vivo studies in mice were performed to assess biodistribution, tumor growth inhibition, and potential toxicity using bioluminescence imaging (IVIS) and histopathological analysis. Specific details include the use of a hydrophobic-based method for Cu nanocube synthesis using CTAB surfactant and PVP, with control over Au atom incorporation via varying HAuCl₄ precursor amounts. SA modification of the nanocubes was also employed to enhance stability. The DFT calculations employed a (100) surface model with hydrogen pre-coverage, considering both hydrogen formation and oxygen adsorption configurations. The implicit solvent model using vaspsol was used in the DFT calculations to mimic the catalytic environment in solution.

The study successfully synthesized Au/Cu⁰ nanocubes with varying Au compositions via galvanic replacement. XAS confirmed the predominant zero-valence state of Au and Cu. Au₀.₀₂Cu₀.₉₈ exhibited significantly enhanced ·OH generation compared to other compositions, attributed to the catalytic activity of single Au atoms. This enhanced ·OH generation was observed via O₂ reduction to H₂O₂, followed by Fenton-like reactions, effectively utilizing ambient oxygen. The nanocubes were found to be degradable, particularly under acidic conditions, suggesting potential for renal clearance. DFT simulations supported the experimental findings, demonstrating that the presence of Au atoms significantly reduces the activation barriers for O₂ reduction and ·OH formation. In vitro studies showed significant cytotoxicity against HepG2 liver cancer cells, with Au₀.₀₂Cu₀.₉₈ demonstrating higher efficacy than other compositions. In vivo studies in mice with orthotopic HepG2-Red-FLuc liver tumors demonstrated that Au₀.₀₂Cu₀.₉₈@SA (with stearic acid surface modification for enhanced stability) significantly inhibited tumor growth. Furthermore, the study showed that Au₀.₀₂Cu₀.₉₈ exhibited excellent biocompatibility with minimal toxicity to major organs in vivo. The SA modification effectively suppressed the premature generation of H₂O₂ and ·OH during blood circulation. Finally, the quantitative analysis via various methods such as colorimetric analysis, fluorescence detection, and ESR spectroscopy provided robust evidence to support the findings. The quantitative data, such as the absorbance intensities, fluorescence emission intensities, and ESR signal amplitudes, were presented and statistically analyzed to support the claims.

This research successfully demonstrates a novel strategy for chemodynamic therapy that overcomes the limitation of relying solely on endogenous H₂O₂. By utilizing ambient oxygen as the primary ROS source, the developed Au₀.₀₂Cu₀.₉₈ nanocubes exhibited enhanced ·OH generation and superior anticancer efficacy. The degradable and biocompatible nature of the nanocubes further enhances their clinical potential, minimizing potential toxicity concerns. The DFT simulations provide a strong mechanistic understanding supporting the experimental results, highlighting the synergistic effect between Au single atoms and zero-valent Cu in catalyzing the O₂ reduction and ·OH generation. The findings have significant implications for the development of advanced nanomaterials for targeted cancer therapy. Specifically, the approach of utilizing readily available oxygen as the source for the generation of reactive oxygen species (ROS) has the potential to overcome the limitations of current chemodynamic therapies. Moreover, the biodegradability and renal clearance characteristics provide a significant advantage over other nanomaterials, reducing the concerns about long-term toxicity.

This study successfully demonstrated the development and application of a novel SAC-based nanomaterial (Au₀.₀₂Cu₀.₉₈) for efficient tumor therapy. The utilization of ambient oxygen as a self-supplying source of H₂O₂ for ·OH generation, coupled with the biodegradability of the nanocubes, addresses key limitations in current chemodynamic therapies. Further research could explore the optimization of nanocube design and surface modifications for enhanced tumor targeting and therapeutic efficacy. Investigating the potential of this approach in other cancer types and exploring different oxygen delivery strategies would also be valuable.

While this study provides strong evidence of the efficacy of Au₀.₀₂Cu₀.₉₈ nanocubes, some limitations should be acknowledged. The in vivo studies were conducted on a specific model of liver cancer. Further studies are needed to evaluate the effectiveness in other cancer models and across different tumor types. Long-term toxicity studies are also necessary to fully evaluate the biocompatibility of this nanomaterial in extended periods. The DFT simulations, while providing valuable mechanistic insights, represent a simplified model of the complex interactions that occur in the biological environment. Further refinement of the computational model might be beneficial.