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

The study addresses the limitation of chemodynamic therapy that relies on endogenous hydrogen peroxide within the tumor microenvironment, where H2O2 levels typically remain below ~100 µM due to redox homeostasis, constraining hydroxyl radical (·OH)-mediated tumor killing. Single-atom catalysts (SACs) have emerged as nanozymes with well-defined coordination environments enabling enzyme-like catalysis (e.g., peroxidase-like). Prior SAC-based tumor therapies commonly used Fe single atoms or peroxidase-mimicking activity to generate ·OH from endogenous H2O2. To overcome the insufficiency of intratumoral H2O2, this work aims to engineer atomically dispersed Au on zero-valent Cu nanocubes via galvanic replacement to catalyze the sequential O2 reduction to H2O2 and subsequent Fenton-like conversion to ·OH, thereby self-supplying H2O2 from ambient O2. The hypothesis is that Au single atoms modulate the electronic structure of Cu0 to boost O2 reduction kinetics and favor ·OH generation, while the degradability of the nanocubes enables renal clearance and minimizes long-term accumulation.

Recent approaches to enhance ROS availability for tumor catalytic therapy include: (1) parallel catalytic behavior to simultaneously generate superoxide (O2−) and ·OH via peroxidase-like activity; (2) co-catalysis using MoS2 supports to accelerate Fe3+ to Fe2+ conversion in Fenton reactions; (3) integrated cascade reactions coupling Fenton- and peroxidase-like activities to concurrently generate ·OH and O2−; (4) MOF-derived flower-like structures offering 3D accessibility of active sites to boost ·OH production; and (5) copper hexacyanoferrate single-site nanozymes catalyzing glutathione oxidation to form H2O2 with concurrent Cu2+ to Cu+ conversion for subsequent Fenton-like ·OH generation. While galvanic replacement has been used to form single-atom alloys (e.g., Cu/Pt(SA), Cu/Pd(SA), Ni/Pt(SA), Pd/Au(SA)) for hydrogenation or glucose oxidation, its application to construct SACs for O2→H2O2→·OH tumor therapy remains underexplored.

Synthesis: Hydrophobic Cu nanocubes were synthesized and transferred to water using CTAB and PVP. Galvanic replacement was performed by adding HAuCl4 (50 mM; 5–100 µL) to a fixed amount of Cu nanocubes (10 ppm, 100 µL) to obtain AuxCuy compositions (e.g., Au0.02Cu0.98, Au0.05Cu0.95, Au0.1Cu0.9, Au0.15Cu0.85). Composition was quantified by atomic absorption. For comparison, Au nanocubes with similar size were prepared. SA (stearic acid) surface modification was conducted (0.5% SA in ethanol/water, sonication, wash) to obtain Au0.02Cu0.98@SA, intended to suppress premature ROS generation during circulation. Characterization: UV–Vis SPR, TEM/HR-TEM, electron diffraction, synchrotron powder XRD with Rietveld refinement for phase quantification and grain size (Scherrer analysis), XAS (XANES/EXAFS) at Au L3-edge and Cu K-edge using SDD detection (Au Lα window) to determine oxidation states and local coordination; XPS for Cu2+ formation upon incubation. ROS assays: H2O2 detection by KMnO4 colorimetry (absorbance at 525 nm) and hydrogen peroxide assay kit (fluorescence λem=510 nm, λex=490 nm) with time- and concentration-dependence; ·OH detection by terephthalic acid probe (λem=425 nm, λex=315 nm) across pH, and ESR using DMPO spin-trapping (PBS, 0.1 M). Stability/degradability: Incubation of Au0.02Cu0.98 and Au0.02Cu0.98@SA in water, PBS pH 7, PBS pH 5.5 at 37 °C up to 7 days with TEM monitoring of morphology; XPS after PBS exposure; evaluation of H2O2 and ·OH suppression by SA coating via KMnO4 and TPA assays. DFT simulations: VASP with PAW-PBE, DFT-D3, spin polarization; plane-wave cutoff 400 eV; slabs of FCC (100) surfaces for pure Cu, Au0.02Cu0.98, Au0.5Cu0.5, pure Au with six layers (bottom three fixed), vacuum 15 Å; Brillouin sampling 10×10×10 (unit cell) and 5×5×1 (supercell); CI-NEB for transition states; implicit solvent (vaspsol, water ε=78.3553). Calculated adsorption energies (side-on vs end-on O2), water dehydrogenation barriers, O2 hydrogenation barriers to OOH and H2O2, and energetics of H2O2 desorption vs ·OH formation; ELF and PDOS analyses. Cell studies: HepG2-Red-FLuc hepatocellular carcinoma and HUV-EC-C endothelial cells cultured under standard conditions; MTT cytotoxicity (up to 100 ppm Cu equivalent); live/dead staining (Calcein-AM/PI); flow cytometry (Annexin V-FITC/PI); intracellular Cu+ (CopperGreen), H2O2 (assay kit), and ·OH (APF) imaging over time; hemolysis assay with 2% RBCs. In vivo studies: Biosafety in C57BL/6 mice (single IV dose 100 µL of 600 ppm Au0.02Cu0.98@SA or PBS), body weight monitoring, serum biochemistry (ALT, ALP, AST, T-Bil, BUN, CREA, UA), H&E of major organs. Biodistribution in female SCID mice by ICP-AES of Cu in organs and urine after IV injection. Orthotopic HepG2-Red-FLuc liver tumor model in NOD-SCID mice; treatments with Au@SA, Au0.05Cu0.95@SA, Cu@SA, or Au0.02Cu0.98@SA; tumor monitoring by IVIS bioluminescence; post-mortem IVIS of livers; IHC for γ-H2AX (Ser139) and cleaved caspase-3 (Asp175).

• Galvanic replacement produced AuxCuy nanocubes with tunable Au content; Au0.02Cu0.98 exhibited atomically dispersed Au on Cu0 surfaces as evidenced by diminished Au(111) XRD intensity and HR-TEM single-atom visualization.
• XANES showed Au and Cu remained predominantly zero-valent after galvanic replacement; EXAFS supported local structures consistent with Au single atoms on Cu.
• ROS generation: Among Cu, Au, and AuxCuy, Au0.02Cu0.98 generated the highest levels of H2O2 (colorimetry at 525 nm; fluorescence at 510 nm) and ·OH (TPA fluorescence at 425 nm; ESR DMPO-OH quartet), with strong enhancement under acidic conditions (pH 5.5). H2O2 production increased over time and with concentration for Au0.02Cu0.98.
• Degradability: Au0.02Cu0.98 nanocubes degraded rapidly in PBS pH 5.5 (near-complete disintegration within 1 day) and showed dissolution in water after 3 days; XPS confirmed increased Cu2+ after 1 day in PBS pH 7. SA coating (Au0.02Cu0.98@SA) suppressed H2O2 and ·OH generation and preserved morphology for at least 7 days across media.
• DFT: O2 side-on adsorption energies (eV): Cu −2.12; Au0.02Cu0.98 −2.00; Au0.5Cu0.5 −1.16; Au −0.42. Hydrogenation barriers (eV) for O2→OOH and OOH→H2O2: Cu 1.32/1.29; Au0.02Cu0.98 with Au site 0.75/0.76 and without Au 0.98/0.99; Au0.5Cu0.5 1.15/0.61; Au 0.48/0.74. Despite low barriers on Au, weak O2 adsorption limits reactivity. Energetics for H2O2 desorption vs ·OH formation (eV): Cu 0.35 vs −0.03; Au0.02Cu0.98 0.34 vs −0.01, indicating thermodynamic favorability for ·OH on Cu and Au0.02Cu0.98 but not on Au0.5Cu0.5 or Au. ELF indicated electron localization changes with Au content, rationalizing activity trends.
• In vitro: Au0.02Cu0.98@SA induced cancer cell death (MTT reduction, live/dead staining) and apoptosis (Annexin V/PI), with time-dependent intracellular Cu+ release, H2O2, and ·OH production in HepG2 cells. Minimal hemolysis at 20 ppm and acceptable viability in endothelial cells were observed.
• In vivo: Au0.02Cu0.98@SA showed favorable biosafety (body weight, serum biochemistry, and H&E normal in major organs). Biodistribution indicated organ accumulation and urinary excretion of Cu. In orthotopic HepG2-Red-FLuc liver tumors, Au0.02Cu0.98@SA significantly reduced tumor bioluminescence compared to controls and other formulations; increased γ-H2AX and cleaved caspase-3 staining supported DNA damage and apoptosis in tumors.

The results demonstrate that incorporating single Au atoms into zero-valent Cu nanocubes optimizes the balance between strong O2 adsorption (Cu-rich surface) and lowered kinetic barriers for O2 hydrogenation (Au-modulated sites), enabling effective conversion of ambient O2 to H2O2 and subsequent Fenton-like ·OH generation without relying on limited endogenous H2O2. The Au0.02Cu0.98 composition uniquely combines favorable adsorption, reduced activation barriers, and thermodynamics that bias ·OH formation over H2O2 desorption, explaining its superior ROS output. The degradable nature of the nanocubes ensures that Cu ions released under acidic tumor conditions participate in oxygen reduction and Fenton-like reactions, while SA coating prevents off-target ROS during circulation, preserving safety until tumor accumulation. In vitro assays confirm intracellular Cu+ increase and ROS generation leading to apoptosis, and in vivo orthotopic tumor studies show significant tumor suppression and apoptosis markers, indicating therapeutic efficacy. Collectively, the findings validate the proposed O2→H2O2→·OH cascade catalyzed by Au0.02Cu0.98 nanocubes as a viable strategy to overcome endogenous H2O2 limitations in chemodynamic therapy.

This work establishes a galvanic replacement route to fabricate atomically dispersed Au on degradable zero-valent Cu nanocubes that self-supply H2O2 from ambient O2 and enhance Fenton-like ·OH production for tumor therapy. The Au0.02Cu0.98 composition exhibits optimal catalytic performance, confirmed by spectroscopy, ROS assays, and DFT analyses, and demonstrates degradability and renal-clearability. SA surface modification suppresses premature ROS generation, improving safety. In vitro and orthotopic in vivo studies indicate potent antitumor efficacy with acceptable biosafety. Future research should optimize dosing and delivery, assess long-term pharmacokinetics and toxicity, explore tumor selectivity and microenvironmental factors (e.g., oxygen levels), expand to additional tumor models, and investigate other SAC architectures or supports to further enhance catalytic efficiency and specificity.

• The therapeutic evaluations were conducted in limited animal numbers (n≈3 per group) and in a single orthotopic tumor model, which may constrain generalizability.
• Long-term biodistribution, clearance, and chronic toxicity were not fully characterized beyond short-term assays.
• The approach relies on available oxygen; efficacy under hypoxic tumor conditions may be reduced and requires further investigation.
• Potential oxidative stress during circulation necessitated SA coating; the stability of the coating in vivo and potential off-target effects over extended periods remain to be comprehensively assessed.
• Detailed quantitative pharmacokinetics and dose–response relationships were not exhaustively reported.