Copper(II) Oxide Spindle-like Nanomotors Decorated with Calcium Peroxide Nanoshell as a New Nanozyme with Photothermal and Chemodynamic Functions Providing ROS Self-Amplification, Glutathione Depletion, and Cu(I)/Cu(II) Recycling

Abstract

Uniform, mesoporous copper(II) oxide nanospindles (CuO NSs) were synthesized via a method based on templated hydrothermal oxidation of copper in the presence of monodisperse poly(glycerol dimethacrylate-co-methacrylic acid) nanoparticles (poly(GDMA-co-MAA) NPs). Subsequent decoration of CuO NSs with a CaO₂ nanoshell (CuO@CaO₂ NSs) yielded a nanozyme capable of Cu(I)/Cu(II) redox cycling. Activation of the Cu(I)/Cu(II) cycle by exogenously generated H₂O₂ from the CaO₂ nanoshell significantly enhanced glutathione (GSH) depletion. CuO@CaO₂ NSs exhibited a 2-fold higher GSH depletion rate compared to pristine CuO NSs. The generation of oxygen due to the catalase (CAT)-like decomposition of H₂O₂ by CuO@CaO₂ NSs resulted in a self-propelled diffusion behavior, characteristic of a H₂O₂ fueled nanomotor. These nanostructures exhibited both peroxidase (POD)-like and CAT-like activities and were capable of self-production of H₂O₂ in aqueous media via a chemical reaction between the CaO₂ nanoshell and water. Usage of the self-supplied H₂O₂ by the POD-like activity of CuO@CaO₂ NSs amplified the generation of toxic hydroxyl (^•OH) radicals, enhancing the chemodynamic effect within the tumor microenvironment (TME). The CAT-like activity provided a source of self-supplied O₂ via decomposition of H₂O₂ to alleviate hypoxic conditions in the TME. Under near-infrared laser irradiation, CuO@CaO₂ NSs exhibited photothermal conversion properties, with a temperature elevation of 25 °C. The combined GSH depletion and H₂O₂ generation led to a more effective production of ^•OH radicals in the cell culture medium. The chemodynamic function was further enhanced by an elevated temperature. To assess the therapeutic potential, CuO@CaO₂ NSs loaded with the photosensitizer, chlorine e6 (Ce6), were evaluated against T98G glioblastoma cells. The synergistic combination of photodynamic, photohermal, and chemodynamic modalities using CuO@CaO₂@Ce6 NSs resulted in cell death higher than 90% under in vitro conditions.

Keywords: Fenton-like reaction; chemodynamic therapy; glutathione depletion; nanozyme; peroxidase-like activity.

Figure 1

Schematic representation of the chemical route used for the synthesis of CuO@CaO₂@Ce6 NSs.

Figure 2

SEM photographs of poly(GDMA-co-MAA) NPs obtained by precipitation polymerization used as a template in the synthesis of CuO NSs. Magnification: (A) 10.0 and (B) 25.0 KX. (C,D) SEM photographs of Cu(OH)₂/polymethacrylate composite nanoparticles. Magnification: 30.0 and 60.0 KX for (C,D), respectively. (E) SEM photograph of CuO NSs synthesized in the presence of poly(GDMA-co-MAA) NPs. Magnification: 20.1 KX. (F,G) SEM photographs of CuO NSs synthesized in the absence of poly(GDMA-co-MAA) NPs. Magnification: 30.0 and 70.0 KX for (F,G), respectively.

Figure 3

(A) SEM photographs of CuO@CaO₂ and CuO@CaO₂@Ce6 NSs: NS type and magnification: (i) CuO@CaO₂, 25.0 KX, (ii) CuO@CaO₂@Ce6, 25.1 KX, (B) EDX spectroscopy images of CuO@CaO₂ NSs. (C) TEM photographs of CuO and CuO@CaO₂ NSs. NS type: (i,ii) CuO, (iii) CuO@CaO₂, and (iv) CaO₂ nanoshell on CuO NSs. The scale bars are given on the photographs.

Figure 4

(A) Hydrodynamic size distributions, (B) XRD patterns, (C) nitrogen adsorption–desorption isotherms, and (D) pore size distribution curves of CuO and CuO@CaO₂ NSs.

Figure 5

XPS of CuO@CaO₂@Ce6 NSs. Core level spectra for (A) Cu 2p scan, (B) O 1s scan, (C) C 1s scan, (D) Ca 2p scan, and (E) N 1s scan.

Figure 6

(A) Trajectory of the motion originated from the nanomotor function of CuO@CaO₂ NSs on XY plane for different NS clusters. (B) Velocity of different NSs.

Figure 7

Sample UV–vis spectra recorded for GSH depletion with (A) CuO NSs and (B) CuO@CaO₂ NSs using DTNB as the reactive probe. Concentration of CuO and CuO@CaO₂ NSs: 0.2 mg/mL. The variation of GSH depleted with the time by (C) CuO NSs and (D) CuO@CaO₂ NSs at different concentrations. GSH initial concentration: 1 mM, reaction volume: 10 mL, temperature: 37 °C.

Figure 8

(A) Fluorescence spectra showing the generation of ^•OH radicals by CuO and CuO@CaO₂ NSs, excitation: 315 nm, emission: 430 nm, fluorescent probe: 2,5-DHTPA. The concentrations of CuO and CuO@CaO₂ NSs: 0.5 and 1.0 mg/mL. Temperature: 25 or 45 °C. (B) Generation of H₂O₂ by CuO@CaO₂ NSs. CuO@CaO₂ concentration: 2.0 mg/mL.

Figure 9

ROS generation behaviors of CuO@CaO₂ and CuO@CaO₂@Ce6 NSs. (A) ¹O₂ radical generation by CuO@CaO₂ and CuO@CaO₂@Ce6 NSs. Probe: DPBF, concentration of CuO@CaO₂ and CuO@CaO₂@Ce6 NSs: 5.0 mg/mL. (B) Intracellular ROS formation with CuO@CaO₂ and CuO@CaO₂@Ce6 NSs with T98G cells. (i) Control, (ii) CuO@CaO₂@Ce6 NSs, and (iii) CuO@CaO₂@Ce6 with NIR laser@808 nm for 5 min + LED@650 nm irradiation for 7 min. T98G cell concentration: 2 × 10⁴ cells/well, concentration of CuO@CaO₂ and CuO@CaO₂@Ce6 NSs: 0.25 mg/mL.

Figure 10

Michaelis–Menten plots for (A) POD-like activities of CuO and CuO@CaO₂ NSs, (B) self-POD-like activity of CuO@CaO₂ NSs without adding H₂O₂ using OPDA as the substrate at pH 7.0, (C) self-POD-like activity of CuO@CaO₂ NSs without adding H₂O₂ using TMB as the substrate at pH 5.0, (D) OD-like activity of CuO NSs using OPDA as the substrate at pH 7.0, (E) OD-like activity of CuO NSs using TMB as the substrate at pH 5.0, and (F) CAT-like activity of CuO NSs. Nanozyme concentration: 2.0 mg/mL. Temperature: 25 °C.

Figure 11

(A) Temperature elevation curves obtained with different concentrations of CuO@CaO₂@Ce6 NSs under NIR laser (808 nm) irradiation: power density: 1.3 W/cm². (B) Change of total temperature increases in 300 s with the concentration of CuO@CaO₂@Ce6 NSs. (C) Consecutive heating/cooling curves with CuO@CaO₂@Ce6 NSs at a concentration of 1.0 mg/mL.

Figure 12

(A) Representative live/dead cell images of T98G cells stained with AO/PI after treatment with CuO, CuO@CaO₂, and CuO@CaO₂@Ce6 NSs under different conditions. The images in the first three columns were obtained without using any light irradiation in the presence of CuO, CuO@CaO₂, and CuO@CaO₂@Ce6 NSs. PDT effect was applied by LED irradiation for 7 min at different concentrations of CuO@CaO₂@Ce6 NSs. The PTT effect was applied by NIR laser irradiation for 5 min at different concentrations of CuO@CaO₂@Ce6 NSs. Simultaneous PDT + PTT effects were applied by LED irradiation for 7 min and NIR laser irradiation for 5 min at different concentrations of CuO@CaO₂@Ce6 NSs. T98G cell density: 2 × 10⁴ cells/well, scale bar: 200 μm. (B) MTT results for the viability of T98G glioblastoma cells after treatment with CuO, CuO@CaO₂, and CuO@CaO₂@Ce6 NSs at different concentrations under LED (650 nm) and NIR laser (808 nm) irradiations. T98G concentration: 2 × 10⁴ cells/well, LED irradiation time: 7 min (0.8 W), NIR irradiation time: 5 min (1.3 W/cm²).