Tuning oxidant and antioxidant activities of ceria by anchoring copper single-site for antibacterial application

Abstract

The reaction system of hydrogen peroxide (H₂O₂) catalyzed by nanozyme has a broad prospect in antibacterial treatment. However, the complex catalytic activities of nanozymes lead to multiple pathways reacting in parallel, causing uncertain antibacterial results. New approach to effectively regulate the multiple catalytic activities of nanozyme is in urgent need. Herein, Cu single site is modified on nanoceria with various catalytic activities, such as peroxidase-like activity (POD) and hydroxyl radical antioxidant capacity (HORAC). Benefiting from the interaction between coordinated Cu and CeO₂ substrate, POD is enhanced while HORAC is inhibited, which is further confirmed by density functional theory (DFT) calculations. Cu-CeO₂ + H₂O₂ system shows good antibacterial properties both in vitro and in vivo. In this work, the strategy based on the interaction between coordinated metal and carrier provides a general clue for optimizing the complex activities of nanozymes.

Introduction

In the past decades, the irrational use of antibiotics worldwide has led to the growing problem of bacterial resistance, which has become one of the major threats to public health¹. Therefore, it is urgent to develop new broad-spectrum antibacterial methods. Recently, the antibacterial application of nanozymes, a new generation of nanomaterials mimicking the catalytic activities of natural enzyme (peroxidase, catalase, oxidase, etc.), has arouse researchers’ great concern^2,3. Among them, some nanozymes are able to catalyze the decomposition of hydrogen peroxide (H₂O₂) by simulating the activity of natural peroxidase (POD) to produce highly toxic hydroxyl radicals (·OH), so as to achieve bactericidal effect^4,5. Of all the massive antibacterial nanozyme families, single site enzymes (SSEs) have been exerting high catalytic POD activities, with maximized atom utilization efficiency^6,7. Since the substrates such as three-dimensional carbon materials exhibit poor catalytic performance, currently reported nanozyme + H₂O₂ system based on atomic level manipulation are mostly focused on modifying metal active sites to obtain POD-like activity^8,9. Yet, the complex catalytic activities of nanozymes in nanozymes + H₂O₂ system should be paid close attention.

As an emerging nanozyme, nanoceria (CeO₂) possesses multiple catalytic properties such as peroxidase-like activity (POD), catalase-like activity (CAT), oxidase-like activity (OXD), superoxide dismutase-like activity (SOD) and hydroxyl radical antioxidant activity (HORAC)^10,11,12,13. When these sophisticated catalytic reaction process coexist, their reaction pathways and outcomes may be antagonistic or competitive with others, resulting in negative antibacterial effect which can be even worse than that of applying H₂O₂ alone¹⁴. In other words, ·OH, superoxide anions (O₂^•−) and H₂O₂, the main toxic by-products of aerobic metabolism, can be scavenged by CeO₂ when employed as a bio-antioxidant^15,16. Obviously, as a peroxidase-mimicking nanozyme, this characteristic is not conducive to the forming of reactive oxygen species (ROS) in the presence of H₂O₂, thus producing a poor physiological activity related to ·OH. Therefore, effective regulation of multiple catalytic properties of CeO₂ and their reaction process towards a favorable direction is key to optimizing CeO₂ + H₂O₂ antibacterial system.

In addition, extensive investigations have been made focusing on the activity of the metal site itself. Meanwhile, interaction between the metal atoms and the substrate with complex intrinsic catalytic activities is of great significance for regulating the nanozyme activities. Both should be carefully considered while investigate the materials with multi-channel activities. CeO₂ has a fluorite-like cubic structure which close-packed cerium atoms are coordinated with eight O^2- ions. It possesses a host of oxygen vacancies on its surface in response to the unique shuttle between Ce³⁺ and Ce⁴⁺ redox states¹⁷. When it comes to SSEs with 100% atomic efficiency^18,19,20, metal-support interaction (MSI) plays a pivotal role in modulating electronic structure on the active site, which favors adsorption and desorption of reactive intermediates^21,22. Such features indicate that its overall performance can be manipulated via tuning the electronic structure of both foreign metal atom and the surrounded Ce atoms. It was reported by Wang²³ that Pt₁/CeO₂ catalyst with an asymmetric Pt₁O₄ configuration displayed exceptional CO oxidation performance relative to square-planar counterpart owing to the tailoring of the local environment of isolated Pt²⁺. Hensen²⁴ illustrated that the high mobility of surface lattice oxygen and oxygen atoms spilled over from Pd-CeO₂ interface which originated from strong MSI contributed to the high stability of oxidized Pd single atoms during CO oxidation. Li also reported that the incorporation of Mn can boost the catalytic performance of the surrounded Ce atoms²⁵. Thus, the incorporation of foreign metals into parent CeO₂ will engineer its surface structure and regulate reaction intermediates, resulting an optimum antibacterial performance.

Copper agent, as a long-standing antibacterial agent, has achieved excellent antibacterial effects through electrostatic adsorption, ion permeation, and disruption of bacterial redox homeostasis^26,27. In recent years, it has attracted widespread attention in simulating natural oxidase and peroxidase for antibacterial purposes^28,29. However, traditional Cu antibacterial agents often possess a high content of Cu, which not only causes waste of Cu catalytic sites in the core, but also poses a risk of causing damage to normal cells after precipitation in the form of Cu ions in practical applications³⁰. The above factors encourage us to seek safer and more efficient Cu antibacterial materials to solve the problems of poor utilization and stability of traditional Cu antibacterial agents. Motivated by the above research findings, we hypothesize that the introduction of Cu single-site into CeO₂ can not only effectively regulate the complex catalytic activities of CeO₂, but also make up for the low utilization of Cu in traditional Cu antibacterial agents while retaining its antibacterial property.

In this work, we deploy a facile strategy to prepare a Cu single site modified CeO₂ nanozyme (Cu-CeO₂) by employing nano-CeO₂ with multiple catalytic activities as the substrate. Benefiting from the modulation of the reaction energy of potential determining step (PDS) of Ce cite by Cu species, Cu-CeO₂ demonstrated an increase in POD-like activity and a decrease in HORAC activity compared to pristine CeO₂. In vitro and in vivo tests demonstrate that CeO₂ can significantly weaken the inherent antibacterial activity of H₂O₂ against Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), which can be effectively reversed by Cu-CeO₂. The above findings indicate that Cu single-site modification can effectively regulate the complex catalytic activities of CeO₂ nanozyme, and Cu-CeO₂ single-site nanozyme has a good prospect in the treatment of drug-resistant bacterial infections.

Results

Synthesis and characterization of Cu-CeO₂ SSE

The fabrication of Cu-CeO₂ catalyst is illustrated in Fig. 1a. First, CeO₂ nanosphere is obtained by a solvothermal approach, followed by calcinating step under air condition. Then, Cu ions could be adsorbed and deposited on ceria substrates in the presence of alkaline solutions. Finally, the obtained composite is heated in air first, and then pyrolyzed under hydrogen atmosphere to get the target Cu-CeO₂ product. Owing to the Cu-O-Ce interaction, Cu species are present as a dispersive state in Cu-CeO₂ sample after calcined in air. The scanning electron microscopy (SEM) image in Fig. 1b and transmission electron microscopy (TEM) image in Fig. 1c clearly reveals that the as-prepared Cu-CeO₂ possesses a spherical morphology with a quite rough surface, which is similar to their parent ones in Supplementary Figs. 1, 2. Supplementary Fig. 3 shows high-resolution TEM (HRTEM) image of Cu-CeO₂ precursor and the positions circled by two rectangular frames are enlarged on the right. Obviously, two representative lattice fringes with a spacing of 0.28 nm are at an angle of 90° in the upper right. The interplanar spacing of the exposed plane with an included angle of 45° is 0.19 nm. As shown in the lower right of Supplementary Fig. 3, the lattice fringes with an interplanar spacing of 0.31 nm correspond to the (111) plane of the sample. Element mapping results in Supplementary Fig. 4 demonstrate the homogeneous distribution of Cu, O and Ce components throughout the sample. The aberration corrected high angle annular dark field-scanning transmission electron microscopy (AC-HAADF-STEM) result in Supplementary Fig. 5 also shows that there are no obvious crystallized copper species in the form of clusters or small particles in the structure. Energy dispersive spectroscopic (EDS) mapping analysis (Fig. 1d) signifies a uniform distribution of Cu component in Cu-CeO₂ catalyst. Figure 1e shows the HRTEM image of Cu-CeO₂. It can be seen that there are no clusters or small particles throughout the structure. The results in Fig. 1f, g indicate that the main interplanar spacing is about 3.2 Å by Z-contrast analysis.

Fig. 1: Schematic illustration and morphology characterization for the Cu-CeO₂ catalyst.

a Schematic illustration of the synthetic procedure of Cu-CeO₂ sample, b Scanning electron microscopy images of Cu-CeO₂ catalyst (scale bar: 200 nm), c Representative TEM image of Cu-CeO₂ catalyst (scale bar: 100 nm), d TEM image and corresponding elemental mapping of Cu, O and Ce recorded on the nanoparticles (scale bar: 100 nm), e HRTEM images of Cu-CeO₂ catalyst, f AC-HAADF-STEM image of Cu-CeO₂ catalyst (scale bar: 5 nm), g Corresponding Z-contrast analysis of region A and B in (f). All experiments were independently repeated three times with similar results.

Figure 2a shows the X-ray diffraction (XRD) patterns of CeO₂ and Cu-CeO₂ samples, respectively. It can be seen that the main diffraction peaks of two samples are indexed to the cubic structure of CeO₂ (JCPDS card no. 34-0394). Except for the diffraction peaks of CeO₂, no agglomerated Cu species are detected, indicating that the structure of CeO₂ is not changed after the introduction of Cu. In addition, the XRD pattern of Cu-CeO₂ sample is fitted in Supplementary Fig. 6, which yields a crystallite size of smaller particle as 9 nm and the unit cell parameters as a = b = c = 5.3912 Å and α = β = γ = 90°. On this basis, the model structural diagram of parent cubic CeO₂ is constructed and illustrated in Supplementary Fig. 7. From the visual point of view in Supplementary Fig. 7b, the lattice fringes which correspond to (200) and (002) facets are perpendicular to each other and the lattice fringes at an included angle of 45° belong to the (220) facet, which is consistent with the above results obtained from the electron microscope analysis. In order to further accurately determine the nanostructure of the material, we also carry out Raman spectroscopy measurements. In Fig. 2b, the peak centered at 460 cm⁻¹ is attributed to the F_2g vibrational mode of CeO₂ crystal³¹. Compared with parent CeO₂, the softening of the F_2g mode of Cu-CeO₂ is accompanied by the appearance of a broad feature centered around 600 cm⁻¹, which belongs to the defect-induced mode (D)³². The appearance of D band is due to the formation of defect species in Ce-O coordination, which leads a consequence that the vibration signal of Ce-O cannot be cancelled in all directions³³. Since there exist a Cu-O-Ce coordination structure in Cu-CeO₂ catalyst, the interaction between Cu-O and Ce-O is not equal, which leads to the difference in the vibration of Ce-O in different directions. Thus, the increasing disordering level of Cu-CeO₂ leads to the variation of D band. In addition, Raman peaks at 290 cm⁻¹ and 340 cm⁻¹, which assigned to CuO phase, are not observed^34,35. The elemental composition and valence states are investigated by X-ray photoelectron spectroscopy (XPS) technique (Supplementary Fig. 8, Fig. 2c). The XPS spectra of Ce 3d (Fig. 2c) are fitted into 10 peaks, which are attributed to the Ce⁴⁺ species at v, v′′, v′′′, u, u′′, u′′′ and the Ce³⁺ species at v₀, v′, u₀, u′, respectively^23,36,37,38. Supplementary Table 1 shows the actual Ce³⁺/Ce ratios of both CeO₂ and Cu-CeO₂ samples. It is obvious that the Ce³⁺ content for Cu-CeO₂ is nearly 18% from XPS analysis, which is lower than that of CeO₂ (23%). These results indicate that the introduction of Cu element into parent CeO₂ would reduce its surface Ce³⁺ concentration. Similar results have also been reported in previous report³⁸. According to the authors’ calculations and structural characterization, the substitution of one Ce³⁺ adjacent to an oxygen vacancy (V_O) by one Cu²⁺ normally accompanies a phenomenon that the other Ce³⁺ would be readily converted to Ce⁴⁺ for the charge balance, and therefore the introduction of Cu²⁺ will reduce the Ce³⁺/Ce⁴⁺ ratio compared with original CeO₂. The O 1s spectra of CeO₂ and Cu-CeO₂ samples are illustrated in Supplementary Fig. 8b. The peak appearing at approximately 529 eV (O_I) can be attributed to the lattice oxygen of Ce⁴⁺ and the feature at 530.7 eV (O_II) corresponds to oxygen vacancies or the lower-coordination lattice oxygen of Ce^3+36,37. Also, the higher binding energy feature at 531.6 eV is also associated with the presence of surface hydroxy-containing groups. It is obvious that parent CeO₂ possesses a larger O_II/(O_I + O_II) ratio (23.3%) than that of Cu-CeO₂ (19.8%), which is in line with the Ce 3d XPS results.

Fig. 2: Structural characterizations of Cu-CeO₂ catalyst and reference materials.

XRD patterns (a), Raman spectra (b) and Ce 3d photoelectron profiles (c) of the CeO₂ and Cu-CeO₂ catalysts, d XANES spectra of Cu foil, CuO and Cu-CeO₂ sample, e Fourier transforms of the Cu K-edge EXAFS oscillations of the materials mentioned above, f FT-EXAFS spectra of Cu-CeO₂ sample at the Cu K-edge and the corresponding Fitting results. Source data are provided as a Source Data file.

Considering that the surface oxygen is prone to be removed under vacuum conditions, the structural information of Cu can be better reflected by means of Synchrotron radiation characterization. X-ray absorption fine structure (XAFS) measurements can effectively detect the local coordination states and electronic structure of copper on CeO₂ support. Figure 2d shows the Cu K edge X-ray absorption near-edge structure (XANES) profiles of Cu-CeO₂, Cu foil, and CuO samples. According to the position of near-edge absorption energy, it can be concluded that the Cu species bear an oxidation valence state in Cu-CeO₂ sample. The Fourier-transformed (FT) extended X-ray absorption fine structure (EXAFS) curve of Cu-CeO₂ (Fig. 2e) exhibits only one prominent peak at approximately 1.9 Å, corresponding to the first shell of Cu-O scattering interaction. No appreciable Cu-Cu coordination characteristic peak is detected at 2.24 Å, signifying that there is no formation of Cu-Cu bond. We also performed CO-probe molecule Fourier transform infrared (FTIR) measurements to investigate the nature of Cu metal sites. In situ diffused reflectance infrared Fourier transform spectroscopy (DRIFTS) was used to investigate the adsorption ability of CeO₂ and Cu-CeO₂. As shown in Supplementary Fig. 9a, Cu-CeO₂ catalyst exhibits a band around 2105 cm⁻¹, assigned to the linear CO adsorbed on Cu⁺ sites (Cu⁺-CO), indicating that CO was adsorbed on the Cu⁺ sites^{34,39,40,41,42}. In addition, the adsorption intensity of the peak gradually increased with the time, and reached saturation adsorption at 480 s with a maximum adsorption peak at 2111 cm⁻¹. Traditionally, the IR band at 2069 cm⁻¹ is regarded as the CO adsorption on Cu⁰ site^40,41. This indicates that there are no Cu⁰ species in this Cu-CeO₂ catalyst. For CeO₂, two obvious gaseous peak of CO are shown in Supplementary Fig. 9b, which indicating that the CO absorption on parent CeO₂ is absent. XANES spectra at the Ce M_5,4-edge were normalized and are shown in Supplementary Fig. 10a. The XANES at Ce M_5,4-edge of CeO₂ based materials correlates with the Ce 3d_3/2 and 3d_5/2 core level transitions into the 4f unoccupied electronic state⁴³. The intense peak S and peak P represent the tetravalent Ce (4f⁰) while the weak peak R indicates the contribution of trivalent Ce (4f¹) states^44,45. As it vividly shows, Cu-CeO₂ sample exhibits a reduction of Ce³⁺ (peak R) compared with parent CeO₂ while an enhancement of Ce⁴⁺ (peak S). In other words, the Ce³⁺/(Ce³⁺ + Ce⁴⁺) ratio of Cu-CeO₂ is lower than that of CeO₂. The result is in good agreement with XPS results (Fig. 2c, Supplementary Table 1). Supplementary Fig. 10b shows the O K-edge XANES spectra. Three main peaks are attributed to the O 2p states that are hybridized with Ce 4f, 5d(e_g) and 5d(t_2g) states, respectively. The intensity variation is attributed to the structural disorders induced by Cu doping owing to the formation of Cu-O-Ce coordination network⁴³. The quantitative FT-EXAFS fitting is conducted to shed light on the structural configuration of Cu (Fig. 2f) and the corresponding coordination parameters are shown in Supplementary Table 2. It can be seen that the coordination number of the center Cu atom is 3.2 and the mean bond length of Cu-O is about 1.94 Å. Supplementary Fig. 11 shows the q-fitting (inverse Fourier transform) curve of Cu-CeO₂, which is consistent with the above R-space fitting results.

Evaluation of Cu-CeO₂ SSE for POD-like and HORAC activity

Multiple enzyme-mimicking activities of Cu-CeO₂ were investigated in vitro. Firstly, the POD-like activity of Cu-CeO₂ was tested based on the principle that H₂O₂ could be catalytically decomposed by Cu-CeO₂ to generate ·OH, and TMB could be oxidized by ·OH to oxidized-TMB (ox-TMB)⁴⁶. The absorbance of the reaction product at the wavelength of 652 nm was read by a microplate reader. Figure 3a shows that the POD-like activity of Cu-CeO₂ was significantly higher than that of pristine CeO₂. Furthermore, steady-state kinetic assay was conducted. According to Lineweaver-Burk equation, the Michaelis-Menten constant (K_m) of CeO₂ and Cu-CeO₂ were 24.34 mM and 30.76 mM, respectively. The maximal reaction velocity (V_max) of CeO₂ and Cu-CeO₂ were 28.05 nM/s and 166.7 nM/s, respectively, and Cu-CeO₂ showed significantly enhanced POD-like activity (Supplementary Fig. 12, Supplementary Table 3). We further investigated the regulation of Cu content on the POD-like catalytic performance of Cu-CeO₂ nanozyme. As shown in Supplementary Fig. 12 and Supplementary Table 3, firstly, all Cu-CeO₂ SSE exhibited significantly enhanced POD-like activity compared to CuO. In addition, Cu content and the V_max of Cu-CeO₂ was positively correlated, which may be caused by the variation of Cu sites involved in the catalytic reaction. However, after calculating the turnover rate, we found that as Cu content increased, the turnover rate showed a gradually decreasing trend. This may be due to the presence of more CuO particles in samples with high Cu content compared to those with low Cu content, and CuO may cause a decrease in the dispersion of active centers. It is worth noting that Cu-CeO₂ with a theoretical Cu content of 5%, which is also the main sample of this study, can achieve a V_max comparable to 10% Cu sample and a turnover rate comparable to 2% Cu sample at the same mass concentration, demonstrating satisfactory catalytic performance. The V_max and the turnover rate of 5% Cu-CeO₂ was 11.78- and 212.51- fold higer than CuO nanozyme, respectively, exhibiting significant enhancement of POD-like activity.

Fig. 3: Catalytic activities and mechanism of CeO₂ and Cu-CeO₂ nanozymes.

a Time-dependent optical density change at 652 nm of 3,3’, 5,5’-tetramethylbenzidine (TMB) in POD reactions. b Time dependent oxygen generation in CAT reactions. c Time dependent optical density change at 652 nm of TMB in OXD reactions. d Time dependent fluorescent intensity of 2,7-Dichlorodihydrofluorescein diacetate (DCFH) in HORAC reactions. e Fluorescent spectra of DCFH after 10 min reaction with H₂O₂ and different nanozymes. f Electron Paramagnetic Resonance (EPR) spectra of DMPO-OH after 10 min reaction with H₂O₂ and different nanozymes. g The calculated model of pristine CeO₂ (left), CeO₂ with Cu3c added on the 3O atoms on the surface (Cu-ad, middle) and CeO₂ with a Ce atom substituted by Cu (Cu-sub, right). The 6 possible reaction sites are highlighted with a tagged arrow. h The reaction mechanism of POD and HORAC process, M indicates the metal sites. i The calculated PDS reaction energy of POD and HORAC processes for different reaction sites with the exact PDS labeled above the bar. j The calculated PDOS, i.e., the d and f band summation of different reaction centers. k The proposed mechanism of regulation of catalytic activities by Cu-CeO₂ single-site nanozyme. Source data are provided as a Source Data file.

Furthermore, the cyclic stability of Cu-CeO₂ was tested, the results showed that after 30 catalytic cycles, the POD-like activity of Cu-CeO₂ was still comparable with the original nanozyme (Supplementary Fig. 13). The ICP-MS analysis showed that Cu accounts for 4.43 wt % of the Cu-CeO₂ sample. Hence, when the concentration of Cu-CeO₂ reaches 200 μg/mL, the total content of Cu is 8.858 μg/mL. The content of Cu in the supernatant after cyclic reaction was below 0.500 μg/mL, much lower than the total amount of Cu in the reaction system. In summary, Cu-CeO₂ nanozyme has good stability, within our test conditions.

As a reaction substrate, H₂O₂ can also be catalytically decomposed into O₂ by Cu-CeO₂, which increases the concentration of dissolved O₂ in the liquid environment. The CAT-like activity of Cu-CeO₂ was investigated subsequently. Figure 3b shows that the CAT-like activity of Cu-CeO₂ was significantly higher than that of pristine CeO₂. In addition, O₂ can be catalyzed by OXD-like activity of Cu-CeO₂ to generate O₂^·-, which oxidizes TMB to ox-TMB⁴⁷. The absorbance of the reaction product at the wavelength of 652 nm was read by a microplate reader, and Cu-CeO₂ showed slightly enhanced OXD-like activity (Fig. 3c). The CAT reaction provides O₂, which further promotes the OXD reaction. The SOD-like activity of Cu-CeO₂ was also tested. Xanthine-xanthine oxidase system was applied to generate O₂^·−, which can reduce nitro blue tetrazolium (NBT) to formazan. In the presence of SOD-mimics, O₂^·− can be catalytically converted to O₂ and H₂O₂, which further provides H₂O₂ for POD reaction. As Supplementary Fig. 14 shows, the SOD-like activity of Cu-CeO₂ was also significantly higher than that of pristine CeO₂. We further tested the consumption of H₂O₂ in CeO₂ + H₂O₂ and Cu-CeO₂ + H₂O₂ systems quantitatively. As shown in Supplementary Fig. 15, as the reaction time prolongs, both systems showed a trend of H₂O₂ consumption, and Cu-CeO₂ maintains a higher consumption than CeO₂ during long-term reaction process. This may be caused by the enhanced multiple catalytic activities of Cu-CeO₂, which accelerate the multiple pathway conversion related to H₂O₂.

Next, the HORAC activity of Cu-CeO₂ was tested. The ·OH generated from H₂O₂ and Fenton reagent can be transformed into H₂O and O₂ through HORAC activity, so as to quench the free radical fluorescent probe⁴⁸. As Fig. 3d shows, the fluorescence intensity of CeO₂ and Cu-CeO₂ groups decreased compared with the reaction baseline, indicating that both nanozymes exerted HORAC activity. However, within 10 min of reaction, the fluorescence intensity of CeO₂ group drastically decreased by ~94.31%, compared with baseline, while Cu-CeO₂ group only decreased by ~1.92%, and the relative fluorescence intensity was higher than that of the H₂O group throughout the entire reaction process, indicating that Cu single-site led to a significant inhibition of HORAC activity of CeO₂, and that Cu-CeO₂ could accelerate the Fenton-like catalytic process. We further used DMPO as the spin trapping agent and measured the changes of ·OH species quantitatively through EPR. As shown in Supplementary Fig. 16 and Supplementary Table 4, after 10 minutes of reaction, both FeCl₂ and FeCl₂ + Cu-CeO₂ group exhibited typical DMPO-OH signal. The signal intensity of FeCl₂ + Cu-CeO₂ group was higher than that of FeCl₂ group, while the signal of FeCl₂ + CeO₂ group was almost invisible, which is consistent with the pattern of the fluorescence results. Quantitative calculation showed that the ·OH scavenging rate of CeO₂ was 97.26% at 10 min, while the spin concentration of DMPO-OH in FeCl₂ + Cu-CeO₂ group reached 158.62% of that in FeCl₂ group. In addition, we investigated the catalytic effect of the physical mixed system of free Cu²⁺ ions/CuO nanozyme with CeO₂ nanozyme. As shown in Supplementary Fig. 17, the initial rate of POD-like reaction of the two physical mixed systems was similar to that of CeO₂. As the reaction continued, the substrate conversion extent of the two groups at the end point of the reaction was similar to that of Cu-CeO₂. However, DCFH fluorescence detection (Supplementary Fig. 18) showed that for physical mixed systems, only Cu²⁺ + CeO₂ group showed significantly higher total ROS generation than CeO₂ group, but was still much lower than that of Cu-CeO₂ group, indicating that in the non-interacting Cu²⁺/CuO + CeO₂ physically mixed system, Cu could not inhibit the HORAC activity of CeO₂ effectively. Through the above results, we reaffirm the enhanced POD-like activity of Cu-CeO₂ nanozyme, and a prominent inhibitory effect of Cu single sites on the HORAC activity of CeO₂. Meanwhile, the interaction between Cu single sites and CeO₂ support also plays a key role in the regulation of catalytic activities. Therefore, by the aid of Cu single sites and its interaction with CeO₂ support, the effective regulation of the redox catalytic pathways of CeO₂ nanozyme was achieved.

Previous studies have shown that structural factors such as Ce³⁺/Ce⁴⁺ ratio⁴⁹, oxygen vacancy¹⁵, defect^11,50, as well as environmental factors such as pH value⁵¹ and ·OH concentration⁵², will affect the type of catalytic reaction and catalytic activity of CeO₂. Among these factors, Ce³⁺/Ce⁴⁺ ratio is one of the most important structural factors which determines the POD-like and HORAC activities of CeO₂^11,15,49. Specifically, increasing the proportion of Ce³⁺ can not only improve the POD-like activity of CeO₂, but also enhance its HORAC activity. In this study, XPS results indicated that the Ce³⁺/Ce⁴⁺ ratio of Cu-CeO₂ was lower than that of CeO₂. Combined with the catalytic activity results, we speculated that the introduction of Cu site inhibits the HORAC activity of CeO₂ carrier, and may also lead to the decrease of its POD-like activity. Under the synergistic effect of the intrinsic activity of Cu site, the overall POD-like activity of Cu-CeO₂ SSE was maintained.

In order to further explore the generation of reactive oxygen species (ROS) in the catalytic decomposition of H₂O₂ by Cu-CeO₂, 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was adopted as total-ROS detecting fluorescent probe. As Fig. 3e shows, Cu-CeO₂ + H₂O₂ group produced strong fluorescence signal, H₂O₂ group produced weak fluorescence signal, while CeO₂ + H₂O₂ group exhibited even lower fluorescence than H₂O₂ group. These results indicated that Cu-CeO₂ + H₂O₂ system can produce abundant ROS efficiently, and CeO₂ + H₂O₂ system demonstrated an outcome of ROS removal. Terephthalic acid (TA) was adopted as ·OH detecting fluorescent probe. The characteristic fluorescent sprectrum of hydroxyterephthalic acid (TAOH) in Cu-CeO₂ group indicated the prescence of ·OH (Supplementary Fig. 17). Next, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was adopted as the spin trap and EPR was used to detect ·OH generation in Cu-CeO₂ + H₂O₂ system. As Fig. 3f shows, no obvious DMPO-OH signal was detected in H₂O₂ or CeO₂ + H₂O₂ group, while Cu-CeO₂ + H₂O₂ exhibited significant DMPO-OH signal. The catalytic generation of O₂·⁻ by Cu-CeO₂ in methanol was also detected. As Supplementary Fig. 18 shows, among all three groups, only Cu-CeO₂ group showed significant DPMO-O₂·^- signal. Combined with the catalytic activity assays above, the results indicated that both CeO₂ and Cu-CeO₂ can catalyze the decomposition of H₂O₂ to generate ·OH through POD-like activity, and Cu-CeO₂ exerts higher POD-like activity, producing more ·OH. CeO₂ may remove ·OH through its high HORAC activity. However, in Cu-CeO₂ + H₂O₂ system, there remain abundant ·OH radicals due to the inhibited HORAC activity of Cu-CeO₂.

Theoretical analysis

Density functional theory (DFT) is employed to elucidate the underlying mechanism for the boost of ROS generation of Cu-CeO₂. Pristine CeO₂ (111) surface (left Fig. 3g) and Cu-CeO₂ (111) surface are built for comparison. Firstly, two configurations of Cu-CeO₂, including one that has a Cu atom directly adhering on the three O atoms on the (111) surface (Cu-ad, middle Fig. 3g), and one that has a Cu atom substitute the surface Ce atom (Cu-sub, right Fig. 3g), are optimized. It should be noted that due to the weak O-binding energy for Cu atom, originally presented surface O3c transformed to O2c in the Cu-sub model, which is with stronger alkalinity and will be protonated in the solution⁵³. Thus, the protonated Cu-sub model is finally adopted. Different reaction sites in these systems are considered. For the pristine (111) surface, only one type of reaction site of Ce7c presents. While for the Cu-ad and Cu-sub surface, two and three types of reaction sites are considered, respectively, as shown in Fig. 3g. In the Cu-ad system, both Cu (Cu@Cu-ad) and Ce (Ce7c@Cu-ad) adjacent to Cu are considered. While for the Cu-sub system, the Ce7c site adjacent to the protonated O atom (Ce7c@Cu-sub) and the Cu site (Cu@Cu-sub), along with the double Ce6c site (diCe6c@Cu-sub) that undergoes lattice oxygen mechanism which involves the protonated O2c during the reaction, are considered. DFT optimizations were then performed for the reaction intermediates on different sites. The optimized geometries are illustrated in Supplementary Figs. 21–26.

In this work, a large amount of ·OH radicals are detected in the experiment and are considered as the major disinfection factor. It is crucial to study the activity of POD that generates ·OH, including reaction P1 to P4 in Fig. 3h.

The reaction energy for the potential determining steps (PDS) of different reaction sites are illustrated in the top of Fig. 3i, with the exact PDS labeled on the bars. The detailed reaction energy along the POD pathway are listed in Supplementary Table 5. It can be found that the Ce7c site on the pristine CeO₂ (111) surface has a poor POD activity due to the huge energy gap of 2.591 eV during reaction P2 as the PDS. Meanwhile, in the Cu-ad system, the Cu@Cu-ad center served as a catalytic site with a drastically decreased energy of 1.358 eV, while the adjacent Ce7c@Cu-ad sites also have an increased activity with PDS energy as low as 1.792 eV. The influence of Cu in the Cu-sub system are, on one hand, greatly increased the activity of the adjacent Ce7c@Cu-sub and especially the diCe6c@Cu-sub sites, with the PDS energy of 1.556 eV and 0.970 eV, respectively. On the other hand, little change of the activity for Cu@Cu-sub center itself has been calculated (2.479 eV). These results indicated that Cu single-site can largely promote the overall POD activities with the activity of itself and via activating the adjacent Ce sites.

The electronic structure analysis, namely the projected density of state (PDOS) analysis, is further conducted for different reaction sites to elucidate the exact mechanism for activity promotion. The summation of d and f bands for the reaction sites are shown in Fig. 3j. Firstly, Cu@Cu-ad can serve as the catalytic center in Cu-ad system due to the fact the site is under-coordinated (Cu3c) and that the d band of the center is concentrated near the fermi-level compared with the Ce7c site, which served a stronger interaction between the centers and the oxidized species, stabilized the *OH intermediates and decreased the energy for the PDS (P2). Cu@Cu-sub on the other hand, due to being fully coordinated (Cu4c), the d-band is far away from the fermi-level, weak binding with oxidized species is anticipated and no better performance is calculated. Secondly, Cu single-site also influenced the performance of adjacent Ce. Comparing with the Ce7c plot, a shift to a higher energy can be spotted in Ce7c@Cu-ad and diCe6c@Cu-sub. Such shift is mainly originated from the less coordinated environment of Ce and will contribute to a higher energy of the anti-bond band of the bond between Ce and adsorbed O, further stabilizing the adsorbed *OH structure. In this case, we see a drastic decrement in reaction energy for reaction P2 of Cu-CeO₂. Meanwhile, tighter Ce-O binding facilitates a harder dehydration process and turns reaction P4 into the PDS for the diCe6c@Cu-sub center. For the Ce7c@Cu-sub site, slightly difference could be told from the PDOS from Ce7c, which indicates that on one hand, the introduction of Cu has little influence on the electronic structure for the adjacent Ce7c@Cu-sub sites. On the other hand, the sharp decline for reaction energy in PDS is mainly contributed by the *OH structure that stabilized with the protonated O atom.

It should be noticed that as reported in the literature, CeO₂ itself can act as an ROS elimination catalyst to prevent oxidative damage, HORAC mechanism (reaction H1 to H6 in Fig. 3h), as an antagonistic pathway of POD, should also be considered. As depicted in Fig. 3i, the PDS for most reaction sites are the dehydration reaction H3, the reaction energies of which are 0.909 eV for pristine Ce7c. Thus, it is anticipated that as the introduction of Cu to the system, the dehydration process should be hindered due to stronger oxidized species adsorption of the reaction sites on and around Cu single-site (except for Cu@Cu-sub), as discussed in the PDOS analysis. To be specific, the diCe6c@Cu-sub site has an increased PDS energy of 0.954 eV for reaction H6, which inhibited the HORAC process. Likewise, the Cu@Cu-ad, Ce7c@Cu-ad, Ce7c@Cu-sub sites all have worse HORAC performance with PDS energy increased to 1.110, 0.924 and 1.258 eV. The PDS energy on Cu@Cu-sub site is 0.652 eV, which indicated a better HORAC activity. Yet, considering the weak interaction between adsorbed ·OH and Cu@Cu-sub center, the HORAC reaction tends to take place on the Ce sites instead of Cu, which limits the promotion effect for Cu@Cu-sub center promotion to the HORAC processes.

In all, Cu single-site influences the reaction activity on and around itself. Except for the drastic energy decrement of POD, the inhibition of HORAC processes also made contributions to the overall ROS generation performance (Fig. 3k).

To elucidate the possible influence of small CuO cluster on the surface, additional DFT calculations were performed. According to previous literatures, small Cu nano-clusters on CeO₂ facets prefer to be in the form of monolayers, a (CuO)₃-CeO₂ model was thus established^54,55. The stable planar (CuO)₃ cluster with similar symmetry of CeO₂ (111) surface was loaded. Considering the aqueous environment of the actual experimental condition, water molecule was added and found dissociated spontaneously on the top of the CuO cluster under the strong influence of 3 under coordinated Cu, forming the hydrated site as shown in Supplementary Figs. 27, 28. Due to the structural complexity of the cluster, there are two possible reaction sites, i.e., the Cu₃ site and the Cu₂Ce site which surround the *OH intermediate. Both HORAC and POD-like mechanisms were calculated on these sites. The results indicated that for POD-like pathway, the energy consumption of the PDS are sharply reduced to 0.796 eV (Cu₃) and 0.915 eV (Cu₂Ce) mainly due to the strong interaction of the intermediates (*OH) with multiple coordination atoms. Thus, (CuO)₃ cluster boosted the activity of ·OH radical generation. While for the HORAC pathway, the strong binding of O₂ with multiple atoms further increase the energy consumption of the PDS (O₂ desorption) to 1.081 eV (Cu₃) and 1.015 eV (Cu₂Ce), which deactivated the HORAC process. The above results suggest that small Cu nano-clusters also possess oxidant activities. Based on the steady-state kinetic results of POD-like activities of Cu-CeO₂ samples with different Cu contents in Supplementary Table 3, when the Cu content increased from 2 to 5%, the V_max increased by ~2.13 folds, and the turnover rate showed a slight decrease, indicating that potential existence of small amount of Cu nanoclusters in Cu-CeO₂ sample may contribute to the overall POD-like activity of the nanozyme. However, when the Cu content continued to increase to 10%, the V_max did not increase correspondingly, but remained comparable to that of the 5% sample. Meanwhile, the turnover rate drastically decreased by ~2.10 folds, suggesting that the presence of a large number of Cu nano-clusters may actually inhibit the overall catalytic activity of the nanozyme. The phenomenon we observed is consistent with that reported in previous literature⁵⁶.

In vitro antibacterial performance of Cu-CeO₂ SSE

After confirming the catalytic activities and underlying ROS generation mechanism of Cu-CeO₂, in vitro and in vivo experiments were carried out to investigate its antibacterial properties. Methicillin-resistant MRSA and E. coli were chosen as representative strains of Gram-positive and Gram-negative bacteria, respectively. The bacterial solution was treated in six groups: PBS, CeO₂, Cu-CeO₂, H₂O₂, CeO₂ + H₂O₂ and Cu-CeO₂ + H₂O₂. After different treatments, the antibacterial effect of each group was evaluated by plate colony-counting. The results showed that except for the Cu-CeO₂ + H₂O₂ group, there was no significant difference colony numbers between the other groups and the control group (P > 0.05) (Fig. 4a, b, g, i). Nanozyme solely showed no obvious antibacterial effect. For MRSA, there was no significant difference in the reduction of colony number after H₂O₂ or CeO₂ + H₂O₂ treatment (~0.12-log), while Cu-CeO₂ + H₂O₂ group completely sterilized ~6.85-log MRSA and showed superior antibacterial properties. Similarly, for E. coli, after H₂O₂ treatment, the colony number decreased by ~0.91-log. It’s noteworthy that after CeO₂ + H₂O₂ treatment, the colony number decreased by ~0.56-log, which was even worse than that of H₂O₂ group. However, Cu-CeO₂ + H₂O₂ group completely sterilized ~7.70-log E. coli, reversing the inhibited antibacterial effect of CeO₂. The bacterial solution of each group was stained and observed using fluorescence microscope, and the results are shown in Fig. 4c, d and Supplementary Fig. 27. Almost all bacteria in groups without H₂O₂ were alive, with only a few dead ones. There were a few dead bacteria in H₂O₂ group and CeO₂ + H₂O₂ group, most of which were living bacteria. For Cu-CeO₂ + H₂O₂ group, no obvious living bacteria were observed. The antibacterial effect of each group was further confirmed by SEM. Except that Cu-CeO₂ + H₂O₂ group showed obvious loss of bacterial membrane integrity, the bacterial morphology of the other groups was intact or slightly wrinkled (Fig. 4e, f, Supplementary Fig. 28). H₂O₂ has been widely used for debridement and disinfection of various infected wounds clinically due to its broad-spectrum antibacterial properties⁵⁷. However, high concentrations of H₂O₂ can be harmful to normal human tissues. In addition, the antibacterial ability of H₂O₂ is relatively weak, and different bacteria perform varied sensitivity to H₂O₂⁵⁸. By the aid of the intrinsic peroxidase-like activity of Cu-CeO₂, the application of Cu-CeO₂ + H₂O₂ system can achieve better bactericidal performance using much lower concentration of H₂O₂ than that in clinic (2.5% ~ 3.5%).

Fig. 4: In vitro antibacterial properties of CeO₂ and Cu-CeO₂ nanozymes.

Bacterial colonies, fluorescent images (scale bar: 50 μm) and SEM images (scale bar: 1 μm) of MRSA (a, c, e) and E. coli (b, d, f) after grouped treatment (I: Phosphate buffered saline (PBS), II: CeO₂, III: Cu-CeO₂, IV: H₂O₂, V: CeO₂ + H₂O₂, VI: Cu-CeO₂ + H₂O₂). Logarithm of colony forming unit (CFU) countings of MRSA (g) and E. coli (i) after grouped treatment. Logarithm of CFU countings of MRSA (h) and E. coli (j) after treatment with different concentrations of nanozymes and H₂O₂. A representative image of three replicates from each group is shown. Data are presented as mean values +/− standard deviation, n = 3 biologically independent replicates. Significance was calculated by two-sided Student’s t-test. Source data are provided as a Source Data file.

To further confirm ROS generation in the Cu-CeO₂ + H₂O₂ antibacterial system, DCFH staining was performed. As shown in Supplementary Figs. 28, 29, for both MRSA and E. coli, the fluorescence of DCFH-DA in Cu-CeO₂ + H₂O₂ group are more obvious than CeO₂ + H₂O₂ group. In addition, the fluorescence was well co-localized with nanozyme-bacteria composite, while planktonic bacteria showed little fluorescence, indicating that the onset of ROS-mediated sterilization in Cu-CeO₂ + H₂O₂ antibacterial system is likely to be located on the nanozyme-bacteria interface.

We further explored the rules of bactericidal changes of CeO₂ and Cu-CeO₂ nanozymes at different concentrations. For MRSA, the arbitrary combinations of CeO₂ + H₂O₂ had no significant antibacterial effect, while Cu-CeO₂ + H₂O₂ groups showed obviously positive correlation between the bactericidal effect and the concentration of nanozyme and H₂O₂ (Fig. 4h, j, Supplementary Figs. 30, 31). For E. coli, due to its high sensitivity to H₂O₂, the colony number showed a decreasing tendency with the increase of H₂O₂ concentration. Notably the antibacterial effect gradually deteriorated with the increase of CeO₂ concentration, which was ever worse than that of H₂O₂ with the same concentration. Cu-CeO₂ + H₂O₂ group still showed a positive correlation between antibacterial effect and nanozyme and H₂O₂ concentration. The phenomenon observed in our study is similar to that reported by Zhu et al.¹⁴. According to their report, the synthesized spherical CeO₂ nanozyme with a particle size of ~150 nm exhibited optimistic POD-like activity at pH = 4.0–6.0. However, the bactericidal effect of CeO₂ + H₂O₂ on E. coli in PBS (pH = 4.0) was significantly weaker than that of using H₂O₂ alone, and no significant ·OH was detected in the CeO₂ + H₂O₂ system. Therefore, they speculated that the ROS scavenging ability of CeO₂ hinders the decomposition of H₂O₂ to generate ·OH, and its POD-like activity did not contribute to the antibacterial effect of the CeO₂ + H₂O₂ system. Combined with the former characterizations of catalytic activities and ROS generation in nanozyme + H₂O₂ system, it can be inferred that the low POD-like activity and high HORAC activity of CeO₂ + H₂O₂ system lead to a relatively low amount of ·OH available for sterilization in the environment. Besides, the concentration of H₂O₂ substrate decreased as POD reaction occurred, resulting in the weakening of its antibacterial effect. In other words, CeO₂ protected bacteria from H₂O₂ and ·OH. In contrast to CeO₂, Cu-CeO₂ + H₂O₂ system showed high POD activity and low HORAC activity. The amount of ·OH in the environment was enough to meet the needs of sterilization.

Human gingival fibroblasts (hGFs) and human periodontal ligament stem cells (hPDLScs) were adopted to test the cytotoxicity of the nanozymes on normal human cells. The CCK-8 results showed that there was no significant difference in the relative activity of cells in each group with the increase of nanozyme concentration (P > 0.05, Supplementary Fig. 32a, b). Meanwhile, Live/Dead cell staining showed that there was no significant number of red stained dead cells in each group (Supplementary Fig. 32c). The maximum concentration of nanozymes used in cell safety test was 200 μg/mL, which was much bigger than the dose required for the antibacterial experiments, indicating that Cu-CeO₂ nanozyme had good biosafety.

In vivo safety of Cu-CeO₂ SSE

To test the in vivo safety of the nanozymes, PBS, CeO₂, and Cu-CeO₂ was intravenously injected into balb/c mice. The mice were observed for 3 days consecutively. As Supplementary Fig. 33 shows, there was no significant differences among the average body weight of the three groups. In addition, the blood routine test results at day 1 and day 3 after administration showed no statistical difference between each group in terms of the average white blood cell (WBC) and red blood cell (RBC) counts (P > 0.05, Supplementary Fig. 34). The hematoxylin-eosin (HE) staining results of the main organs on day 1 and day 3 indicate no significant histological differences between CeO₂, Cu-CeO₂ and PBS group (Supplementary Fig. 35). In summary, both CeO₂ and Cu-CeO₂ nanozymes showed good in vivo safety.

In vivo antibacterial performance of Cu-CeO₂ SSE

According to previous literature, the pH value of normal intact skin tissue is weakly acidic (pH = 4–6), while in the case of wound infection, due to inflammatory stimulation, the local pH value tends to be weakly alkaline (pH≈7.4)⁵⁹. It was confirmed through in vitro experiments that Cu-CeO₂ catalytically decomposed H₂O₂ efficiently to generate ROS under pH = 7.4, achieving excellent antibacterial effects and good cyclic stability. These inspired us to further explore the therapeutic effect of Cu-CeO₂ + H₂O₂ on in vivo infected skin wounds. For in vivo bactericidal experiment, we established a mouse skin wound infection model, and the schematic procedure is shown in Fig. 5a. A full-thickness skin defect with a diameter of ~6 mm was made on the back of BALB/c mice, and the wound was contaminated with MRSA. After 24 h of infection, the wounds were treated in six groups (PBS, CeO₂, Cu-CeO₂, H₂O₂, CeO₂ + H₂O₂ and Cu-CeO₂ + H₂O₂, n = 4), and the healing of skin wounds was observed daily. As Fig. 5b shows, 24 h after MRSA inoculation (Day 0), pyogenic infection occurred locally in the wounds of each group. After grouped treatments, the wounds in each group showed a trend of scab formation and contraction, and Cu-CeO₂ + H₂O₂ group showed the fastest wound healing speed (Supplementary Fig. 36). On day 7, the wounds in Cu-CeO₂ + H₂O₂ group were completely closed and the scabs fell off. The mice were sacrificed on day 7, and tissue sections of the skin around the wound were prepared. The results of HE staining showed that the epithelial structure of Cu-CeO₂ + H₂O₂ group was approximately intact and continuous with no signs of infection, while the epithelium of the other groups was discontinuous. The epithelial defects showed varying degrees of deep dyeing inflammatory cells infiltration and extensive unstructured necrosis. Masson’s trichrome staining was used to observe the distribution of collagen fibers in skin tissues of each group. There were a large number of coarse, blue stained newly formed collagen fibers under the intact epithelium in Cu-CeO₂ + H₂O₂ group, and the collagen fibers were uniformly distributed. In the other groups, however, the collagen fibers were scattered, inconsistent in thickness and disordered in structure. A certain amount of skin tissue around the wound of each group were taken to prepare homogenates, and the expression of three important pro-inflammatory factors, tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin 1β (IL-1β) was detected by enzyme-linked immunosorbent assay (ELISA) kit to reflect the severity of infection⁶⁰. After H₂O₂ or CeO₂ + H₂O₂ treatment, the level of inflammatory factors in skin tissue homogenates decreased compared with the control group, but the difference was not statistically significant (P > 0.05). However, the level of inflammatory factors in Cu-CeO₂ + H₂O₂ group was significantly lower than the control group (P < 0.001) (Fig. 5c–e). Immunohistochemistry staining was further performed to evaluate the expression of TNF-α and IL-1β in the skin tissues, and the results are shown in Supplementary Figs. 37–40, which were consistent with the ELISA results. The above results indicated that Cu-CeO₂ + H₂O₂ system showed good antibacterial activity in vivo and effectively reduced the inflammatory response caused by wound infection.

Fig. 5: In vivo antibacterial properties of CeO₂ and Cu-CeO₂ nanozymes.

a Schematic procedure of skin wound infection model and treatment. b Photographs, HE staining and Masson staining of the infected skin wounds after grouped treatment. Concentrations of TNF-α (c), IL-6 (d) and IL-1β (e) in skin tissue homogenates after grouped treatment. A representative image of three replicates from each group is shown. Data are presented as mean values +/− standard deviation, n = 3 biologically independent replicates. Significance was calculated by two-sided Student’s t-test. Scale bar: 500 μm. Source data are provided as a Source Data file.

Discussion

In this study, a Cu-CeO₂ SSE was synthesized by a facile method. The enzyme-mimicking activities of CeO₂ were regulated by Cu single site modification. Foreign Cu sites not only reduced the reaction energy of the PDS drastically during POD-like process at Ce site, but also increased its reaction energy of the PDS during HORAC process, resulting in higher POD-like activity and lower HORAC activity of Cu-CeO₂ than pristine CeO₂. Therefore, Cu-CeO₂ + H₂O₂ system successfully reversed the protective effect of CeO₂ on bacteria and demonstrated superior antibacterial properties. This study not only expands the existing antibacterial SSE family, but also reveals the regulation mechanism of metal single site on carriers with complex catalytic activities, which has important enlightenment for designing and developing smart nanocatalysts in the future.

On the other hand, CeO₂, as an excellent antioxidant nanozyme, has received widespread attention in promoting injury healing^61,62. In this study, although the ·OH scavenging capacity of Cu-CeO₂ was selectively inhibited, the scavenging acitivites of common ROS (i.e., H₂O₂ and O₂·⁻) in the injury microenvironment were enhanced compared to CeO₂. This may be one of the advantages of Cu-CeO₂ in treating infected wounds. Specifically, since Cu-CeO₂ generates a large amount of ·OH only when applied simultaneously with H₂O₂, and does not generate excess toxic ROS in the absence of H₂O₂, when H₂O₂ is completely consumed, the residual Cu-CeO₂ nanozyme may continue to scavenge endogenous ROS in the wound microenvironment. It is clear that within the scope of this study, Cu-CeO₂ + H₂O₂ system plays an important role in the early anti-infectious process. However, its ability to accelerate wound healing in the subsequent antioxidant and tissue healing processes has not been thoroughly studied, which is also a limitation of this study.

Methods

Chemicals and materials

All chemicals were used as received without further purification. Cerium nitrate hexahydrate (Ce(NO₃)₃·6H₂O, 99.95%), copper nitrate trihydrate (Cu(NO₃)₂·3H₂O, 99.99%), TMB (C₁₆H₂₀N₂, 98%), Ferrous chloride (FeCl₂, 99.5%), Sodium acetate (CH₃COONa, NaAc, 99.0%) and TA (C₈H₆O₄, 99.0%) were obtained from the Aladdin chemical reagent company. Sodium carbonate (Na₂CO₃, 99.8%), ethylene glycol ((CH₂OH)₂, 99.5%) and acetic acid (CH₃COOH, 99.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Deionized (DI) water from Milli-Q System (resistivity: 18.2 MΩ·cm, Millipore, Billerica, MA) was used in all experimental processes. CCK-8 and DMPO were obtained from Dojindo Laboratories Co., Ltd. LIVE/DEAD™ Viability/Cytotoxicity Kit, LIVE/DEAD™BacLight™ Bacterial Viability/Counting Kit and IL-1 beta Polyclonal Antibody (P420B) were obtained from ThermoFisher Scientific Co., Ltd. Mouse TNF-α, IL-6, IL-1β ELISA kit were obtained from 4ABio Co., Ltd. Recombinant anti-TNF alpha antibody (ab307164) was obtained from Abcam Co., Ltd.

Synthesis of Cu-CeO₂

In a typical preparation of CeO₂ nanosphere, 1.0 g of Ce(NO₃)₃·6H₂O was dispersed in a mixed solvent of water (1 mL), acetic acid (1 mL) and ethylene glycol (25 mL) to form a uniform solution. Then, the mixture was transferred into a steel reactor followed by a solvothermal reaction at 180 °C for 200 min. Then the precipitate was washed by centrifuging at 19,319 × g for 10 min with water for several times. After drying, the resultant products were calcinated in air at 400 °C for 4 h with a heating rate of 2 °C min⁻¹ to acquire the final CeO₂ nanospheres. 200 mg of the obtained CeO₂ powder was dispersed in 30 mL of deionized water and sonicated for 30 min to form a uniform dispersion. After that, copper nitrate aqueous solution was slowly added to the above suspension under stirring conditions. The copper content in weight percent was 5%. Five minutes later, 0.5 M sodium carbonate aqueous solution was added dropwise to ensure the pH value of the solution at ca. 9. Then the precipitates were aged at ambient temperature for 6 h, followed by centrifugation (16,099 × g, 3 min) and washed with water and ethanol several times. Subsequently, the product was dried at 80 °C overnight to acquire Cu-CeO₂ precursor. The precursor was calcined at 400 °C for 4 h in air to get the sample Cu-CeO₂ air. The Cu-CeO₂ air sample was then annealed at 250 °C for 1 h under a 5% H₂/95% Ar atmosphere to obtain the target product Cu-CeO₂.

Characterization

Transmission electron microscopy (TEM, Hitachi H-7650) was used to obtain the morphologies of the samples. The high-resolution TEM (JEOL JEM-2100F) and elemental mappings were operated at an accelerating voltage of 200 kV. Atomically resolved HAADF-STEM images were recorded on a JEM-ARM200F transmission electron microscope with a spherical aberration corrector operated at 200 kV. Field-emission scanning electron microscopy measurements were carried out on a SU-8200 scanning electron microscope. The powder XRD patterns were obtained on a Japan Rigaku RU-200b X-ray diffractometer using Cu-Kα radiation with λ = 1.5406 Å. The theoretical X-ray diffraction pattern was simulated using MAUD version 2.8 (Materials Analysis Using Diffraction). XPS experiments were conducted on a ULVAC PHI Quantera microscope. Raman measurements were carried out with a Renishaw inVia micro-Raman spectrometer with a 532 nm excitation laser. All of the DRIFTS spectra were collected by using a Bruker Vertex 70 FTIR spectrometer. The XAFS of Cu K-edge spectra were measured on the beamline BL11B station in Shanghai Synchrotron Radiation Facility (SSRF). The acquired XAFS data was processed using the ATHENA module in the Demeter 0.9.25 software package. Soft XAS spectra of Ce M_4,5-edge and O K-edge were measured at soft X-ray magnetic circular dichroism end station (XMCD) of National Synchrotron Radiation Laboratory (NSRL) in University of Science and Technology of China (USTC).

Measurements

This research complies with all relevant ethical regulations, and the study protocols are approved by the biomedical ethics committee of Peking University.

Peroxidase-like activity of Cu-CeO₂

The POD-like activity was tested according to the principle that H₂O₂ was catalytically decomposed by Cu-CeO₂ and TMB was oxidized in 0.1 M NaAc/HAc buffer (pH = 4.5). TMB was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 41.6 mM before use. The generation of water-soluable ox-TMB was measured by a multimode plate reader (PerkinElmer EnSpire^TM), and the absorbance at 652 nm was recorded. For time-dependent catalytic assay, the final concentrations of Cu-CeO₂, H₂O₂ and TMB in the system were 50 μg/mL, 44.1 mM and 1040 μM, respectively. The result was recorded every 60 s, 1200 s in total. For steady-state kinetic assays, the system contains 50 μg/mL Cu-CeO₂, 208 μM TMB with 15-150 mM H₂O₂. K_m and V_max were calculated by Lineweaver-Burk equation. The turnover rate of the nanozymes were calculated as follow:

$${{{{{\rm{Turnover}}}}}}\,{{{{{\rm{rate}}}}}}={V}_{\max }/[E]$$

(1)

Where [E] represents the total concentration of Cu atoms in the reaction system.

Catalase-like activity of Cu-CeO₂

The CAT-like activity was tested according to the principle that H₂O₂ was catalytically decomposed by Cu-CeO₂ to generate O₂ in PBS buffer (pH = 7.4). The final concentrations of Cu-CeO₂ and H₂O₂ in the system were 20 μg/mL and 0.6% (w/v). The concentration of dissolved O₂ was measured with a dissolved oxygen meter (JPSJ-606L, Leici China).

Oxidase-like activity of Cu-CeO₂

The OXD-like activity of Cu-CeO₂ was tested according to the principle that O₂ was catalytically converted by Cu-CeO₂ and TMB was oxidized in 0.1 M NaAc/HAc buffer (pH = 4.5). TMB was dissolved in DMSO at the concentration of 41.6 mM before use. The generation of water-soluable ox-TMB was measured by a multimode plate reader, and the absorbance at 652 nm was recorded. The final concentrations of Cu-CeO₂ and TMB were 50 μg/mL and 1040 μM. The result was recorded every 60 s, 900 s in total.

DMPO was used as the spin trap of O₂^·-, and the DMPO-O₂^·- spin in methanol was measured by EPR (Bruker Magnettech ESR5000) at the following parameters: microwave power of 2.5 mW, modulation field frequency of 100 kHz, amplitude of 0.2 mT, central magnetic field of 316.5 mT and scanning speed of 20 mT/min. A typical experimental system includes 10 mM DMPO and 50 μg/mL Cu-CeO₂. The reaction system was incubated at room temperature for 10 min.

Superoxide-dismutase-like activity of Cu-CeO₂

According to the manufacturer’s instructions, the SOD activity detection kit was used to detect the SOD-like activity of Cu-CeO₂. Xanthine/xanthine oxidase system was used to generate superoxide anion (O₂^·−), which reduced nitro blue tetrazolium (NBT) to formazan. O₂^·- could be removed through the SOD-like activity of Cu-CeO₂ so that the formation of methyl was inhibited. The absorbance at 560 nm was recorded.

H₂O₂ consumption of Cu-CeO₂

H₂O₂ consumption of the nanozymes were determined by spectrophotometry. In a classic assay, the final concentrations of nanozyme and H₂O₂ in the system were 100 μg/mL and 10 mM, respectively. After incubated at 37 °C for different time, samples were taken and the concentration of H₂O₂ was measured using a hydrogen peroxide content assay kit (Solarbio BC3595).

Hydroxyl radical scavenging ability of Cu-CeO₂

FeCl₂ was used as the Fenton reagent to decompose H₂O₂ to generate ·OH, and DCFH-DA was chosen as the free radical fluorescence probe. Through the HORAC activity of CeO₂ and Cu-CeO₂, ·OH can be removed so as to quench the fluorescence. The final concentrations of nanozymes, DCFH-DA, FeCl₂ and H₂O₂ were 100 μg/mL, 5 μM, 4 mM and 4 mM, respectively. Immediately after the reaction, the fluorescence intensity was measured with a multimode plate reader (λ_em = 490 nm, λ_ex = 525 nm) and recorded every minute, and the final fluorescence intensity was compared with the initial fluorescence intensity (baseline) of each group. The relative change of fluorescence intensity was calculated as follow:

$${{{{{\rm{Relative}}}}}}\,{{{{{\rm{change}}}}}}=|{F}_{0}-{F}_{{{{{{\rm{t}}}}}}}|/{F}_{0}\times 100\%$$

(2)

Where F₀ represents the initial fluorescence intensity (baseline), and F_t represents the fluorescence intensity after certain time of reaction.

Quantitative analysis of ·OH scavenging was performed using EPR (Bruker Magnettech ESR5000). DMPO was used as the spin trap of ·OH, and FeCl₂ was used as the Fenton reagent to decompose H₂O₂. The final concentrations of nanozymes, DMPO, FeCl₂ and H₂O₂ were 50 μg/mL, 10 mM, 1 mM and 1 mM, respectively. The detection parameters are as follow: microwave power, 2.5 mW; modulation field frequency, 100 kHz; amplitude, 0.2 mT; central magnetic field, 336.5 mT and scanning speed, 20 mT/min.

Detection of ROS

DCFH-DA was used as the fluorescence probe of ROS. ROS generation during the process of catalytic decomposition of H₂O₂ in PBS was measured and recorded by a multimode plate reader (λ_em = 490 nm, λ_ex = 525 nm). A typical experimental system included 5 μM DCFH-DA, 50 μg/mL Cu-CeO₂ and 10 mM H₂O₂. The reaction system was incubated at room temperature in the dark for 10 min.

DMPO was used as the spin trap of ·OH, and the catalytic decomposition of H₂O₂ by Cu-CeO₂ in 0.1 M NaAc / HAc buffer (pH = 4.5) was measured and recorded by EPR (JEOL JES FA200) at the following parameters: microwave power of 1 mW at the frequency of 9.0–9.1 GHz, modulation field frequency of 100 kHz, amplitude of 0.1 mT, time constant of 0.1 s, central magnetic field of 322.5 mT and scanning speed of 10 mT/min. A typical experimental system includes 100 mM DMPO, 50 μg/mL Cu-CeO₂ and 10 mM H₂O₂. The reaction system was incubated at room temperature for 10 min.

Cyclic stability of Cu-CeO₂

200 μg/mL Cu-CeO₂ and 10 mM H₂O₂ were mixed in PBS. After certain time of reaction, the precipitate was recovered by centrifugation at 20,627 × g. Cu-CeO₂ after 10, 20 and 30 cycles were collected and POD-like activities of the samples were tested. The content of Cu element in the supernatant after reaction was determined using ICP-MS (Thermo Scientific XSeries 2).

Calculation details

The DFT calculations were conducted with the Vienna Ab-initio Simulation Package (VASP 6.1.0)^63,64 with the Projected Augmented Waves (PAW) basis and Perdew-Burke-Ernzerhof (PBE)⁶⁵ exchange-correlation functional. DFT + U correction with the effective U-J value of 5 eV⁶⁶ and 3 eV⁶⁷ were adopted to describe the exchange interaction of Ce 4f and Cu 3d orbitals, respectively. Spin-polarization was applied throughout the calculations along with the Grimme’s DFT-D3⁶⁸ method accounting for the long-range van der Waals interaction. A 4-layered 3 × 3 CeO₂ 111 supercell was built to represent the pristine CeO₂ surface. 20 Å vacuum space was added along the z direction to avoid interaction between the adjacent layers. Two Cu-doped CeO₂ surface, namely Cu-ad and Cu-sub were built on the basis of the pristine surface to investigate the exact doping configuration. The 400 eV energy cutoff and 3 × 3 × 1 Gamma centered k-point mesh was adopted during the calculation. The geometries for the reaction intermediates were optimized with the bottom two CeO₂ layers fixed until the residual force less than 0.03 eV/Å.

In vitro antibacterial properties of Cu-CeO₂

E. coli and MRSA were cultured in brain heart infusion (BHI) broth to logarithmic growth phase, and the absorbance of bacterial solution at 630 nm wavelength was measured by a microplate reader. The bacterial solution was centrifuged at 9167 × g for 3 min, and the precipitation was resuspended with PBS buffer. In a typical experimental system, the concentrations of bacteria, nanozyme and H₂O₂ were ~10⁶ CFU/mL, 25 μg/mL and 4 mM for MRSA, and ~10⁷ CFU/mL, 25 μg/mL and 4 mM for E. coli, respectively. After incubation at 37 °C for 1 h, gradient dilutions were inoculated on BHI agar dish, incubated in an air incubator at 37 °C overnight, and the colonies were counted.

Bacterial live/dead staining (ThermoFisher LIVE/DEAD BacLight Bacterial Viability Kit) were performed according to the manufacturer’s instructions. SYTO-9 and propidium iodide (PI) were added to the reaction system after co-culture, incubated in dark for 15 min. The bacterial solution was observed with a fluorescence microscope (λ_em = 480 nm, λ_ex = 500 nm for SYTO-9, and λ_em = 535 nm, λ_ex = 615 nm for PI).

For SEM observation, the bacterial solution after co-culture was fixed with 4% paraformaldehyde-PBS and dehydrated with gradient ethanol. The samples underwent gold sputtering at 10 mA and 40 mBar vacuum, and the morphology of the bacteria was observed.

In vitro cytotoxicity of Cu-CeO₂

HGFs and hPDLScs were inoculated in 96 well plates at 10⁴/well and cultured with Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). The nanozymes were added for co-culture, and the final concentrations were 0, 3, 12, 50, 100 and 200 μg/mL, respectively. After incubation for 24 h, proliferation medium containing 10% Cell Counting Kit-8 (CCK-8) reagent was added. After incubation for 1 h, the absorbance at 450 nm was measured using a microplate reader. For live/dead cell staining, after co-cultured with nanozymes, the cells were washed with PBS, and calcein AM/PI (Keygen Biotech KGAF001) were added according to the manufacturer’s instructions. After incubation for 15 min, the cells were observed with a fluorescence microscope (OLYMPUS U-RFL-T λ_em = 490 nm, λ_ex = 515 nm for calcein AM, and λ_em = 535 nm, λ_ex = 615 nm for PI).

In vivo experiments

The animal experiments were carried out under the approval of the biomedical ethics committee of Peking University (approval number: LA2021473). Mice were kept in constant temperature (22 °C), constant humidity (55%) and cyclic lighting (12 h light/12 h dark). For in vivo safety analysis, eighteen 6-week-old BALB/c female mice were purchased from SiPeiFu Biotechnology Co., Ltd, Beijing, and were injected with PBS, CeO₂, Cu-CeO₂ through i.v., respectively. The body weights were recorded consecutively for 3 days. At day 1 and day 3 after administration, peripheral blood and main organs were collected for further analysis. For in vivo antibacterial experiment, twenty-four 6-week-old BALB/c female mice were purchased from SiPeiFu Biotechnology Co., Ltd, Beijing. MRSA was cultured to logarithmic growth stage in BHI liquid, and the bacterial solution was adjusted to 10⁶ ~ 10⁷ CFU/mL with PBS. After anesthesia, a full-thickness wound with a diameter of ~6 mm was made on the dorsal skin of the mice through aseptic operation. MRSA solution was injected locally to make an infected wound model. After 24 h of infection, the wounds were treated in groups (PBS, CeO₂, Cu-CeO₂, H₂O₂, CeO₂ + H₂O₂ and Cu-CeO₂ + H₂O₂), and covered with Band-Aids. The Band-Aids were changed every 24 h. The concentrations of nanozymes and H₂O₂ were 25 μg/mL and 4 mM, respectively. The wound healing was observed, and the wound size were measured. On the 7th day after infection, the mice were sacrificed and local skin tissue was obtained. 50 mg samples were obtained from each tissue and lysed with Radio immunoprecipitation assay buffer (Ripa): Phenylmethanesulfonyl fluoride (PMSF) = 100:1 mixed lysate to prepare tissue homogenate. The supernatant was obtained under 14,000 × g centrifugation for 10 min at 4 °C. The protein concentration was detected by Bicinchoninic acid (BCA) method. IL-6, IL-1, and TNF-α contents were detected using ELISA kit. The skin tissues of each group were fixed with 10% neutral formalin solution for 24 h. The tissues were dehydrated and paraffin embedded. Tissue sections were made and HE Masson and immunohistochemistry staining were used for histological analysis. For immunohistochemistry staining, recombinant anti-TNF alpha antibody (dilution of 1:1000) and IL-1 beta Polyclonal Antibody (dilution of 1:500) were used as primary antibodies. Detailed information (supplier name, catalog number) about the antibodies are listed in Reporting Summary.

Statistical analysis

The experimental data were analyzed using IBM SPSS Statistics 24.0 software. One-way ANOVA was used for comparisons, and Games-Howell test was used for post hoc multiple comparisons.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.