A ruthenium single atom nanozyme-based antibiotic for the treatment of otitis media caused by Staphylococcus aureus

Staphylococcus aureus (S. aureus) infection is a primary cause of otitis media (OM), the most common disease for which children are prescribed antibiotics. However, the abuse of antibiotics has led to a global increase in antimicrobial resistance (AMR). Nanozymes, as promising alternatives to traditional antibiotics, are being extensively utilized to combat AMR. Here, we synthesize a series of single-atom nanozymes (metal-C₃N₄ SANzymes) by loading four metals (Ag, Fe, Cu, Ru) with antibacterial properties onto a crystalline g-C₃N₄. These metal-C₃N₄ display a rob-like morphology and well-dispersed metal atoms. Among them, Ru-C₃N₄ demonstrates the optimal peroxidase-like activity (285.3 U mg^–1), comparable to that of horseradish peroxidase (267.7 U mg^–1). In vitro antibacterial assays reveal that Ru-C₃N₄ significantly inhibits S. aureus growth compared with other metal-C₃N₄ even at a low concentration (0.06 mg mL^–1). Notably, Ru-C₃N₄ acts as a narrow-spectrum nanoantibiotic with relative specificity against Gram-positive bacteria. Biofilms formed by S. aureus are easily degraded by Ru-C₃N₄ due to its high peroxidase-like activity. In vivo, Ru-C₃N₄ effectively eliminates S. aureus and relieves ear inflammation in OM mouse models. However, untreated OM mice eventually develop hearing impairment. Due to its low metal load, Ru-C₃N₄ does not exhibit significant toxicity to blood, liver, or kidney. In conclusion, this study presents a novel SANzyme-based antibiotic that can effectively eliminate S. aureus and treat S. aureus-induced OM.

1 Introduction

Otitis media (OM) is a prevalent disease that is frequently diagnosed and treated by pediatricians. It is estimated that nearly half of all children will experience at least one episode of ear infection by the time they reach their second birthday (Paul and Moreno, 2020). The global incidence rate of OM is approximately 16% (Elzinga et al., 2021), underscoring the significant impact of OM on pediatric health and the urgent need for effective treatment strategies. OM can be manifested in the form of acute OM (AOM), OM with effusion, persistent as recurrent AOM (rAOM) and chronic suppurative otitis media (CSOM) (Hoberman et al., 2021). If left untreated, AOM will progress to CSOM, leading to serious complications such as mastoiditis, labyrinthitis, facial nerve palsy, meningitis, and cerebral abscesses (Holm et al., 2020). These complications are particularly problematic in low-income countries (Peñaranda et al., 2023), where an estimated 21,000 people die annually from OM complications (Monasta et al., 2012). The estimated global prevalence of OM patients with hearing loss is approximately 30 (ranging from 0.7 to 95) per 10,000 individuals (Li et al., 2022).

The management of uncomplicated OM typically involves the cleansing of external auditory canal and antibiotics application. Topical aminoglycosides and quinolones are commonly used as first-line antibiotics (Nawaz et al., 2023). However, the overprescription of these drugs significantly contributes to the development of AMR (Dadgostar, 2019; Huemer et al., 2020). It is estimated that 300 million individuals will experience premature mortality due to AMR within the next 35 years (Ahmed et al., 2024). The Gram-positive bacterium Staphylococcus aureus (S. aureus) infection is recognized as a primary cause of OM(Mohammed and Abdullah, 2020), constituting 13% (171 out of 1309) of total isolates (Roland et al., 2005). During infection, planktonic S. aureus invades the middle ear via the eustachian tube, leading to the inflammation of the middle ear mucosa and tympanic cavity. Independent S. aureus can form biofilms within the middle ear and attach to the surface of middle ear mucosal cells (Abu Bakar et al., 2018). In this biofilm state, the resistance of S. aureus to antibiotics significantly increases. Antibiotics tend to accumulate within the biofilm matrix, a barrier that inhibits their penetration into the deeper bacterial layers. Consequently, this restriction hampers antibiotics mobility within the matrix, thereby compromising their delivery to the inner layers of the biofilms (Coppens et al., 2023). For instance, vancomycin is the first-line drug for treating infections associated with S. aureus. In recent years, there has been a notable reduction in the sensitivity of S. aureus to vancomycin due to its increased usage and dosage (Cong et al., 2020). Notably, the resistant phenotype of S. aureus can rapidly switch to a susceptible phenotype when the biofilms are disrupted (Parastan et al., 2020). Destructive enzymes have been proposed as an effective agent for eliminating adhesions and preventing bacterial accumulation within the biofilm structure. Protease, hyaluronidase, lysostaphin, and peroxidase can destroy bacteria-formed biofilms through various mechanisms (Rosenthal et al., 2014; Wei et al., 2021; Song et al., 2024b). However, certain intrinsic limitations such as high cost, low catalytic stability, and complex preparation process of natural enzymes restrict their practical application in the antibacterial field (Zhang et al., 2020).

Nanozymes are emerging as promising alternatives to natural enzymes due to their inherent enzymatic properties and activities. They exhibit kinetics and catalytic mechanisms that closely mimic those of natural enzymes. Unlike conventional antibiotics, nanozymes induce the development of AMR at a lower frequency, attributed to their excellent membrane permeability and catalytic activity (Mei et al., 2021; Feng et al., 2022). Furthermore, their enzyme-like properties effectively eliminate bacterial biofilms (Gao et al., 2014; Liang et al., 2020; Xu et al., 2021). Antibacterial nanozymes encompass metal- or nonmetal-based nanoparticles, nanoclusters, and single-atom nanozymes (SANzymes) (Xiong et al., 2019; Meng et al., 2022; Lan et al., 2023). Among these categories, SANzymes display optimal catalytic activity due to their uniformly distributed active sites and maximum atomic utilization (nearly 100%) (Tang et al., 2022). Compared to conventional nanoparticles, the low metal atom loading also improves the biocompatibility of SANzymes (Zhu et al., 2023). Recently, a variety of metal-based nanozymes have been demonstrated to have promising antibacterial efficacy, such as Fe, Ru, Ag, Cu, Pt and Ir (Mei et al., 2021). Compared to other enzyme-based therapeutic approaches, nanozymes can generate a higher intensity of reactive oxygen species (ROS) to easily disrupt biofilms and induce bacterial death. Therefore, the design and synthesis of efficient antibacterial nanozymes hold significant potential for addressing AMR.

In this study, we successfully synthesized four SANzymes with similar geometries and well-defined atomic metals to replace natural peroxidase in the treatment of S. aureus-induced OM. We selected Ag, Fe, Cu, and Ru as the metal catalytic sites due to their superior antibacterial properties (Gao et al., 2017; Huang et al., 2017; Xi et al., 2020; Wang et al., 2021). The crystalline g-C₃N₄ nanorods were synthesized and utilized as the carrier of single atoms to improve the metal loading and catalytic activity. Meanwhile, its excellent biosafety further expanded its application (Pourmadadi et al., 2023). Characterization data showed that four metal-C₃N₄ exhibited a rod-like structure, with monodispersed metal atoms on the C₃N₄ carrier. We then conducted a systematic investigation of the peroxidase-like properties of these metal-C₃N₄, including catalytic activity, catalytic efficiency, substrate selectivity, and ROS production capacity. Among these SANzymes, Ru-C₃N₄ demonstrated superior peroxidase-like activity (285.3 U mg^–1), comparable to that of HRP (267.7 U mg^–1). Furthermore, Ru-C₃N₄ exhibited optimal efficacy in inhibiting S. aureus growth and disrupting biofilms. We finally conducted a comprehensive evaluation of the therapeutic effect of Ru-C₃N₄ on OM induced by S. aureus in vivo (Scheme 1). In summary, this study provides an effective SANzyme for combating S. aureus and its associated OM.

Scheme 1

Scheme 1. The schematic illustration of metal-C₃N₄ preparation and their treatment of S. aureus-induced OM model.

2 Results and discussion

2.1 Synthesis and characterization of metal-C₃N₄

Four metal-C₃N₄ (the metal denoting Ru, Fe, Cu, and Ag) were prepared by heating the mixture of crystalline C₃N₄ and metal salts in an aqueous solution according to previous methods (Wang et al., 2019). As crystalline C₃N₄ provided strong coordination sites for metal ions, the metal can be effectively incorporated into this carrier. The morphologies of these metal-C₃N₄ and free C₃N₄ were investigated by transmission electron microscopy (TEM). As shown in Figure 1A and Supplementary Figure S1A–D, both the metal-C₃N₄ and free C₃N₄ exhibited very similar rod-like structure, indicating that there was no substantial morphological alteration following the integration of four metal ions.

Figure 1

Figure 1. The characterization of Ru-C₃N₄. (A) TEM image of Ru-C₃N₄. Scale bar = 200 nm. (B) SEM image of Ru-C₃N₄. Scale bar = 200 nm. (C) EDS mapping images of Ru-C₃N₄. Scale bar = 80 nm. (D–E) HAADF-STEM image and the enlarged image of Ru-C₃N₄. Scale bar = 2 nm. (F) Corresponding intensity image of Ru-C₃N₄ in (E). (G) XPS analysis of C₃N₄ and Ru-C₃N₄. (H) High-resolution XPS spectra of N 1s and (I) C 1s (or Ru 3d).

The Ru-C₃N₄ was thoroughly studied due to its superior catalytic activity and antibacterial effect compared to other metal-C₃N₄, as demonstrated by our subsequent analysis. Scanning electron microscope (SEM) measurement clearly displayed the rod-like morphology of Ru-C₃N₄ (Figure 1B). Energy dispersive spectroscopy (EDS) mapping image revealed that Ru, C and N were uniformly distributed throughout the entire C₃N₄ architecture (Figure 1C; Supplementary Figure S2). For comparison, EDS mapping images of other metal-C₃N₄ were showed in Supplementary Figures S3A–C. Specifically, the concentrations of metal atoms anchoring on C₃N₄ were described as Ru (1.12 wt%), Fe (1.88 wt%), Cu (0.95 wt%) and Ag (0.66 wt%) quantified by inductively coupled plasma optical emission spectrometry (ICP-OES) (Supplementary Table S1). Additionally, high-angle annular dark-field image-scanning transmission electron microscopy (HAADF-STEM) measurements were conducted to provide the atomic information of metal-C₃N₄ (Figures 1D–F; Supplementary Figure S4). Figures 1D–F clearly showed single Ru atoms fixing across the C₃N₄ carrier, suggesting that no metal nanoparticle was formed during the heating of the synthesis process. Similarly, the HAADF-STEM images of other metal-C₃N₄ (Supplementary Figure S3) revealed that the metals were atomically dispersed on C₃N₄.

Next, the chemical composition and oxidation state of Ru-C₃N₄ were studied by X-ray photoelectron spectroscopy (XPS). The survey spectra of Ru-C₃N₄ and free C₃N₄ showed two peaks with binding energy around 287 eV and 398 eV, which belonged to C 1s (or Ru 3d) and N 1s, respectively (Figure 1G). The high-resolution N 1s spectra of Ru-C₃N₄ and free C₃N₄ were shown in Figure 1H. The N1s spectrum of free C₃N₄ was deconvoluted into two subpeaks centered at 398.15 and 399.69 eV, corresponding to sp²-hybridized pyridinic nitrogen (C-N=C) and sp³-hybridized tertiary nitrogen (N-(C)₃), respectively. By contrast, the binding energy peak of C-N=C was observed at 398.32 eV, and the blue shift by 0.17 eV could be attributed to the electron transfer from nitrogen atoms of C₃N₄ to Ru ions for Ru-C₃N₄. Furthermore, Figure 1I provided the C 1s (or Ru 3d) spectra of Ru-C₃N₄ and free C₃N₄. The peaks at 284.60 and 287.84 eV in the C 1s spectrum of free C₃N₄ were attributed to the N-C=N bound and C-C bound, respectively. Notably, the peak around 284 eV in the C 1s spectrum of Ru-C₃N₄ was enhanced compared with free C₃N₄ due to the overlap of the C 1s and Ru 3d_3/2 peaks (Chen et al., 2018). Moreover, the Ru 3d spectrum was deconvoluted into two subpeaks at 281.84 eV and 285.63 eV, which were attributed to Ru 3d_5/2 and Ru 3d_3/2, respectively, suggesting that Ru atoms in Ru-C₃N₄ were present in the Ru²⁺ oxidation state.

2.2 Peroxidase-like activity evaluation of metal-C₃N₄

ROS generation is a mainstream mechanism for the antibacterial activity of nanozymes (Song et al., 2022; Xu et al., 2023). Therefore, we next evaluated the peroxidase-like activity of four metal-C₃N₄ by catalyzing the generation of free radicals from H₂O₂ to achieve the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). As shown in Figure 2A, Ru-C₃N₄ showed robust peroxidase-like activity, while the catalytic activity of Ag-C₃N₄ was close to that of Fe-C₃N₄, and both were weaker than that of Ru-C₃N₄. Conversely, the Cu-C₃N₄ and C₃N₄ carrier demonstrated no peroxidase-like activity, suggesting that the catalytic activity of metal-C₃N₄ resulted from their single-atom metals. For the color reaction, we employed different concentrations of Ru-C₃N₄ to further confirm its catalytic properties. Supplementary Figure S5 showed that the ability of Ru-C₃N₄ to catalyze the color reaction of TMB increased with its concentration.

Figure 2

Figure 2. Peroxidase-like activity characterization of metal-C₃N₄. (A) Reaction-time curves of the TMB colorimetric reaction catalyzed by metal-C₃N₄. (B) The optimal temperature and (C) pH value of Ru-C₃N₄ for the catalytic activity. (D) ESR spectra showed the generation of ·OH and (E) ·O₂⁻ by Ru-C₃N₄. (F,G) Michaelis-Menten curves and kinetics parameters for Ru-C₃N₄. (H,I) The specific activities (U mg⁻¹) of Ru-C₃N₄ and HRP.

The peroxidase-like activity of Ru-C₃N₄ was systematically analyzed under different reaction temperatures and pH values. With the increase of temperature (15°C–60°C), the catalytic activity of Ru-C₃N₄ showed a trend of increasing and then decreasing. Its optimal reaction temperature was close to 35°C, which was comparable with that of horseradish peroxidase (HRP) (Figure 2B; Supplementary Figure S6A). Further, Ru-C₃N₄ could maintain higher relative activity over a wider temperature range compared with HRP, indicating that Ru-C₃N₄ had greater potential for medical applications than the natural enzyme. Figure 2C and Supplementary Figure S6B exhibited that the optimal reaction pH values for Ru-C₃N₄ and HRP were 5.0 and 4.5, respectively. Under this condition, we evaluated Ru-C₃N₄ as a SANzyme-based antimicrobial to generate ROS in the presence of H₂O₂. The results of electron spin resonance (ESR) assays showed that Ru-C₃N₄ induced a strong intensity of two ROS, including ·OH and·O₂⁻. In contrast, its C₃N₄ carrier failed to catalyze H₂O₂ to produce ROS (Figures 2D, E).

The steady-state kinetic analyses of Ru-C₃N₄ and HRP were next compared comprehensively. Figure 2F and Supplementary Figure S7A demonstrated that the catalytic curves of Ru-C₃N₄ and HRP with H₂O₂ as the reaction substrate and TMB as the oxidation substrate followed the Michaelis-Menten equation. Figure 2G and Supplementary Figure S7B showed that the K_M value of Ru-C₃N₄ was 7.65 × 10⁻⁵ M, which was smaller than that of HRP (9.42 × 10⁻⁵ M) under the optimal reaction conditions, suggesting that the affinity of Ru-C₃N₄ for H₂O₂ was higher than that of HRP. Furthermore, other kinetics parameters of Ru-C₃N₄ and HRP, including V_max, k_cat, and k_cat/K_M, showed similar levels (Supplementary Table S2). To further characterize the enzymatic properties of Ru-C₃N₄, we calculated its peroxidase-like specific activity. The calculated protocols following the previous protocols (Jiang et al., 2018; Wang et al., 2024a). A length of 60 s was chosen for the initial rate period because the R² coefficient was close to 1.0 during this period after a linear-regression analysis (Figure 2H). As shown in Figure 2I, the mean peroxidase-like specific activity of Ru-C₃N₄ was 285.3 U mg^–1, which was comparable to that of HRP (267.7 U mg^–1). The outstanding peroxidase-like property of Ru-C₃N₄ SANzyme aligned with the previously reported enzymatic characteristics of Ru cluster nanozyme (Fan et al., 2024), indicating that Ru element is a promising material for the construction of high peroxidase-like activity nanozymes. In this work, we loaded single atomic Ru onto a crystalline C₃N₄, thereby enhancing its biosafety and contact area with bacteria. Additionally, the storage stability is an essential condition as antibacterial agents. Therefore, we finally investigated the catalytic stability of Ru-C₃N₄ under room temperature. Supplementary Figure S8 showed that there was no significant change in the peroxidase-like activity of Ru-C₃N₄ after the storage at room temperature for 20 weeks. Collectively, these data provide reliable evidence that Ru-C₃N₄ has excellent peroxidase-like activity.

2.3 Evaluation of inhibition of S. aureus growth by metal-C₃N₄ in vitro

Catalytic activity studies have shown that Ru-C₃N₄ possessed stronger peroxidase-like activity than other metal-C₃N₄. However, which metal-C₃N₄ possesses the best antibacterial properties was unclear. To address this, we initially evaluated the inhibitory effects of four metal-C₃N₄ on S. aureus growth in vitro. As shown in Figure 3A, four metal-C₃N₄ exhibited different anti-S. aureus activities. Compared to control group, the bactericidal efficiency of Ru-C₃N₄ reached more than 99.9% when the concentration was 0.06 mg mL^–1 and the incubation time was 0.5 h (Figure 3B). Under the same condition, other metal-C₃N₄ also showed significant antibacterial activity compared to control group. The antibacterial efficiency was Cu-C₃N₄ (76.6%), Fe-C₃N₄ (98.4%), and Ag-C₃N₄ (98.5%) respectively, which was largely positively correlated with their peroxidase-like activity. As a result, Ru-C₃N₄ was selected for further investigation.

Figure 3

Figure 3. Evaluating the anti-S. aureus efficiency of metal-C₃N₄ in vitro. (A,B) The inhibition efficiency and the statistics of metal-C₃N₄ on S. aureus growth. Control represented only 10 mM H₂O₂ treatment, while other groups were treated with 10 mM H₂O₂ + metal-C₃N₄. (C,D) Anti-S. aureus efficiency the statistics of Ru-C₃N₄ at different concentrations (0.01–0.09 mg mL^–1). Control represented only 10 mM H₂O₂ treatment, while other groups were treated with 10 mM H₂O₂ + 0.01–0.09 mg mL^–1 Ru-C₃N₄. (E,F) Antibacterial efficiency and statistics of Ru-C₃N₄ against Gram-positive (S. aureus, S. agalactiae, and P. aeruginosa) and Gram-negative bacteria (E. coli and K. pneumoniae). Control represented only treated with 10 mM H₂O₂ for Gram-positive bacteria and 1 mM H₂O₂ for Gram-negative bacteria.

Next, we optimized the administrated dosage (0.01–0.09 mg mL^–1) of Ru-C₃N₄ to better understand its antibacterial effect. The results showed that the bactericidal effect of Ru-C₃N₄ progressively enhanced with increasing concentration (Figure 3C). The anti-S. aureus efficiency of Ru-C₃N₄ at 0.01, 0.03, 0.06, and 0.09 mg mL^–1 was 25.1%, 96.3%, over 99.9%, and over 99.9%, respectively (Figure 3D). Therefore, we considered that 0.06 mg mL^–1 was the optimal concentration of Ru-C₃N₄ for anti-S. aureus investigation. According to many current studies, some nanozymes with peroxidase-like activity have been attempted for the killing of S. aureus. For instance, the Au@ZIF-8 presented enhanced peroxidase-like activity under near-infrared laser (NIR). Au@ZIF-8 achieved S. aureus clearance rate up to 97% (Ren et al., 2023). MoWS₂ nanozyme could achieve efficient anti-S. aureus activity (98.84%) and biofilm clearance through hyperthermia and reactive oxygen species under NIR-II irradiation (Du et al., 2024). Liang et al. designed a NiCo₂O₄ nanozyme with self-adaptive hierarchical nanostructure that can capture bacteria of various morphotypes via the physico-mechanical interaction between the nanostructure and bacteria. 1 mg mL⁻¹ NiCo₂O₄ nanozyme exhibited superior bacterial inhibition rates of >99.99% against S. aureus (Song et al., 2024a). Compared with these nanozymes, low dose of Ru-C₃N₄ (0.06 mg mL^–1) could achieved satisfactory antibacterial effect (>99%).

The broad-spectrum antibiotics exhibited potent antimicrobial properties, their application presented several drawbacks, including the propensity to select multiple bacterial species and the propagation of resistance. Additionally, they may disrupt the host microbiome’s diversity (Melander et al., 2018). If the specific cause of the infection is identified, the utilization of narrow-spectrum antimicrobials had the capability to alleviate the certain challenges (Casadevall, 2009; Alm and Lahiri, 2020). Therefore, we finally evaluated the antibacterial selectivity of Ru-C₃N₄. Three Gram-positive bacteria (S. aureus, Staphylococcus agalactiae, and Pseudomonas aeruginosa) and two Gram-negative bacteria (E. coli and K. pneumoniae) were cultured and treated with Ru-C₃N₄ at 0.06 mg mL^–1. As shown in Figures 3E, F, Ru-C₃N₄ showed significant antibacterial effects against all strains. However, the killing effect of Ru-C₃N₄ on Gram-positive bacteria was significantly higher than that on Gram-negative bacteria (Supplementary Figure S9). The antibacterial efficiency of Ru-C₃N₄ against S. aureus, S. agalactiae, and P. aeruginosa was over 99.9%, 99.7%, and 93.0%, while that against E. coli and K. pneumoniae was 72.5% and 65.5%, respectively. Hence, Ru-C₃N₄ is relatively selective against Gram-positive bacteria.

2.4 Ru-C₃N₄ disrupts S. aureus-formed biofilms

Biofilm formation is a survival strategy for bacteria to adapt to their living environment, especially in the hostile environment (Wu et al., 2015). Bacterial biofilms play a critical role in the development of numerous persistent diseases (Mah, 2012). The structure and physiological characteristics of biofilms provide them with a natural resistance to antimicrobial agents (Donlan and Costerton, 2002). Once the biofilms become incomplete, bacteria will lose their shield and return to their planktonic state, becoming more sensitive to antibacterial agents (da Silva et al., 2021; Goodman and Bakaletz, 2022; Ji et al., 2022). Thus, we systematically evaluated the disrupted effect of Ru-C₃N₄ on S. aureus-formed biofilms. As shown in Figure 4A, S. aureus attached themselves to the surface of cell crawling sheets by using their sticky appendages, which resulted in the formation of microaggregation. Initially, filamentous junctions were formed between bacteria during biofilms formation. With the increase of culture time, bacteria-secreted polysaccharide matrix, fibrin, lipid protein, and other substances formed a bacteria gathered membrane-like substance. We then compared the effect of Ru-C₃N₄ at different concentrations (0.01–0.09 mg mL^–1) on the destruction of biofilms. Figure 4B exhibited that C₃N₄ carrier was unable to cause disruption of S. aureus-formed biofilms. By contrast, Ru-C₃N₄ at 0.01 mg mL^–1 could cause biofilm destruction moderately. With the increase of Ru-C₃N₄ concentration, the destruction effect of Ru-C₃N₄ on biofilms became more significant. 0.03 mg mL^–1 Ru-C₃N₄ induced obvious biofilm destruction. S. aureus lost their shield and returned to planktonic state. Ru-C₃N₄ at 0.09 mg mL^–1 achieved complete elimination of S. aureus and its biofilms. Therefore, the single atom Ru on Ru-C₃N₄ determines its antimicrobial activity. In the bacterial microenvironment, Ru-C₃N₄ first catalyzed H₂O₂ to produce large amounts of ROS. These free radicals effectively destroyed S. aureus-formed biofilms. Finally, the growth and structure of planktonic S. aureus were severely disrupted under excessive ROS condition.

Figure 4

Figure 4. The destructive effect of Ru-C₃N₄ on the biofilms formed by S. aureus. (A) The process of biofilm formation by S. aureus in 1–7 days. Scale bar = 1 μm. (B) Destructive effects of Ru-C₃N₄ at different concentrations on S. aureus-formed biofilms. Control represented only 10 mM H₂O₂ treatment, while other groups were treated with 10 mM H₂O₂ + 0.01–0.09 mg mL^–1 Ru-C₃N₄ or Ru-C₃N₄ carrier. The scale bars for the upper and lower parts of Ru-C₃N₄ and C₃N₄ treated groups are 10 μm and 5 μm, respectively.

2.5 Evaluation of anti-S. aureus effect of Ru-C₃N₄ in vivo

We next investigated the anti-S. aureus effect of Ru-C₃N₄ in vivo. The OM model was constructed using C57BL/6J mice according to previous studies (Davidoss et al., 2018; Li et al., 2023). The histological morphology and infected process of the middle ear mucosa were observed by SEM system at different time points (0–9 days). As shown in Figure 5A, the middle ear mucosa of OM mouse model presented a typical physiological morphology at day 0. By contrast, many small bacterial colonies of S. aureus were found on the surface of middle ear mucosa at day 3. At day 6, S. aureus significantly formed interbacterial plasmodesmata and secreted abundant substances. Furthermore, S. aureus formed thick biofilms on the surface of middle ear mucosa after 9 days modelling, with the entire surface being completely covered, suggesting the successful construction of OM model. Subsequently, we evaluated the destruction effects of Ru-C₃N₄ for biofilms in vivo. Figure 5B showed that C₃N₄ carrier failed to inhibit the biofilm formation of S. aureus. On the contrary, Ru-C₃N₄ exhibited effective biofilm destruction and S. aureus killing effects after 7 days of treatment. Very few S. aureus were able to survive on the middle ear mucosa.

Figure 5

Figure 5. Anti-S. aureus effect of Ru-C₃N₄ in OM model. (A) The growth and biofilm formation of S. aureus in middle ear mucosa of C57BL/6 mouse. The scale bar = 50 μm. (B) Ru-C₃N₄ effectively disrupted the growth and biofilm formation of S. aureus in vivo. The scale bar = 10 μm. (C) Live/dead cells staining for the middle ear mucosa of OM model. The scale bar = 100 μm. (D) H&E staining for the middle ear. The scale bar = 1 mm. (E) Inflammatory cytokines detection in the middle ear lavage fluid of OM model.

To investigate the therapeutic effect of Ru-C₃N₄ on OM model, the live/dead cell assay using calcein AM/propidium iodide (PI) staining was performed. At day 0 of Ru-C₃N₄ treatment, many dead cells (red) accumulated on the middle ear mucosa, indicating that the integrity of mucosal cells were severely damaged in OM model. With the extension of treatment time, the dead cells gradually disappeared and middle ear mucosal cells were fully recovered (green). At day 10 of Ru-C₃N₄ treatment, the dead mucosal cells of middle ear almost disappeared (Figure 5C), suggesting the mucosal destruction caused by S. aureus has been fully repaired. In addition, hematoxylin and eosin (H&E) staining was performed to further confirm the therapeutic effect of Ru-C₃N₄ on OM model. In the middle ear tissue of normal mouse, every type of cells was stained uniformly and packed tightly. Moreover, there was no obvious necrosis, bleeding, and inflammation in the ear tissues. The laminae propria connective tissue in normal ear tissues was equally distributed. As shown in the middle ear tissue of OM model, connective tissue hyperplasia was appeared, accompanied by eosinophilic exudate in the laminae propria. Epithelium exfoliation locally, new capillaries in the deep mucosa, and bone destruction were observed clearly. Meanwhile, several inflammatory features were present, including the infiltration of neutrophils and lymphocytes. These pathological features were not effectively improved in the C₃N₄ carrier treated group. By contrast, typical OM features of ear tissues almost disappeared after Ru-C₃N₄ treatment, accompanied by gradual healing of mucosal epithelium, alleviation of edema symptoms and inflammation, and repair of connective tissue (Figure 5D). Several inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were obviously increased in the middle ear lavage fluid of OM model at day 0 (Supplementary Figure S10). Following the initial administration of Ru-C₃N₄, a significant decrease in the levels of all three inflammatory factors was observed. As the duration of treatment extended, their levels continued to decrease, eventually reverting to those in the normal group. However, C₃N₄ carrier had no significant effects on these inflammatory factors (Figure 5E).

2.6 Ru-C₃N₄ relieves S. aureus-induced hearing damage

With the excellent anti-S. aureus activity of Ru-C₃N₄ both in vitro and in vivo, we subsequently evaluated the efficacy of Ru-C₃N₄ in the treatment of hearing impairment caused by S. aureus. The auditory brainstem response (ABR) threshold of OM model was systematically tested. In vivo, the hearing function of normal mice under certain frequencies (8, 16, 24, 32 kHz) was not impaired by the treatments of Ru-C₃N₄ and C₃N₄ carrier (Figure 6A), suggesting the well biocompatibility of Ru-C₃N₄ SANzymes. By contrast, the ABR threshold of S. aureus-induced OM model was significantly decreased compared to normal mice, further suggesting the effectiveness of the established OM model.

Figure 6

Figure 6. Ru-C₃N₄ relieves S. aureus-induced hearing damage. (A) C₃N₄ and Ru-C₃N₄ treatment did not affect the hearing function of normal mice. (B–E) Changes in hearing performance of OM mice after different treatments. (F) The statistics of ABR threshold for (B–E).

We next conducted a systematic examination of the impact of Ru-C₃N₄ on auditory improvement in OM model. Figures 6B–E illustrated the ABR waveforms at frequencies of 8, 16, 24 and 32 kHz from both PBS- and Ru-C₃N₄-treated OM models. The ABR thresholds for the PBS-treated group were recorded as 70, 65, 65 and 70 dB at these frequencies, while those for the Ru-C₃N₄-treated group were found to be 35, 30, 20 and 40 dB. Compared with PBS-treated group, the average ABR thresholds at frequencies of 8, 16, 24 and 32 kHz decreased by amounts of 30, 43, 38 and 29 dB respectively in Ru-C₃N₄-treated group (Figure 6F). These results suggested that S. aureus infection caused a marked inflammatory response in the middle ear tympanic part. Inflammatory fluid limited the transmission of mechanical waves to the inner ear, resulting in conductive hearing loss. However, the structure and function of the hair cells were not affected. Therefore, S. aureus-induced hearing loss is temporary and reversible. After Ru-C₃N₄ treatment, S. aureus is effectively eliminated, which lead to the alleviation of inflammation in the middle ear and gradual restoration of hearing.

The biosafety of nanomedicines is critical to their further application. We finally examined the toxicity of Ru-C₃N₄ to cells and mice. We introduced two common human cell lines, named human umbilical vein endothelial cell (HUVEC) and human skin fibroblast cell (HSF), to evaluate the biosafety of Ru-C₃N₄ in vitro. The results of calcein AM/PI staining assay showed that Ru-C₃N₄ with 100–500 μg mL^–1 did not cause significant cell death compared to PBS group (Supplementary Figure S11). In vivo evaluation, 10 mg kg^–1 C₃N₄ or Ru-C₃N₄ was intravenously administered to normal mice on day 0, 4, 8, 12, 16, 20, 24, and 28. As shown in Supplementary Figure S12, the growth curves of C₃N₄- and Ru-C₃N₄-treated mice exhibited no obvious difference compared to that of PBS-treated group. We then determined the bleeding and clotting time to reflect the hematotoxicity of Ru-C₃N₄. Supplementary Figures S13A, B illustrated that the bleeding and clotting times for normal mice were approximately 80.49 s and 92.04 s, respectively. In contrast, the C₃N₄- and Ru-C₃N₄-treated mice exhibited bleeding time of 80.41 s and 80.45 s, and clotting time of 91.95 s and 92.03 s, respectively. To evaluate the effects of Ru-C₃N₄ on liver and kidney function, we tested the levels of relevant indicators in mouse blood. Following treatment with C₃N₄ or Ru-C₃N₄, there were no significant changes observed in the levels of alanine transaminase and aspartate aminotransferase (two liver injury indicators). Similarly, no significant alterations were noted in the levels of two kidney injury indicators, blood urea nitrogen and creatinine (Supplementary Figure S14). Therefore, Ru-C₃N₄ and its carrier exhibited high biosafety, which provides sufficient guarantee for its clinical applications.

3 Conclusion

In this study, four antibacterial metals (Cu, Fe, Ag, and Ru) were coordinated on a crystalline C₃N₄ carrier. The synthesized metal-C₃N₄ expanded the contact area with their surroundings, thereby enhancing its efficiency in binding to substrates and microorganisms. Among these metal-C₃N₄, Ru-C₃N₄ demonstrated the highest peroxidase-like activity. Utilizing its catalytic activity, Ru-C₃N₄ effectively inhibited S. aureus growth and destroyed S. aureus-formed biofilms, exhibiting robust anti-S. aureus efficiency (>99.9%). In the S. aureus-induced OM model, Ru-C₃N₄ successfully eliminated S. aureus, reducing inflammation in the ear microenvironment, and finally promoting the repair of middle ear mucosal cells and hearing recovery. Additionally, the low loading of Ru atoms on C₃N₄ carrier ensured the excellent biosafety of Ru-C₃N₄. In summary, this work provides valuable insights into the design of highly efficient antibacterial SANzymes. The selected Ru-C₃N₄ serves as an effective nanoantibiotic for treating S. aureus-induced OM.

4 Materials and methods

4.1 Preparation of C₃N₄

The C₃N₄ nano-crystals were achieved via an ionothermal synthesis approach according to our previous work (Wu et al., 2023). Typically, 2 g melamine were dispersed in 10 mL deionized water by vigorous agitation, whereafter ultrasonicated for 30 min. The mixture was subsequently refluxing for 3 h and dehydrated at 80°C for 8 h. The dried samples were ground and placed on the corundum porcelain boat of muffle furnace for 4 h of heating at 500°C with 5°C min⁻¹ rate. After cooling, the sample was ground with a mixture of LiCl/KCl (molar ratio = 59: 41) in a mortar, followed by heating at 550°C for 4 h with 2.5°C min⁻¹ rate under a N₂ flow in the tubular furnace. After cooling to room temperature naturally, the resulting sample was washed with boiling deionized water to remove residual salt and air-dried at 60°C for 8 h.

4.2 Preparation of metal-C₃N₄

To synthesize Ru-C₃N₄, 10 mg of C₃N₄ were first dispersed into 10 mL of ultrapure water. Subsequently, 2 mL RuCl₃ solution (2 mg mL⁻¹) was added into the above solution, which was then refluxed at 100°C for 4 h. The Ru-C₃N₄ was obtained by centrifuging and washing with water twice to remove the excess RuCl₃. Fe-C₃N₄, Cu-C₃N₄, Ag-C₃N₄ were prepared in a similar procedure where equivalent mass of FeCl₃, CuCl₂, or AgNO₃ precursors were used instead of RuCl₃. The temperature of reaction system was 80°C, 100°C, and 80°C, respectively.

4.3 The characterization of metal-C₃N₄

Transmission electron microscopy (TEM) was performed by a FEI-Talos F200X microscope at an acceleration voltage of 200 kV (FEI, United States). Scanning electron microscope (SEM) was carried out by GeminiSEM 300 at 5 keV (ZEISS, Germany). High-angle annular dark-field image-scanning transmission electron microscopy (HAADF-STEM) was conducted at 200 kV on a JEM ARM 200F instrument (JEOL, Japan). X-ray photoelectron spectroscopy (XPS) spectra were acquired on an Escalab 250Xi (Thermo Scientific, United States). Inductively coupled plasma optical emission spectrometry (ICP-OES) was performed on ICPOES730 (Agilent, United States).

4.4 The peroxidase-like activity of metal-C₃N₄

The peroxidase-like activity of SANzymes was described in our previous studies (Wang et al., 2024a; Wang et al., 2024b). In brief, the activity testing of the four metal-C₃N₄ was performed within a 100 μL system, including 10 μL TMB (2.08 mM), 10 μL H₂O₂ (0.5 M), 10 μL metal-C₃N₄ (2 μg mL⁻¹), and 70 μL HAc-NaAc (0.02 M, pH = 4.5). The absorbance of oxTMB was recorded within a time-scan mode at 652 nm via SpectraMax M4 spectrophotometer (Molecular Devices, United States). Since Ru-C₃N₄ had the optimal peroxidase-like activity, we then tested its optimal temperature and pH value. The optimal temperature was tested within a 100 μL system containing 10 μL Ru-C₃N₄ (2 μg mL⁻¹) or HRP (0.9 μg mL⁻¹), 10 μL TMB (2.08 mM), 10 μL H₂O₂ (0.5 M) and 70 μL HAc-NaAc (0.02 M, pH = 4.5). The absorbance at 652 nm was recorded when the temperature ranged from 15°C to 60°C. The optimal pH of Ru-C₃N₄ was also detected in the same 100 μL system at 25°C when the pH value ranged from 3.0 to 7.0.

For the steady-state kinetic analysis, 100 μL reaction system containing 10 μL Ru-C₃N₄ (2 μg mL⁻¹) or HRP (0.9 μg mL⁻¹), 10 μL TMB (2.08 mM), 70 μL HAc-NaAc (pH = 5.0, 0.02 M), and a series of concentrations of H₂O₂ range from 0 to 1000 μM. The Michaelis-Menten constant was calculated using the following formula:

$v=\left(v_{\mathit{max}}\times \left[S\right]\right)/\left(K_{\mathrm{M}}+\left[S\right]\right).$

The catalytic constant ($k_{cat}$) was calculated as follows:

$k_{cat}=v_{\mathit{max}}/\left[\mathrm{E}\right]$

where $v$ represents the initial velocity, $K_{\mathrm{M}}$ represents the Michaelis constant, $\left[S\right]$ represents the substrate concentration, and $v_{\mathit{max}}$ represents the maximal reaction velocity.

The specific activities (U mg⁻¹ Ru atoms) were calculated by the formula [V/(Ɛ × Ɩ) × (ΔA/Δt)]/[C], where V denotes the total volume of reaction solution (μL); Ɛ denotes the molar absorption coefficient of the colorimetric substrate, which is typically maximized at 39,000 M⁻¹ cm⁻¹ at 652 nm for TMB. Ɩ denotes the path length of light traveling in the cuvette (cm); A denotes the absorbance after subtraction of the blank value; ΔA/Δt denotes the initial rate of change in absorbance at 652 nm; and [C] denotes the concentration of HRP or Ru-C₃N₄ (mg).

4.5 Free radical identification

The generation of ·OH and ·O₂⁻ was assessed through ESR analysis using dimethyl pyridine nitrogen oxide (DMPO) spin-trapping adduct. Glass capillary tubes with internal diameters of 1 mm were filled with 50 μL C₃N₄ or Ru-C₃N₄ and then sealed. These tubes were then inserted into the cavity of an ESR instrument (Bruker, Germany), with parameters set as follows: 1 G field modulation, 100 G scan range, and 20 mW microwave power. The spin trap DMPO was used to verify the formation of ROS during H₂O₂ degradation in presence of C₃N₄ or Ru-C₃N₄. Using the peak-to-peak height of the second line of the ESR spectra, the ROS production was estimated based on the signal intensity of the DMPO/·OH and DMPO/·O₂⁻.

4.6 Bacterial culture and in vitro antibacterial activity

The S. aureus monoclonal was seeded to 5 mL of liquid LB medium and cultured for 8 h at 37°C and 180 rpm, respectively. When the bacterial solution reached the logarithmic growth cycle, it was diluted to 10⁶ colony-forming units (CFU) mL^–1 for antibacterial testing. The S. aureus solution (10⁶ CFU mL^–1) was treated with 1% PBS +10 mM H₂O₂ (Control), and different metal-C₃N₄ (0.06 mg mL^–1) + 10 mM H₂O₂ at 37°C and 180 rpm for 0.5 h. Then the bacterial suspension was diluted with 1% PBS and distributed on solid medium and cultured at 37°C for 16 h to evaluate the antibacterial activity of metal-C₃N₄. To evaluate the optimal concentration of Ru-C₃N₄, we treated S. aureus with different concentrations of Ru-C₃N₄ (0, 0.01, 0.03, 0.06, 0.09 mg mL^–1) and 10 mM H₂O₂ in NaAc buffer. The antibacterial efficiency of nanozymes was calculated as: (number of clones in experimental group/number of clones in control group) × 100%.

Next, three Gram-positive bacteria (S. aureus, S. agalactiae, and P. aeruginosa) and two Gram-negative bacteria (E. coli and K. pneumoniae) were used to evaluate the relative selectivity of Ru-C₃N₄. The concentration of H₂O₂ was 10 mM for Gram-positive bacteria and 1 mM for Gram-negative bacteria according to previous study (Xi et al., 2019). 0.06 mg mL^–1 Ru-C₃N₄ + H₂O₂ were used to evaluate its antibacterial activity for the five bacteria. The detailed culture conditions were the same as described above.

4.7 In vitro destruction of biofilms

To investigate the destructive ability of Ru-C₃N₄ on biofilms, the bacterial suspension of S. aureus was cultured in the 24-well plate covered with cell crawling sheets. After 3–4 days of daily culture with fresh medium, a thin biofilm could be seen on the cell crawling sheets. Then different concentrations of Ru-C₃N₄ (0.01–0.09 mg mL^–1) + 10 mM H₂O₂ were applied to treat the biofilms for 72 h. The destruction of biofilms was imaged by using S-4800 SEM system.

4.8 Biosafety analysis of Ru-C₃N₄

In vitro, HUVEC and HSF cells were cultured to 80% confluence and then treated with 1% PBS or 100–500 ug mL^–1 Ru-C₃N₄ for 24 h. Next, the cells in ever group were stained with calcein AM/PI to distinguish live and dead cells. Finally, a laser scanning confocal microscope (LSM 800) was used to take pictures.

In vivo, C57BL/6J mice were intravenously administered a dose of 10 mg kg^–1 C₃N₄ or Ru-C₃N₄ via the tail vein on day 0, 4, 8, 12, 16, 20, 24, and 28. Prior to each injection, body weight was meticulously measured. A scalpel was used to create a transverse incision over the lateral vein, following which the tail was immersed in 1% PBS at 37°C. The duration from the initiation of the incision until bleeding ceased was recorded as bleeding time. Blood samples were procured from each mouse’s orbital plexus using a glass capillary. The time required for fibrin threads to emerge between the fractured capillaries was documented as clotting time. The functional indexes of liver and kidney in mice, including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA), were detected by their commercial ELISA kits.

4.9 OM model construction and therapy

All the animal procedures were approved by the Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (Approval number: 20230727-A1682). The research adheres to globally accepted animal welfare standards, and all animal-related studies are also in compliance with the ARRIVE guidelines and the AVMA euthanasia guidelines 2020. The C57BL/6 J mice with 6–8 weeks old were utilized to construct OM model to evaluate the antibacterial efficacy of metal-C₃N₄ in vivo. All the mice underwent an otoscopic examination and an ABR test to confirm that their tympanic membranes were normal and no middle ear effusion was present. The external auditory canal of mouse was sterilized before modelling. The suspension of S. aureus (1.0 × 10⁶ CUF mL^–1, 25 µL per ear) was injected through tympanic membrane puncture under sterile conditions. The OM mice were divided into three groups and injected with 1% PBS +10 mM H₂O₂, 0.2 mg mL^–1 C₃N₄ + 10 mM H₂O₂, or 0.2 mg mL^–1 Ru-C₃N₄ + 10 mM H₂O₂. The morphological changes of biofilms were evaluated with an S-4800 SEM system.

The tissues were collected and rinsed 3 times with 1% PBS after varied treatments. The samples were fixed with 2.5% glutaraldehyde overnight at 4°C. Subsequently, the samples were treated with an EDTA decalcifying solution (PH = 7.2) to soften the tissues in preparation for further testing. After 1% PBS washing, the samples were dehydrated with ethanol solutions of several concentrations (30%, 50%, 70%, 80%, 95% and 100%). Finally, the samples were dried in a desiccator for 3 h and fixed on the plate with conductive glue and then sprayed with gold for 1 min. The result was obtained with an S-4800 SEM system.

4.10 Live/dead cells staining assay

After different treatments, the middle ear mucosa of OM model was separated and stained with calcein AM/PI. Commercially available dyes calcein AM and PI were used to distinguish live and dead cells. Calcein AM could result in green fluorescence when staining live cells. PI is a membrane-impermeable red dye. Finally, the living and dead cells were imaged with a laser scanning confocal microscope LSM 800.

4.11 Histological staining

Typical H&E staining was used to observe the histomorphology of the ears. Briefly, the ear tissues were obtained from the normal mice and different treated OM models. 4% paraformaldehyde was used to fix the tissues. Then the tissues were embedded with paraffin for further study. 5 μm pathological sections were obtained and stained with H&E.

4.12 Detection of TNF-α, IL-1β, and IL-6 in middle ear lavage fluid

The middle ear lavage fluids were collected from the normal mice and different treated OM models. The concentrations of TNF-α, IL-1β, and IL-6 in middle ear lavage fluids were quantified by mouse TNF-α, IL-1β, and IL-6 highly sensitive ELISA kits, respectively. All the measurements were strictly performed following the procedures provided by manufacturers.

4.13 Statistical analysis

Results were presented as the mean ± SD, derived from a minimum of three independent experiments, unless otherwise specified. Statistical analysis was conducted using the paired Student’s t-test, while comparisons involving more than two groups were evaluated through ANOVA. Graph plotting was executed utilizing GraphPad Prism 8.0 software. A significance level of p < 0.05 was adopted for statistical significance. *p < 0.05, **p < 0.01, and ***p < 0.001.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement

The animal study was approved by the Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (Approval number: 20230727-A1682). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

JW: Data curation, Methodology, Writing–original draft. RG: Conceptualization, Methodology, Writing–review and editing. MY: Methodology, Writing–review and editing. XW: Methodology, Writing–review and editing. ZL: Methodology, Writing–review and editing. HH: Data curation, Methodology, Writing–original draft. XY: Project administration, Writing–review and editing. DW: Conceptualization, Writing–review and editing.

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was financially supported by the National Natural Science Foundation of China (No. 32200744), the Natural Science Foundation of Guangdong Province (Nos. 2021A1515110028, 2022A1515220147, and 2022A1515110858), the Science and Technology Program for Basic Research in Shenzhen (No. JCYJ20210324103015039).

Acknowledgments

The authors thank Meng Wang (Guangdong Mobile Communication Co., LTD.) for her help in graph editing.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2024.1439039/full#supplementary-material

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