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Alternative Copper-Based Single-Atom Nanozyme with Superior Multienzyme Activities and NIR-II Responsiveness to Fight against Deep Tissue Infections
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Jiaxiang Bai1, Yonghai Feng2, *, Wenming Li1, Zerui Cheng2, Jessica M. Rosenholm3, Huilin Yang1, Guoqing Pan2, *, Hongbo Zhang3, 4, *, Dechun Geng1, *
Research. Vol 6 Article ID 0031
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Research. Vol 6 Article ID 0031
• Research Article •
Alternative Copper-Based Single-Atom Nanozyme with Superior Multienzyme Activities and NIR-II Responsiveness to Fight against Deep Tissue Infections
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Jiaxiang Bai1, Yonghai Feng2, *, Wenming Li1, Zerui Cheng2, Jessica M. Rosenholm3, Huilin Yang1, Guoqing Pan2, *, Hongbo Zhang3, 4, *, Dechun Geng1, *
Affiliations
  • 1Department of Orthopedic Surgery, Orthopedic Institute, The First Affiliated Hospital, Medical College, Soochow University, Suzhou, Jiangsu 215006, P. R. China.
  • 2Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, P. R. China.
  • 3Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Turku 20520, Finland.
  • 4Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku 20520, Finland.
Published: 2023-01-20 doi: 10.34133/research.0031
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Nanozymes are considered to represent a new era of antibacterial agents, while their antibacterial efficiency is limited by the increasing tissue depth of infection. To address this issue, here, we report a copper and silk fibroin (Cu-SF) complex strategy to synthesize alternative copper single-atom nanozymes (SAzymes) with atomically dispersed copper sites anchored on ultrathin 2D porous N-doped carbon nanosheets (CuNx-CNS) and tunable N coordination numbers in the CuNx sites (x = 2 or 4). The CuNx-CNS SAzymes inherently possess triple peroxidase (POD)-, catalase (CAT)-, and oxidase (OXD)-like activities, facilitating the conversion of H2O2 and O2 into reactive oxygen species (ROS) through parallel POD- and OXD-like or cascaded CAT- and OXD-like reactions. Compared to CuN2-CNS, tailoring the N coordination number from 2 to 4 endows the SAzyme (CuN4-CNS) with higher multienzyme activities due to its superior electron structure and lower energy barrier. Meanwhile, CuNx-CNS display strong absorption in the second near-infrared (NIR-II) biowindow with deeper tissue penetration, offering NIR-II-responsive enhanced ROS generation and photothermal treatment in deep tissues. The in vitro and in vivo results demonstrate that the optimal CuN4-CNS can effectively inhibit multidrug-resistant bacteria and eliminate stubborn biofilms, thus exhibiting high therapeutic efficacy in both superficial skin wound and deep implant-related biofilm infections.

Jiaxiang Bai, Yonghai Feng, Wenming Li, Zerui Cheng, Jessica M. Rosenholm, Huilin Yang, Guoqing Pan, Hongbo Zhang, Dechun Geng. Alternative Copper-Based Single-Atom Nanozyme with Superior Multienzyme Activities and NIR-II Responsiveness to Fight against Deep Tissue Infections[J]. Research, 2023 , 6 (1) : 0031 - . DOI: 10.34133/research.0031
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Introduction
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Compared with superficially exposed skin wounds, infections that occur at deeper sites, such as osteomyelitis [1] and implant-related and deep soft tissue infections [2–4], are exceedingly more difficult to treat with antibiotics since they are deeper and often associated with methicillin-resistant Staphylococcus aureus (MRSA) [5,6]. To this end, incision, drainage, and long-term high-dose antibiotic administration are usually needed. However, these courses of action are more likely to promote bacterial evolution into super strains [7]. Meanwhile, owing to inherently high reproduction and strong adherence, biofilms, known as bacterial communities, can easily form and are much harder to eliminate on the surfaces of deep sites such as implants [8,9]. The failure rate of antibiotic treatment continuously increases, and even persistent systemic infections to human hosts inevitably take place. Although antibiotic-loaded nanocarriers are able to enhance the therapeutic efficiency of antibiotics, they usually require a long time (several days) for antibiotic-administered therapeutics [10]. Therefore, it is imperative to develop antibiotic-free, in situ, and effective strategies to fight deep tissue infections.
Alternatively, nanozyme-based antibacterial therapy has emerged as a new era of powerful weapons to combat bacterial infections without causing antimicrobial resistance (AMR), since the antibacterial mechanism is based on enzyme-mimetic catalytic generation of highly toxic agents, such as reactive oxygen species (ROS) [11–13]. These agents can irreversibly and rapidly damage the cell wall, membrane, DNA and proteins of bacteria, as well as extracellular DNA and polysaccharides in biofilms [14]. Peroxidase (POD)- or oxidase (OXD)-like nanozymes have been widely developed for antibacterial applications due to the conversion of hydrogen peroxide (H2O2) or oxygen (O2) into hydroxyl (•OH) or superoxide anion (•O2−) radicals under biological conditions [15]. However, in deep tissues [5], the catalytic ROS generation of nanozymes certainly decreases due to limited substrate diffusion. In this case, it requires the nanozymes to possess higher activity to produce sufficient ROS up to the inhibitory levels under lower substrate concentrations when facing deep infections.
Compared to traditional noble-metal or transition-metal oxide/sulfide-based nanozymes, single-atom nanozymes (SAzymes) possess atomically dispersed metal atoms with a maximum atom utilization efficiency, remarkably enhancing the enzyme-like activities [16]. Different from noble-metal single atom-based SAzymes [17,18], transition-metal single atom and nitrogen codoped porous carbon material (MNx-C)-based SAzymes generally have similar active centers to those of natural metalloenzymes, which consist of metal and nitrogen coordination (MNx, M = Fe, Zn, Cu), resulting in extremely high or superior POD- or OXD-like activities even compared to their natural counterparts [19]. By the merits of the coordinated Cu atom serving as cofactor or active center in copper-containing metalloenzymes (e.g., laccase, urate oxidase, amine oxidase, and azurin) [20], it is interesting to find that in addition to high POD-like activity, the CuNx-C SAzyme also possesses excellent OXD-like activity, meaning it can promote ROS generation through the parallel reactions of converting H2O2 into •OH and O2 into •O2− [21]. On the other hand, the Cu decoration in defective porous carbon materials can further broaden the optical absorption in the near-infrared (NIR) region and improve the photothermal conversion efficiency of the nanohybrid (48.5%) [22,23]. These results suggest that CuNx-C SAzymes are potential candidates with multiple enzyme-mimicking activities and NIR responsiveness for deep infection treatment. It is noted that the activity of CuNx sites for the CuNx-C single site catalysts significantly depends on the N-coordination number due to their different electron structures affecting their catalytic abilities [24]. However, regulating the coordination environment of CuNx-C SAzymes is vital for enzyme-mimicking reaction but still remains a challenge.
It is interesting to note that silk fibroin (SF) from Bombyx mori silkworm not only is a natural protein rich in nitrogen (N) and carbon (C) but also can specifically chelate Cu2+ to form different Cu and SF (Cu-SF) complexes, with central Cu2+ coordinated with different N numbers changing from 4 to 1 when the pH is reduced from 8.0 to 4.0 [25]. Inspired by this, Cu-SF complexes can be used as precursors to synthesize different CuNx-C SAzymes with tunable N coordination numbers. Thus, in this work, we developed an alternative Cu-SF complex pyrolysis strategy to fabricate alternative CuNx-C SAzymes with tunable N coordination numbers, namely, ultrathin two-dimensional (2D) N-doped porous carbon nanosheet supported Cu single atoms (CuNx-CNS, x = 2 or 4), as depicted in Fig. 1A. The CuNx-CNS SAzymes possess triple enzyme-like activities [POD, OXD, and catalase (CAT)] and high NIR-II-responsive photothermal activity (40.9% photothermal conversion efficiency). Experimental studies and theoretical calculations unambiguously identified that both CuN4 and CuN2 sites exhibit similar POD-like activity, but the CuN4-CNS SAzyme favors superior CAT- and OXD-like activity, implying more ROS generation. Therefore, the synergy between enhanced ROS generation and photothermal ablation of CuN4-CNS SAzyme contributes to preeminent bactericidal efficacy against Escherichia coli, MRSA, and biofilms. The in vivo experiments further prove that the synergistic antibacterial effect promotes wound disinfection and implant-related biofilm removal (Fig. 1B). This work highlights the engineering of efficient SAzymes for combating bacterial infections from superficial to deep tissues.
Results and Discussion
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Synthesis and characterization
The fabrication procedure of CuNx-CNS based on a Cu-SF complex pyrolysis strategy is depicted in Fig. 1. Generally, regenerated SF aqueous solution was first prepared by dissolving SF, which was extracted from the B. mori cocoon by the classical protocol [26]. After the addition of Cu(II) salt, a Cu-SF complex with square planar coordination modes of Cu-2N2O [Cu-SF(I)] was formed due to the chelation between Cu2+ ions and SF at a pH value of 5.0, whereas a Cu-SF complex with Cu-4N mode [Cu-SF(II)] was formed at a pH of 11.0. Meanwhile, the solution color changed from light blue to dark purple (Fig. S1A) [25]. The Cu-SF complex with Cu-4N coordination was confirmed by the ultraviolet–visible (UV–Vis) absorption spectrum, which displayed a characteristic absorption peak of ca. 540 nm (Fig. S1B). SF can self-assemble into a lamella-like layer structure after the treatment of all-aqueous extraction [27]. Thus, following freeze-drying and pyrolysis, the Cu-SF complexes were in situ transformed into 2D isolated Cu atoms and CuNx-CNS. Finally, CuNx-CNS were obtained after the removal of soluble salts and copper nanoparticles (NPs) by acid etching.
Figure 2A shows the transition electron microcopy (TEM) of CuN4-CNS with a sheet-like 2D structure. Atomic force microscopy (AFM) demonstrates that the thickness of CuN4-CNS is approximately 1.8 nm (Fig. S2), implying a multilayer structure. Meanwhile, a number of disordered pores are clearly formed on the sheet surface because excess metal salts can provide a hypersaline environment to activate the pyrolysis process [27]. Consequently, CuN4-CNS displays a larger specific surface area than does the pure N-doped carbon nanosheets (N-CNS), along with a narrow pore size distribution (Fig. S3 and Table S1). As shown in the high-resolution TEM image (Fig. 2B), some irregular fringes are assigned to a few graphene layers and no obvious Cu NPs are observed. Abundant white bright dots, some circled in red, are observed on the carbon nanosheet surface from the corresponding aberration-corrected high angle annular dark field-scanning transmission electron microscope (HAADF-STEM) image (Fig. 2C), proving the presence of atomically isolated Cu single atoms on the carbon matrix. The HAADF-STEM image (Fig. 2D) and its energy-dispersive spectroscopy mappings (Fig. 2E to G) reveal that the Cu, N, and C elements are homogeneously distributed on the carbon nanosheet. In addition, the Cu content was determined to be approximately 1.5 wt% by inductively coupled plasma–optical emission spectrometry (ICP-OES) (Table S1). The x-ray diffraction (XRD) pattern (Fig. S4) of CuN4-CNS is similar to that of pure N-CNS, suggesting the NP-free feature of CuN4-CNS. X-ray photoelectron spectroscopy (XPS) demonstrates the existence of C, N, and O elements, and the high-resolution Cu 2p spectrum verifies the presence of copper species in CuN4-CNS (Fig. S5A). Compared to the N-CNS spectrum, the N 1s spectrum of CuN4-CNS can be deconvoluted into 4 obvious peaks centered at 398.2, 398.93, 400.5, and 404 eV, corresponding to pyridinic, cupric, pyrrolic, and graphitic-N species, respectively (Fig. S5B), suggesting the presence of the so-called nitrogen-coordinated Cu atom (CuNx) sites [28]. The Raman spectra (Fig. S6) show that the intensity ratios of D band to G band (ID/IG) (D, disorder; G, graphite) are calculated to be 1.03 and 0.99 for N-CNS and CuN4-CNS, respectively, indicating that CuN4-CNS is less defective than N-CNS due to the introduction of copper species, but the relatively high intensity of the D band implies that CuN4-CNS still has a highly defective structure [29].
X-ray absorption near-edge structure (XANES) was further used to elucidate the chemical state and coordination environment of isolated Cu atoms in CuNx-CNS. Figure 2H shows the normalized XANES curves of the Cu K-edge for CuN2-CNS, CuN4-CNS, Cu foil, Cu2O, and CuO. The XANES profiles of CuNx-CNS (CuN2-CNS and CuN4-CNS) are relatively smoother than those in other references, and the positions of the absorption edges are located between those of Cu2O and CuO, suggesting positive charges of copper valence between +1 and +2, probably caused by N doping [21]. Moreover, the Cu 2P3/2 XPS spectrum of CuN4-CNS can be deconvoluted into 2 peaks, at 933.9 and 932.5 eV, assigned to Cu(II) and Cu(I) species, respectively, which is confirmed by the corresponding Auger electron spectroscopy spectrum of Cu LMM, demonstrating that the valence state of Cuδ+ (1 < δ < 2) is closely correlated to the XANES analysis (Fig. S5C and D). Subsequently, extended x-ray absorption fine structure (EXAFS) was employed to determine the local coordination structure of the Cu sites of CuNx-CNS (Fig. 2I). Compared to the Cu foil reference, no scattering of the Cu–Cu coordination signal at 2.24 Å but a dominant Cu–N coordination at approximately 1.5 Å is detected in the Fourier transformed EXAFS spectrum of CuN4-CNS (or CuN2-CNS) (Fig. 2J). Additionally, the wavelet transforms of the Cu K-edge EXAFS oscillations obviously demonstrate the good atomic dispersion of Cu species in CuN4-CNS (Fig. 2K), with one intensity maximum of ca. 3.9 Å−1 assigned to the Cu–N coordination present. No intensity maximum (6.7 Å−1) related to Cu–Cu coordination of Cu foil (Fig. 2K) is observed. The quantitative EXAFS curve fitting (Fig. 2J and M) evidently presents a main peak related to Cu–N first shell coordination with a coordination number of ca. 3.9 ± 0.1 N atoms at a mean bond length of 2.04 ± 0.02 Å in CuN4-CNS (Table S2). Thus, the coordination model of Cu atoms in CuN4-CNS can be dominantly identified as one Cu atom coordinated by 4 N atoms (CuN4) confined in the carbon matrix. For CuN2-CNS, the morphology and structure are similar to those of CuN4-CNS (Figs. S2 and S7), but it has a slightly lower N content, and the CuN2 sites composed of one Cu atom coordinated with 2 N atoms are confirmed by quantitative EXAFS curve fitting. The above results demonstrate that the N coordination numbers of CuNx-CNS can be precisely modulated by facilely changing the coordination mode of Cu-SF complex before pyrolysis.
The UV–Vis–NIR absorption spectra (Fig. S8A) demonstrate that CuN4-CNS, CuN2-CNS, and N-CNS exhibit broad absorption ranging from the UV to the second NIR (NIR-II) biowindow, which possesses deeper tissue penetration, a higher skin tolerance threshold, and less energy dissipation compared to the first NIR (NIR-I) [30]. Significant enhancement in the optical absorption, especially in the NIR-II region, is observed for both CuN4-CNS and CuN2-CNS compared to the pure N-CNS. Therefore, upon NIR-II light (1,064 nm, 1 W cm−2) irradiation for 8 min, both CuN4-CNS and CuN2-CNS aqueous dispersions (200 μg ml−1) exhibit similar maximum temperature elevations, reaching ca. 53.3 °C from 31 °C, much higher than that of N-CNS (47.2 °C). The temperature elevation is concentration dependent (Fig. S8B), suggesting the remarkable NIR-II light-responsive photothermal activity of CuNx-CNS with a photothermal conversion efficiency of 40.9% (Fig. S8C and D). The photothermal performance of CuN4-CNS hardly changes during 4 heating/cooling cycles, demonstrating good photothermal stability (Fig. S8E).
Multienzyme-mimicking activity and mechanism
The POD-like property of CuNx-CNS was first investigated by the typical chromogenic reaction of catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidized TMB (ox-TMB) in the presence of H2O2. As shown in Fig. 3A, a much higher time-dependent increase in the absorbance at 652 nm, attributable to the formation of ox-TMB, is observed for both CuN4-CNS and CuN2-CNS compared to that of N-CNS, demonstrating that both the CuN4 and CuN2 sites are highly active sites for catalyzing H2O2 decomposition into •OH. The absorbance value at 652 nm of ox-TMB obviously decreases (Fig. 3D) after adding isopropanol as an •OH scavenger into the CuN4-CNS (or CuN2-CNS)/H2O2/TMB solution, implying that •OH is produced as the active intermediate. Terephthalic acid (TA) is further used as an indicator to determine the generated •OH since it can convert the nonfluorescent TA into fluorescent 2-hydroxy terephthalic acid (HTA). Figure 3E shows that the fluorescence intensity of HTA for CuN4-CNS + H2O2 at room temperature is much higher than that of H2O2 alone, demonstrating the enhanced production of •OH, which can be further confirmed by the obvious electron spin resonance (ESR) signal for CuN4-CNS + H2O2 (Fig. 3F). Based on the steady-state kinetics of CuN4-CNS, the values of KM and Vmax for TMB were calculated to be 0.22 mM and 9.5 × 10−8 M s−1, while for H2O2, they were 5.6 mM and 138 × 10−8 M s−1, respectively (Fig. S9A to D). It is noted that the KM values of CuN4-CNS for TMB and H2O2 are comparable to those of previously reported nanozymes, but the Vmax value for catalyzing H2O2 is much higher than that of reported Cu-C-N SAzymes [31,32], even comparable to that of 2D sheet-like Fe–C–N SAzymes [33] (Table S2). As shown in Fig. 3F, the ESR signal of CuN4-CNS + H2O2 + NIR-II is 1.7 times higher than that without NIR-II irradiation, indicating the enhanced production rate of •OH due to the elevation of the reaction temperature. This can be proven by the significantly enhanced fluorescence intensity of HTA after TA treatment by CuN4-CNS + H2O2 + 50 °C (Fig. 3E). Figure S9F shows that even under weakly acidic conditions (pH 5.5), CuN4-CNS can still maintain high activity, which can be attributed to the participation of H+ ions during the reactions.
In parallel to POD, CAT can catalyze the decomposition of H2O2 to O2 and H2O. Thus, monitoring the O2 generated from H2O2 decomposition can reflect the CAT-like activities of CuNx-CNS. Figure 3B shows the performances of O2 generation for N-CNS, CuN2-CNS, CuN4-CNS, and Pt/C. Almost no O2 is generated without catalyst. With N-CNS as the catalyst, the dissolved O2 concentration only increased from 8.4 to 10.2 mg l−1 after reacting for 300 s. For CuN2-CNS, the dissolved O2 concentration reached 19.2 mg l−1 at 300 s, while it reached 25.2 mg l−1 for CuN4-CNS. Note that the dissolved O2 concentration of CuN4-CNS is close to that (31.4 mg l−1) of the commercial Pt/C catalyst, which is known to possess typical CAT-like activity [34], suggesting that CuN4 sites are more reactive than CuN2 sites. Furthermore, the oxygen generation velocities of the first 60 s are shown in Fig. S10, and the results indicate that the catalytic activity has a positive correlation with the CuN4-CNS concentration.
The OXD-like property of CuNx-CNS with the capacity to reduce O2 can also be detected by using TMB as a •O2− capturer [35]. The •O2− generation can be confirmed by using dihydroethidium (DHE) as the indicator (Fig. S11). As shown in Fig. 3C, the absorbance at 652 nm of CuN4-CNS is much stronger than that of CuN2-CNS, indicating that CuN4 sites have a stronger O2 reduction ability. In the presence of N2, the absorbance strength at 652 nm remarkably decreases, whereas it is evidently enhanced with continuous O2 supply. Based on the velocity of TMB oxidation by the OXD-like reaction, the steady-state kinetics of CuN4-CNS are obtained, giving KM = 0.57 mM and Vmax = 6.5 × 10−8 M s−1 for TMB (Fig. S12), comparable to those of previously reported CeO2 NPs, a typical OXD-like nanozyme with KM and Vmax values of 0.42 mM and 10 × 10−8 M s−1, respectively [36].
To further investigate the mechanisms of the triple enzyme-like reactions catalyzed by CuNx-CNS and clarify the effect of the N coordination number on the activities, density functional theory (DFT) calculations were used for the prediction and evaluation, which were represented in terms of the variation in free energy (Fig. 3G to I). First, 3 typical CuN4, CuN2, and NC structures in a graphene matrix were adopted as the optimized calculation models (Fig. S13). Figure S14 reveals the obvious difference in the bonding charge distributions between the central Cu coordinated with N2 or Cu coordinated with N4. Obviously, the catalytic decomposition of H2O2 into •OH over CuNx-CNS is supposed to follow a pathway of * + H2O2 → *H2O2 (i), *H2O2 → 2*OH (ii), 2*OH → •OH + *OH (iii) (Fig. 3J). The energy diagram (Fig. 3G) demonstrates that the CuN4 site has the highest capacity for capturing H2O2 molecules since its H2O2 adsorption energy is the lowest (−0.45 eV). Moreover, CuN4 also has the lowest energy barrier for the rate-determining step (RDS) (1.61 eV); thus, it is a more active site than CuN2 for catalyzing H2O2 decomposition into •OH. Figure 3K shows the proposed CAT-like catalytic pathway of H2O2 + * → *H2O2 (i), *H2O2 → *OOH + H+ + e− (ii), *OOH → *O2 + H+ + e− (iii), *O2 → O2 (iv). Combined with the energy diagram (Fig. 3I), it is clearly observed that the initial intermediate OOH* for O2 production has a more negative adsorption energy for CuNx sites (ii), suggesting that the reaction is spontaneous. Compared with the CuN2 site, the CuN4 site has a lower RDS barrier energy for forming intermediate *O2 (iii), and it also has easier desorption of O2 (iv), which can be closely associated with the higher O2 production from H2O2 decomposition over CuN4-CNS. To elucidate the enhanced OXD-like activity for CuN4-CNS compared to CuN2-CNS, the free energy diagrams for CuN2, CuN4, and NC sites are calculated on the basis of the proposed 4-electron pathway of O2 + * → *O2 (i), *O2 + H+ + e− → *OOH (ii), *OOH + H+ + e− → *O + H2O (iii), *O + + H+ + e− → *OH (iv), *OH + H+ + e− → H2O2 (v) [19], as shown in Fig. 3l. It is clearly observed that the initial O2 adsorption is the RDS for the whole O2 reduction reaction process under acidic conditions. Among them, CuN4 displays the lowest O2 adsorption energy, indicating that it has the strongest capacity to activate the O2 molecule. Moreover, the last step, the conversion of *OH into H2O (v), is more difficult for the CuN2 site than for the CuN4 site. As a result, the OXD-like activity of CuN4-CNS for TMB oxidation with O2 as an oxidant is superior to that of CuN2-CNS.
In general, the parallel POD- and OXD-like reactions or cascaded CAT- and OXD-like reactions contribute to enhanced ROS generation over CuNx-CNS SAzymes. However, the difference between the bonding charge distributions of CuN2 and CN4 sites causes the difference in their intrinsic enzyme-like activities, which affects the final production of ROS. Notably, despite similar POD-like activity, the CAT- and OXD-like activities of CuN2-CNS are much lower than those of CuN4-CNS. Therefore, CuN4-CNS can be the more desirable SAzyme for antibacterial therapy.
Antibacterial performance
The catalytic antibacterial performances of CuN4-CNS against gram-negative bacteria, E. coli, and gram-positive bacteria, MRSA, were examined by the classical plate count method and live and dead bacterial staining method (Fig. S15A and Fig. 4A). Compared to the bacteria treated with CuN4-CNS (II) (200 μg ml−1) or H2O2 (200 μM) (III) alone, the viabilities of E. coli and MRSA were significantly reduced to 35.6% and 46.8% (Fig. S15B and Fig. 4B), respectively, when they were treated with CuN4-CNS + H2O2 (IV), which can be attributed to the catalytic ROS generation of CuN4-CNS inhibiting the growth of bacteria. When the laser is applied to CuN4-CNS, group (V), the temperature of the bacterial suspension is elevated to 48 °C upon 10 min of NIR irradiation (1,064 nm, 1 W cm−2), resulting in viability inhibition to 37.6% and 43.8% for E. coli and MRSA, respectively. It is noted that, compared to E. coli, the higher viability of MRSA can be attributed to the thicker cell walls or membranes of MRSA [37], endowing them with stronger tolerance to ROS attack. Interestingly, almost no E. coli or MRSA survived when they were treated with CuN4-CNS + H2O2 + NIR(VI). This can be attributed to the enhanced ROS generation of CuN4-CNS by an external NIR stimulus in the presence of H2O2, but more importantly in combination with local hyperthermia (even mild temperature). However, for the CuN4-CNS + H2O2 group (IV) or CuN4-CNS + NIR group (V), completely killing the bacteria requires a much higher substrate concentration (600 μM H2O2), CuN4-CNS loading (600 μg ml−1), and NIR laser power density (2 W cm−2) (Fig. S16). Thus, it can be seen that complete bacterial death can be achieved under the optimal conditions of 200 μg ml−1 CuN4-CNS and 200 μM H2O2 with the laser power density of 1 W cm−2. This enhanced antibacterial behavior of CuN4-CNS was further confirmed by scanning electron microscopy (SEM) and TEM. The SEM images (Fig. 4C and Fig. S15C) show that for the heathy bacterial strains treated with phosphate-buffered saline (PBS), the typically spherical or rod-shaped bacteria with smooth and intact cell walls are observed, consistent with the corresponding TEM images (Fig. 4D and Fig. S15D). However, after a single treatment of CuN4-CNS + H2O2 for 10 min, the bacterial surfaces of E. coli rapidly became rough, wrinkled, obscured, and even ruptured, and the cellular integrity could not be maintained due to cytoplasmic leakage. Despite becoming rough and obscure, the damage extent of the MRSA surface was much less than that of E. coli, probably due to the thicker cell membrane. For the E. coli treated with CuN4-CNS + NIR for 10 min, the bacterial surfaces became rather rough and uneven along with many holes, which resulted in serious loss of intracellular substrates, making the cell membranes crash down. For the photothermal therapy (PTT)-treated MRSA, although the outlines of the cell wall remained intact and clear, their surfaces became extremely wrinkled and looked severely dehydrated. These results demonstrate that the bactericidal mechanism of ROS attack is quite different from PTT, and the damage extent is also dependent on the type of bacteria. Interestingly, when combined (CuN4-CNS + H2O2 + NIR), both E. coli and MRSA were heavily damaged, and rather rough, uneven, wrinkled, and obscure membranes along with a number of holes were observed, therefore resulting in complete bacterial death.
Antibiofilm performance
Considering that implant-related biofilm infections (IRIs) usually occur in deep tissue with intrinsic AMR much stronger than that of planktonic bacteria (>1,000 times) [38], it is worth investigating the catalytic antibiofilm capacity of CuN4-CNS. As shown in Fig. 4E and F, when the biofilm was incubated with pure CuN4-CNS(II), lower bacterial viability was observed compared to those treated with PBS (I) or H2O2 (III) alone (Fig. S17), which could be attributed to the OXD-like activity of CuN4-CNS, triggering some bacterial death in the biofilm. However, owing to the very limited generation of •O2− through catalytic O2 reduction by CuN4-CNS, a great number of living bacteria remain inside the biofilm, which can be confirmed by the corresponding confocal 3D image. Nevertheless, with extra H2O2 addition, CuN4-CNS with POD-like activity can effectively catalyze the decomposition of H2O2 into abundant •OH, leading to more bacterial death inside the biofilm due to enhanced ROS generation. For the treatment with CuN4-CNS + H2O2 + NIR, the multienzyme activities of CuN4-CNS are activated and further enhanced, thus achieving significant enhancement in biofilm elimination with almost total death of bacteria. Therefore, CuN4-CNS can be introduced as an effective antibacterial material that not only kills planktonic bacteria but also eliminates mature biofilms.
Biocompatibility
In addition to creating an antibacterial effect at local infection sites, the residual CuN4-CNS can also be exposed to healthy tissues and cells in other sites of the body. Therefore, it is also important to analyze the biocompatibility of CuN4-CNS to mammalian cells. Since laser and H2O2 are only applied locally, in this case, we did not study the +laser and +H2O2 groups. The results showed that no obvious viability change was observed when mammalian cells were treated with CuN4-CNS (<600 μg ml−1) (Fig. S18A), and the hemolysis measurements demonstrated a low hemolysis ratio in vitro for CuN4-CNS (<2%; Fig. S18A).
Superficial skin wound healing
An infected rat skin wound model was subsequently constructed with MRSA and used to evaluate the therapeutic efficacy of superficial infection by CuN4-CNS in vivo. Figure 5A and B shows the treatment process and the healing results of superficial skin wounds treated under different conditions: PBS (I), CuN4-CNS (II), H2O2 (III), H2O2 + CuN4-CNS (IV), CuN4-CNS + NIR (V), or CuN4-CNS + H2O2 + NIR (VI) at different times (days 0, 4, 7, 10, and 14). In the gross view, a large amount of yellow pus was dispersed in the wound bed on day 4 for the control group treated with PBS, while on day 7, yellow pus decreased but could still be observed. In contrast, for all the treated groups, particularly CuN4-CNS + H2O2 + NIR, the wound was apparently smaller and started to dry and scab on day 7 without emerging inflammation. Especially on day 10, as illustrated in Fig. 5C to E, the wound becomes much smaller, with a relative wound area of 8%, and neither ulceration nor suppuration occurs during the treatment. On day 14, the wound was fully healed. Apart from that, the quantitative analysis of bacterial colonies from LB plates shows that the number of residual bacteria obviously decreases after treatment with CuN4-CNS + H2O2 + NIR (Fig. 5F) and is much lower than those of the other treated groups (II to V). It is noteworthy that the wound healing efficacy treated with CuN4-CNS + NIR (day 14, group V) was obviously lower than that treated with CuN4-CNS + H2O2, which can be attributed to the relatively low temperature, which reaches only 45 °C after NIR-II irradiation for 10 min (Fig. S19). Therefore, it is suggested that the remarkable wound healing efficacy of CuN4-CNS + H2O2 + NIR can predominantly originate from the enhanced ROS production of CuN4-CNS rather than photothermal activity when using an external NIR-II stimulus.
Hematoxylin and eosin (H&E) staining results indicated that keratinocytes migrated from the edge of the wound into the wound site in all treatment groups, especially in the CuN4-CNS + H2O2 + NIR group. Inflammatory cells become observably lower, granulation length significantly increases, and the epidermis gradually grows back and thickens in the normal skin sections on day 7 (Fig. 5G and H). Masson’s trichrome staining further showed that the established collagen fibers and dermal layers of wounds in the CuN4-CNS + H2O2 + NIR group were significantly better than those in the control group, and the collagen fibers were denser, thicker, and better arranged on day 14 (Fig. 5I and J). Figure 5K shows that the expression of tumor necrosis factor-α (TNF-α) was highest in the control group (with only bacterial injection) on day 7 and remained high after 14 days. Comparatively, the expression of TNF-α significantly decreased after treatment with CuN4-CNS + H2O2 + NIR. Based on this, it is inferred that the CuN4-CNS SAzyme can effectively alleviate the inflammatory response under infection conditions, accelerating wound healing and tissue regeneration, which may be due to the promoting effects of copper species in CuN4-CNS for cell migration, angiogenesis, and collagen deposition [26].
Deep implant-associated infection treatment
Compared with superficial skin wound infections, deep infections, including deep tissue abscesses and medical implant-associated infections (IRIs), are more troubling and concerning due to the formation of highly recalcitrant surface-associated biofilms [3,39]. Therefore, we further established a rat IRI model to validate the antibiofilm effect of CuN4-CNS in deep tissue in vivo. Figure 6A describes the comprehensive procedure of IRI treatment. As generally observed in the gross specimens, the implants of the untreated groups were covered by a large amount of pus and residual biofilm, especially on the 10th day after the surgery (Fig. 6B). In contrast, for the corresponding treated groups, especially with CuN4-CNS + H2O2 + NIR treatment, residual biofilms or pus are obviously reduced on the surfaces of implants. Figure 6C shows that the media in the CuN4-CNS + H2O2 + NIR group were clear and transparent, and the number of bacterial colonies was significantly lower than that in the other 3 groups. In addition, the bacterial residues on Ti plates and bacterial infiltration in the peri-implant tissues were also evaluated by estimating the number of colony-forming units (CFUs). As expected, the number of bacterial colonies in the CuN4-CNS + H2O2 + NIR group (IV) was considerably lower than that in the other groups, which further substantiates the prominent antibiofilm activity of CuN4-CNS (Fig. 6D and E). IRIs often accompany the overexpression of proinflammatory chemokines and reactions, which delay tissue repair processes. Typical acute infection signs in the control group were present, including infectious inflammatory exudation in the H&E staining (Fig. 6F) as well as myeloperoxidase (MPO) immunofluorescence staining (Fig. 6J) and biochemical analysis (Fig. 6H). Comparatively, after treatment with CuN4-CNS + H2O2 + NIR, an obvious decrease is observed in inflammatory infiltration. These results are consistent with the TNF-α expression by enzyme-linked immunosorbent assay (ELISA) (Fig. 6I), suggesting that CuN4-CNS can effectively reduce the inflammatory response. Meanwhile, numerous colonies of bacteria were visualized in the control group by Giemsa staining (Fig. 6G). In contrast, nearly no bacteria were observed in the CuN4-CNS + H2O2 + NIR group, but considerable bacterial colonies remained in the other 2 groups (CuN4-CNS + H2O2 or CuN4-CNS + NIR). Altogether, these results confirm that CuN4-CNS injection in situ with laser irradiation and H2O2 can effectively treat IRIs and have a robust inflammation resolution effect, while CuN4-CNS or CuN4-CNS + H2O2 injection can only partially reverse further deterioration of infection.
Conclusion
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In this study, a novel Cu-SF complex pyrolysis strategy was developed for the construction of CuNx-CNS SAzymes with tunable N coordination structures. The atomically dispersed CuNx sites, 2D ultrathin structure, large specific surface area, and porosity endow CuNx-CNS with excellent triple enzyme-mimetic activities, significantly promoting ROS generation through parallel POD- and OXD-like or cascaded CAT- and OXD-like catalytic pathways. The stronger O2 adsorption capacity and electron push effect of CuN4 sites contribute to the higher enzyme-mimetic activities of CuN4-CNS compared to those of CuN2-CNS. Owing to the inherently strong NIR II absorption of CuNx-CNS, using an external NIR stimulus can further promote catalytic ROS generation. As a result, the optimal CuN4-CNS exhibits broad-spectrum bactericidal properties and effectively inactivates the bacteria embedded in stubborn biofilms in vitro while displaying high therapeutic efficacy in treating superficial skin wound infections and deep IRIs in vivo. This work provides a new perspective for the rational design of multiple enzyme-mimicking SAzymes with great potential to combat bacterial infections from superficial to deep tissues.
Materials and Methods
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Chemicals
B. mori cocoons were purchased from Huangshan Huasheng Silk Group Co. Ltd. Anhydrous sodium carbonate (Na2CO3), lithium bromide (LiBr), copper chloride dihydrate (CuCl2·2H2O), hydrochloric acid (HCl), H2O2 (30 wt%), acetic acid (HAc), and isopropanol were supplied by Sinopharm Chemical Reagent Co. Ltd., and p-phthalic acid (TA), 3,3',5,5'-tetramethylbenzidine (TMB), 2,2-dimethyl-3,4-dihydropyrrol-1-ium-1-olate (DMPO), and DHE were purchased from Shanghai Aladdin Bio-Chem Technology Co. Ltd.
Synthesis of CuNx-CNS
Extracted SF was first prepared by degumming the cocoons with 1 wt% Na2CO3 solution for approximately 40 min, which was then dissolved into LiBr solution (9.3 M) for 4 h and then dialyzed for 3 days, yielding a 10 wt% regenerated silk fibroin (RSF) solution. A given amount of CuCl2 was added to 5 wt% RSF solution with a Cu/RSF mass ratio of 1:20. In particular, by adjusting the pH value of the mixture (5.5 or 11.0), the chelation mode between RSF and Cu2+ ions can be tailored. The solution was subsequently freeze-dried and pyrolyzed at 900 °C for 1 h (3 °C min−1) under a nitrogen atmosphere. Finally, the CuNx-CNS SAzymes were obtained after etching by HCl (1 M) at 120 °C for 12 h.
Characterization of CuNx-CNS
The morphology of CuNx-CNS was determined by TEM (Tecnai G2 F30), AC-HAADF-STEM (FEI, USA), and AFM (MultiMode VIII SPM, Bruker). The composition and chemical states of CuNx-CNS were measured by XRD (Bruker, Germany), XAFS spectra (Shanghai Synchrotron Radiation Facility), and XPS (Kratos, England). The Cu contents were determined by using the ICPtechnique (VISTA-MPX). N2 adsorption–desorption isotherms were used to determine the specific surface area of CuNx-CNS on a physical adsorption apparatus (NOVA 2000e). All UV–vis and fluorescence measurements were acquired by a UV 3600 spectrophotometer and Hitachi F-4600 spectrophotometer, respectively.
Evaluation of photothermal conversion performance of CuNx-CNS
An infrared camera (BritIR) was used to record the temperature variation of CuNx-CNS suspensions irradiated by a 1,064-nm NIR laser under different conditions. The photothermal conversion efficiency (η) of CuNx-CNS was calculated by Roper’s calculating method [40].
Evaluation of the POD-like activity of CuNx-CNS
POD-like catalysis assays were performed in a NaAc-HAc solution mixed with H2O2 and TMB (pH 5.5). The POD-like activity of CuNx-CNS was determined by the reduction of TMB to ox-TMB, which has a characteristic absorption peak at 652 nm and exhibits blue color. The kinetic experiments were conducted by varying the concentrations of TMB and H2O2 and calculated by a typical Michaelis–Menten curve. When using TMB as substrate, the mixture solutions contained 100 μg ml−1 CuNx-CNS, 1 mM H2O2, and 0 to 0.2 mM TMB, while toward H2O2, the mixture solutions contained 100 μg ml−1 CuNx-CNS, 0.2 mM TMB, and 0 to 2.55 mM H2O2. All kinetic measurements were performed in a time-sweep fashion at 52 nm by a Shimadzu UV spectrophotometer and repeated 3 times. For pH-dependent experiments, the POD-like activities of CuNx-CNS were performed in pH of 3.0 to 8.0. Electron paramagnetic resonance (EPR) spectrometer (Bruker A300-10/12) was used to confirm the •OH generation with DMPO as spin-trapping adduct.
Evaluation of CAT-like activity of CuNx-CNS
The CAT-mimicking property of CuNx-CNS was conducted by measuring the O2 produced by H2O2 dissociation using a dissolved oxygen meter (JPB-607A, INESA). Specially, 10 μl of CuNx-CNS (1 mg ml−1) and 50 μl of 10 M H2O2 were added into 10 ml of PBS buffer. The typical CAT-like nanozyme, namely, active carbon supported Pt NPs (2%Pt/C), was used for comparison.
Evaluation of the OXD-like activity of CuNx-CNS
OXD-like activities were also determined by colorimetric assays with TMB as substrate. Typically, CuNx-CNS (50 μg ml−1) were added to TMB (800 μM) air-saturated buffer (2.0 ml, pH 5.5). The resulting ox-TMB with absorption peak at 652 nm was monitored. DHE can be used as a fluorescent indicator toward •O2−. In the test, a mixture composed of CuNx-CNS (1 mg ml−1) and DHE (5 μM) was reacted in air-saturated buffer (pH 5.5) for 10 min. Then, the fluorescence spectrum of DHE was recorded.
In vitro and in vivo experiment
The experimental details were demonstrated in the Supplementary Materials.
Statistical analysis
All data were raw data and expressed as the mean ± standard deviation (SD) with at least 3 replicates for every experimental sample. One-way analysis of variance with Tukey’s test was used for statistical analysis. The statistical analysis was conducted using Origin 9.0 software. Statistical differences were defined as *P < 0.05, **P < 0.01, and ***P < 0.001.
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Appendix
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Year 2023 volume 6 Issue 1
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Article Info
doi: 10.34133/research.0031
  • Receive Date：2022-11-01
  • Online Date：2026-10-27
  • Published：2023-01-20
Article Data
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History
  • Received：2022-11-01
  • Accepted：2022-12-05
Affiliations
    1Department of Orthopedic Surgery, Orthopedic Institute, The First Affiliated Hospital, Medical College, Soochow University, Suzhou, Jiangsu 215006, P. R. China.
    2Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, P. R. China.
    3Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Turku 20520, Finland.
    4Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku 20520, Finland.

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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