Efficient nanozyme engineering for antibacterial therapy

As mentioned, many kinds of nanomaterials have been found to possess POD-like characteristics. However, the active centers of these POD mimics, especially for noble metal and carbon-based nanomaterials, are remarkably different from natural enzymes, thus leading to relatively low activity and selectivity compared with natural enzymes. Fortunately, the emerging of SACs bridges the gap between nanozymes and enzymes, not only because they possess atomically isolated metal sites with ultrahigh activity, but more importantly, they possess similar active centers to metalloenzymes composed of metal–nitrogen coordination (M–N_x ). By integrating advanced single-atom technology with intrinsic active centers of metalloenzymes, a variety of bioinspired single-atom nanozymes (SAzymes) resembling the active centers in metalloenzymes have been designed and explored for boosting the enzyme-like activity [62–66, 73].

Benefiting from the MOFs pyrolysis strategy for SACs synthesis, a series of POD SAzymes with FeN₄ [61, 63], ZnN₄ [66], CuN₄ [64, 73] sites, which simulate the active centers of horseradish peroxidase (HRP) [74], Zn-cytochrome c [75], and type-2 Cu depleted laccase [76], respectively, have been atomically dispersed and anchored on the surfaces of N doped porous carbon, as shown in figure 1. Owing to the elaborately mimicking the structures of the active site of metalloenzyme, taking the atomically isolated FeN₄ sites on nitrogen-doped amorphous carbon (SAF NCs) for example, the active FeN₄ site resembles the active center of HRP composed of Fe atom coordinated with four iminazole groups of histidine residues (figure 1(A-i)). The transmission electron microscope (TEM) image shows no copper nanoparticles present on the NCs surfaces (figure 1(A-ii)), while the high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image demonstrates the Fe atoms densely dispersed on the NCs surface (figure 1(A-iii)), demonstrating the successful synthesis of single Fe atoms, which endows the SAF NCs with a V_max value of 2.23 × 10⁻⁷ M s⁻¹, about 76.4 folds higher than one of the most favorable POD-like Fe₃O₄ NPs. This can be attributed to the FeN₄ sites mimicking the active center of HRP (figure 1(A-ii)) [63]. Consequently, the SAF NCs could produce abundant ‧OH for noticeably eliminating Escherichia coli and Staphylococcus aureus by critical cell membrane destruction, and in vivo the SAF NCs displayed highly effective eradication of E. coli and S. aureus pathogens and achieved better wound healing efficacy [63]. More interestingly, inspired by the active centers of cytochrome P450, Dong's group built a novel SAzymes (single-atom nanozyme with carbon nanoframe–confined FeN₅ active centers (FeN₅ SA/CNF)) with axial FeN₅ as active sites based on a bottom-up strategy [62]. The FeN₅ SA/CNF exhibited extremely high oxidase (OXD)-like activity, about 17 times higher than that of square planar FeN₄ catalyst. Thus, it directly converted O₂ into abundant ‧OH for effectively killing bacterial pathogens and promoting the healing of the infected wound [62]. Shi's team also predicted by density functional theory (DFT) calculation, as compared to the CuN₄–C sites, CuN₅ sites incorporated with a fifth nitrogen atom could enhance the adsorption of single *OH, largely reducing the apparent barrier energy, thus they believed the CuN₅ would be a more reactive POD-like center [73]. Based on these, it can be seen that rationally mimicking the enzymic active centers by the booming SAC technology can obtain highly active catalytic sites for boosting the catalytic performance of nanozymes.

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Moreover, for enzyme simulation, apart from merely resembling the enzymic active centers, simultaneously creating a spatial microenvironment for the active centers similar to that in natural enzymes is also crucial for designing highly active nanozymes, since the high catalytic efficiency of enzymatic active centers is derived from its nearby favorable hydrophobic spatial microenvironment offered by the assembly hierarchical structure of the enzyme itself [77]. Recently, Qu's group has designed a highly efficient metal–organic frameworks and covalent organic frameworks hybrid material (MOF@COF) nanozyme, in which the MOF of NH₂-MIL-88B (Fe) was used as the POD mimic, and the superficial COF_TP-TTA was constructed with the ligands possessing phenol and triazine functional groups [58]. The hierarchical nanocavities with tailored surface functional groups from COF_TP-TTA offered the enzyme-like binding pockets around the active sites, remarkably enriching H₂O₂ via non-covalent interactions. Owing to this unique bioinspired design, the MOF@COF nanozyme exhibited a significant enhancement in bacterial inhibition as compared to the parent NH₂-MIL-88B (Fe) [58].

Overall, mimicking enzymatic active centers integrated with spatial microenvironments pave a new way to the design and construction of highly active nanozymes for antibacterial therapy applications.

Besides lacking highly-active catalytic centers, due to the large and solid spatial structures, active atoms available on the nanozyme surface are only a very small fraction. So, the low atomic utilization efficiency is another important reason for causing the low catalytic activity of nanozymes. Downsizing the nanoparticles to ultrasmall clusters with size of less than 2 nm can be an effective strategy to improve the catalytic activity of nanozymes due to significantly increased specific surface area and quantum confinement effects of clusters as compared to the larger counterparts. For example, the Michaelis–Menten constant (K_m ) to the H₂O₂ substrate for silica-supported Au NPs (4.5 nm) toward the POD enzymatic reaction was about 15.8 mM [78], while for ultrathin 2D MOFs supported Au clusters (1.96 nm), the K_m was determined as 7.94 mM, indicating a much stronger affinity to H₂O₂ [20]. Consequently, the UsAuNPs/MOFs nanozyme could convert low dosage of H₂O₂ into abundant ‧OH for killing both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, and exhibit an enhanced wound healing efficacy. Interestingly, further downsizing the metal clusters to atomic-level to form the SACs, such as Fe [61, 63], Zn [66], Cu [64], Pt [27, 67], and Pd [79]-based SAzymes, can even boost their catalytic performance comparable to natural enzymes by virtue of the maximum atom utilization efficiency. Moreover, the well‐defined active centers of SAzymes allows us to interpret their catalytic mechanism more precisely based on DFT calculations. During the catalytic decomposition of H₂O₂ into ‧OH over SAzymes, the H₂O₂ molecule is first adsorbed on the active M–N₄ sites (M = Fe, Co, Zn), then the activated H₂O₂ molecule is homogeneously dissociated into two absorbed OH*, one of them will finally desorb from the M–N₄ site, generating the active ‧OH, while the other reacts with H⁺ under acidic conditions to form an H₂O molecule (figure 2(A-i)). Compared to CoN₄ and ZnN₄ sites, the FeN₄ exhibited less electron transfer to the adsorbed OH*, which is beneficial for FeN₄–C SAzymes reducing the energy barrier of ‧OH formation (figure 2(A-ii, iii)), and therefore showing enhanced PDO-like activities [80]. However, due to the easy migration of high-surface-energy single metal atoms to form metal nanoparticles, the metal loading on SACs is extremely low (∼1%), obviously hindering their catalytic activities. To address this issue, Jiao et al have explored a universal salt-template strategy for fabricating 2D MN₄–C SAzymes (M = Zn, Co, and Fe) with highly dense metal atoms, of which the Fe concentration of FeN₄–C SAzymes was up to 13.5 wt%, showing a far exceeding POD-like activity as compared to its counterpart SAzymes with a Fe mass of 1.3 wt%, as shown in figure 2(A-iv) [80]. In general, downsizing nanoparticles to atom levels can not only allow us to obtain high-efficiency antibacterial nanozymes but also to investigate the catalytic mechanism in depth, and in turn rationally design more powerful nanozymes.

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Despite small amounts, the active sites, mainly derived from some coordinatively unsaturated atoms in the sites of steps, edges, kinks and defects; play the key role in offering nanozymes with inherent enzymatic properties. Therefore, besides mimicking enzymatic active centers, creating more edges or defects on the nanomaterial surface can be another promising approach to improve the intrinsic catalytic ability of nanozymes due to more highly active centers available, which is particularly crucial for the large-sized metal oxide/sulfide-based nanozymes. Inspired by the defect-rich engineering protocol for catalytic activity improvement, Qu's group fabricated a unique MoS₂ POD nanozyme (R-CMs (R-CMs have MoS₂ nanosheets wrapped around the Cu nanowires)) with rough surfaces and defect-rich active edges (figure 2(B-i, iii)) through the control growth of MoS₂ nanosheets on the copper nanowires by a one-pot hydrothermal approach [43]. The reaction energy of MoS₂, S-defect MoS₂, and MoS₂ edge in the whole reaction (H₂O₂ → 2OH* → OH* → ‧OH) was 1.92, ‒0.82, and ‒0.26 eV, respectively (figure 2(B-vi)), suggesting both S-defect MoS₂ and MoS₂ edge facilitate the production of ‧OH radical in the H₂O₂ decomposition. As a result, the MoS₂ with defect rich edges endowed the nanozymes with higher intrinsic POD-like activity than pristine structure (figure 2(B-iv, v)). Moreover, the rough surface originated from MoS₂ nanosheets is beneficial for trapping the bacteria. Therefore, increased antibacterial activity against both drug resistant E. coli and S. aureus and wound healing efficacy was obviously observed due to the enhanced production of ‧OH localizing on the bacterial surfaces [43]. Subsequently, Wang et al developed a microwave-assisted hydrothermal method for constructing a defect-rich adhesive MoS₂/rGO vertical heterostructure (VHS) [81]. This MoS₂/rGO VHS could obtain much more active edge sites, allowing them with triple OXD-, POD-, CAT-like activities, thereby showing efficiently catalytic disruption of drug-resistant E. coli and S. aureus in vitro and in vivo. More recently, Wei et al have also taken advantage of defect-rich-edge MoS₂ to construct recoverable Fe₃O₄@MoS₂-Ag nanozyme for enhanced catalytic antibacterial ability [82]. The combination of Fe₃O₄ and MoS₂ could further increase the intrinsic POD-like properties. In addition, the rough MoS₂ shell favored the capture of bacteria, amplifying the synergistic attack of ‧OH and Ag⁺ on bacteria surface. Meanwhile, the magnetism of Fe₃O₄ could be used to recycle the nanozyme after antibacterial treatment [82].

Composition regulations including hetero atom doping, multi-active sites design, and support hybridization, and so forth are promising ways to take advantage of the synergy between different components or alter the lattice structure of nanozymes to tune the catalytic activities. For instance, a B-GDY nanosheet shows enhanced POD-like activity [8]. N-SCSs displayed enhanced bacterial disruption and accelerated infected wound recovery due to the synergistic effect between its excellent multiple enzyme-like activities [55]. Copper doping endows the degradable phosphate-based glass (PBG) bio-glass with excellent POD-like activity to achieve high antibacterial effects [83]. Owing to the synergy between binary metals, the bimetallic CuCoS₄ NPs nanozymes possessed higher POD-like activity than monometallic CuS and CoS, and even offered high activity in neutral pH [84]. As for bimetallic Pd–Ir nanocubes nanozyme, the introduction of more active Ir component into Pd nanocubes significantly improved the catalytic efficiency, giving a 20- and 400-fold higher activity than monometallic Pd nanocubes and HRP, respectively [85]. The ultrathin 2D MOFs supported ultrasmall AuNPs (UsAuNPs/MOFs) hybrid exhibited a remarkable POD-like catalytic antibacterial performance and could effectively facilitate wound healing at a low dosage of H₂O₂ [20]. Moreover, besides active centers, the enzyme-mimicking activity of nanozymes is also strongly related to their own morphology. For example, the POD mimetic activity of truncated octahedron shaped Fe₃O₄ NPs was superior to that of spherical-shaped ones [86]. The bamboo-like nitrogen-doped carbon nanotubes encapsulating Co nanoparticles (N-CNTs@Co NPs) possessed higher OXD-like activity than its spherical precursor CoHCF due to that the bamboo-like structure could prevent Co NPs leakage and improve its dispersion and stability [87]. Notably, the chemical state of copper in carbon supported copper nanohybrids also plays an important role in regulating the enzyme-mimicking activity of nanozymes [28]. Overall, rationally regulating the compositions of nanozymes can improve their catalytic and antibacterial efficiency.

In general, the activities of most POD nanozymes are pH dependent. As for Fe₃O₄ nanozymes, better catalytic performance is obtained in an acidic medium (pH = ∼4) [93], whereas in a neutral environment the PDO activity can be dramatically reduced and even changed [94]. Studies have shown some chronic wound infections are around 6.5–8.5 [95], which hinders the effective antibacterial activity of Fe₃O₄ NPs due to the near neutral pH. In this regard, Vallabani et al have recently developed an alternative ATP-triggering strategy to enhance the Fe₃O₄ NPs catalytic activity and antibacterial efficiency at neutral pH [89]. With the aid of ATP, owing to its ability of participation in single electron transfer reaction through complexation with Fe₃O₄ NPs [96, 97], the ‧OH generation catalyzed by Fe₃O₄ NPs could be significantly accelerated, resulting in enhanced bactericidal efficiency in presence of H₂O₂ at neutral pH [38] (figure 3(A)). To break the limitations of local pH and H₂O₂ under physiological conditions, instead of ATP, Chen et al have constructed a versatile glucose-stimulated nanozymes composed of aptamer-functionalized platinum nanozymes (Apt-PtNZ), glucose oxidase (GOX) and hyaluronic acid (HA) (denoted as APGH) for treating diabetic infections (pH = 7.0–8.0) [90]. With aptamer recognition, APGH could first adhere to bacterial surface, then glucose oxidase would catalyze the glucose oxidation to generate H₂O₂ and lower the local pH, and finally the POD-mimicking Pt NPs in-situ converted the H₂O₂ into ‧OH on bacteria surfaces, subsequently enhancing the antibacterial effect in vitro and in diabetic wounds [90] (figure 3(B)).

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For most antibacterial nanozymes, their continuously generating ROS-mediated antibacterial action may allow opportunistic bacteria to generate resistance, similar to antibiotics. Therefore, nanozymes with 'on-demand' antimicrobial activity could be highly desirable. Considering light possessing great tenability and spatial controllability along with no physical contact, Karim et al have developed visible-light-active CuO nanorods (NRs) using a highly basic tertiary amine [39]. The visible light illumination obviously facilitated the binding affinity of CuO NRs to H₂O₂, 4.35 times higher than that in dark, and activated the CuO NRs with a band gap of 1.44 eV, remarkably accelerating the ROS production (figure 3(C)). As a result, the photoactive CuO NRs exhibited a significant enhanced antibacterial performance against E. coli even at a low H₂O₂ concentration [39]. Similarly, Chen's work also demonstrated visible light illumination improved ROS generation for MoS₂/rGO VHS, thereby resulting in high bactericidal efficiency and wound healing promotion with the assistant of H₂O₂ and light [81]. A TiO₂NTs@MoS₂ nanozyme composed of TiO₂ nanotubes coated with MoS₂ nanoflowers was also reported to display visible light-stimulated enhancement in the POD-like activity of MoS₂ due to the combination with the semiconductor TiO₂NTs [45]. Accordingly, the as-obtained TiO₂NTs@MoS₂ could produce much more ‧OH, showing outstanding antibacterial effect against drug-resistant bacteria under the visible light and excellent wound healing efficacy.

As mentioned above, light has become an attracting trigger to tailor the enzyme-like activity. Comparing with visible light, NIR with wavelength ranging from 780 to 2500 nm is featured with deeper tissue penetration and invasiveness [98]. Thus, NIR-based PTT has been extensively explored for anti-infective therapy [99–101]. The photothermal nanoagents can absorb the NIR and convert the optical energy into heat, which then initiates the cell membrane breakage and protein denaturation in bacteria, finally leading to their thermal ablation [102–104]. Based on this, NIR-responsive nanozymes, such as Au nanoplates [26], Au@Pt nanodots [32], Au NRs/CeO₂ hybrid [91], rough carbon/Fe₃O₄ nanohybrids [102], cationic chitosan@RuO₂ hybrid [105], MoS₂ nanozymes [43, 82, 106], N-doped carbon nanozyme [55], PEG@Zr-Fc MOF hydrogel [107], and Fe- and Cu–N–C SAzymes [61, 63, 64], have been developed. Under the NIR laser irradiation, these NIR-active nanozymes can not only produce local photothermal effect, but more importantly the elevated temperature certainly accelerates catalytic reaction rate, thereby realizing the enhanced synergic CDT and PTT for effectively treating drug-resistant bacterial infections. Actually, for NIR responsive metal-semiconductor hybrid nanozymes, the NIR excitation can not only induce local photothermal effect but also transfer of hot electrons simultaneously, of which the enzyme-like function is dominantly dependent on the hot electrons' transfer. Taking the gold–silver nanocages loaded nitrogen-doped graphdiyne quantum dots (N-GDQDs/AuAg NC) heterostructures as an example [35], as shown in figure 3(D), under NIR excitation, the N-GDQDs was induced to produce heat and hot carriers (e^‒), which was then injected into AuAg, remarkably accelerating the decomposition of H₂O₂ into ‧OH. Taking advantage of NIR-enhanced POD-like activity, the N-GDQDs/AuAg NC nanozyme possessed efficient broad-spectrum antibacterial activity under NIR irradiation. Similar enhanced effect can be found for the Au/CeO₂ plasmonic nanohybrids [91] and graphdiyne nanowalls wrapped hollow copper sulfide nanocubes (CuS@GDY) [50] with strong LSPR absorption and fast hot electrons transfer efficiency. Overall, the NIR stimuli will be a promising spatiotemporal way to enhance the catalytic process of nanozymes.

Iron-based nanozymes are one of the most promising POD mimics for antimicrobial therapy. However, their unsatisfactory catalytic efficacy is still limiting their practical applications. Thus, developing proper external stimuli to modulate the catalytic activity of iron-based nanozymes may give an opportunity in enhancing their antibacterial treatment. To address this issue, Zhao's recent work reported an alternative strategy of utilizing alternating magnetic field (AMF) to regulate the activity of amorphous iron nanoparticles (AIronNPs) in POD-like catalytic reaction [92]. Due to the rapidly ionized AIronNPs and the AMF augmented chemodynamic effect, abundant ferrous iron ions were released from AIronNPs with AMF exposure (figure 3(E)), which significantly accelerated the catalytic reaction rate of converting H₂O₂ to ‧OH as compared to AIronNPs alone, thus exhibiting excellent broad-spectrum antibacterial properties. Moreover, for in vivo infected model the AIronNPs combined with AMF synergistically promoted the formation of granulation tissue and facilitated the wound healing. This work paves a new way for spatiotemporally modulating the catalytic activity of magnetic nanozymes in the antibacterial field.

Rough surface engineering has been recognized as a powerful tool to enhance the bacterial adhesion of nanozymes. This is because bacteria covered with plenty of flagella and pili are easily trapped by the rough nanostructures, but normal cells will not be attracted [108, 109]. Moreover, benefiting from rough surfaces, larger surface area and more edge-defect active sites are available on the nanozyme surface, resulting in enhanced intrinsic catalytic activity [43, 70, 81, 82, 102]. Remarkable works have been done by fabricating defect-rich MoS₂ nanosheets on the surface of one-dimension Cu nanowires (R-CMs) (figure 4(A-i, ii)) [43], two-dimension rGO (MoS₂/rGO VHS) (figure 4(B-i, ii)) [81], and zero-dimension Fe₃O₄ nanoparticles (Fe₃O₄@MoS₂-Ag) (figure 4(C-i, ii)) [82] to develop rough-surface nanozymes. As for 1D R-CMs hybrid, it possessed dual antibacterial actions of POD-like catalytic attack and NIR-based photothermal lysis [43], while 2D MoS₂/rGO VHS exhibited triple POD, OXD, and CAT-like properties with visible light enhanced ROS generation [81], and the 0D Fe₃O₄@MoS₂-Ag could generate dual antibacterial actions of ‧OH and Ag⁺ [82]. Therefore, the rough MoS₂ nanosheets facilitated the enhanced bacterial adhesion (figures 4(A-iii, iv), (B-iii, iv) and (C-iii, iv)) due to special interaction between roughness of MoS₂ nanosheets and flagella and pili of bacteria, which made the generated antibacterial actions surrounding the bacterial surface rather than normal cells, resulting in the enhanced catalytic therapeutic effect without potential hazards. Lately, Xu et al reported a carbon–iron oxide hybrid nanozymes with rough surfaces (RCF) (figure 4(D-i, ii)), which also provided increased bacterial adhesion for the nanozyme (figure 4(D-iii, iv)), thereby benefiting the catalytic and photothermal therapeutic outcome for wound infection [102]. Similarly, engineering sea urchin-like PdCu nanoparticles can also produce rather rough surfaces for enhancing the bacterial adhesion ability of the nanozymes and achieve improved antibacterial activity [33]. In general, rough surface engineering paves a new way to design strong bacteria-adhesive nanozymes with enhanced catalytic activity.

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Inspired by the branch-like pseudopodia mechanism of neutrophil trapping bacteria in the human immune system [110], Qu's group have designed a MOF@COF nanozyme with a pseudopodia-like surface and tailored microenvironment (figure 5(A)). The spiky COFs (figure 5(B)) offer multivalent topological interactions for the nanozyme, thus allowing them to strongly capture bacteria (figure 5(C)). Then, integrating with active sites generated by the hierarchical nanocavities of COFs for binding substrate molecules, the ROS could be in-situ produced around the bacteria, significantly amplifying the catalytic and therapeutic efficiency (figure 5(D)) [58]. Overall, this bioinspired strategy provides an alternative way to design novel strong bacteria-adhesive nanozymes.

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