Catalytically Potent and Selective Clusterzymes for Modulation of Neuroinflammation Through Single-Atom Substitutions

Emerging artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have long been pursued with sustained efforts. The majority of current candidates rely on noble metals or transition metal oxides with rather poor catalytic activity compared with natural molecules. To tackle this limitation, we strategically designed a novel artificial enzyme based on a structurally well-defined Au25 cluster, namely clusterzyme, which is endowed with intrinsic high catalytic activity and selectivity driven by single-atom substitutions with modulated bond lengths. The 3-mercaptopropionic acid (MPA)-stabilized Au24Cu1 and Au24Cd1 clusterzymes exhibit 137 and 160 times higher antioxidant capacities than the natural trolox, respectively. Meanwhile, the clusterzymes each demonstrate preferential enzyme-mimicking catalytic activities with compelling selectivity: Au25 exhibits superior glutathione peroxidase-like (GPx-like) activity; Au24Cu1 shows a distinct advantage towards catalase-like (CAT-like) activity by its Cu single active site; Au24Cd1 preferably acts as a superoxide dismutase-like (SOD-like) enzyme via the Cd single active site. This unique diversified catalytic landscape manifests distinctive reactions against inflammation in brain. Au24Cu1 behaves as an endogenous multi-enzyme mimic that directly decreases peroxide in injured brain via catalytic reactions, while Au24Cd1, catalyzes superoxide and nitrogenous signal molecules by preference, and significantly decreases inflammation factors such as IL-1\b{eta}, IL-6, and TNF{\alpha}, indicative of an important role in mitigating neuroinflammation.


Introduction
Due to their exclusive catalytic activity and selectivity, artificial enzymes are exploited as promising tools for wide-reaching biomedical implications, [1][2][3][4][5][6][7][8] particularly as advanced diagnostics 9,10 and therapeutics [11][12][13][14][15][16] of diseases. Earlier studies shed light on the oxidase-and peroxidase-like activities of noble metals. 17 Gold-based materials were unraveled to possess versatile enzyme-like activities such as nuclease, glucose oxidase (GOD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD). 17,18 The Michaelis-Menten constant (Km) to the H2O2 substrate of gold nanoparticles towards the POD enzymatic reaction is below 1 mM, but the catalytic activity is weak. 19 In contrast, Pt-based materials generally confer a high overall catalytic activity but it can only show a good H2O2 substrate affinity when Km is up to 16.7 mM, 14,20 and modulation of selective catalysis often needs to be purposely realized through rationally designed combination with other catalysts. 21 Meanwhile, metal oxides have also revealed great potentials as enzyme mimetics. 22 Typically, Fe3O4 nanoparticles display the PODlike activity, 23,24 but are limited by their affinity to the H2O2 substrate (Km at ~ 154 mM) and a maximal reaction rate (5.9 µM/min) that do not meet expectations. Mn3O4 nanoparticles concurrently exhibit SOD-, CAT-and GPx-like activities via the redox switch between Mn 3+ and Mn 4+ with a maximum reaction rate reaching 6-125 mM/min at nanomolar levels, which is unfortunately still inferior to natural enzymes. 25 Thus, the development of catalytic artificial enzymes with exceptional activity, adequate selectivity and satisfactory stability remains a major challenge for any foreseeable practical applications.
As is well known to all, most brain injuries involve enzyme-related catalytic processes and continuous neuroinflammation. [26][27][28] However, it is largely unclear yet which specific catalytic route(s) can be selectively targeted to inhibit neuroinflammatory responses, primarily because brain injuries simultaneously trigger various kinds of multi-enzymatic reactions between free radicals and numerous bioactive molecules. 29 Therefore, exploration of versatile artificial enzymes with different catalytic routes and desirable selectivity is beneficial to establish the relationship between oxidative stress and inflammation, and to reveal the underlying molecular pathways of catalysis. [30][31][32] Atomic-level catalysts suffice a viable solution for the unmet need of improved catalytic activity and precisely modulated selectivity in a controllable manner, with lots of Fe-and Ptbased single-atom nanozymes developed. [33][34][35][36][37][38][39][40] In particular, Au contains excessive transition metal electronic states and rich electronic energy levels, which provide a solid basis for designing atomic-scale enzyme. Nevertheless, hindered by uncontrollable syntheses and complicated spatial coordination, it is difficult to reveal their electronic structures accurately, which can further influence the catalytic activity and prevent researchers from understanding exact catalytic mechanisms at atomic levels. 41 Herein, an exemplified Au-based clusterzyme was rationally designed at atomic precision with ultrahigh catalytic activity and superiority over natural antioxidants, and favorable enzymatic selectivity can be achieved via exquisite singleatom substitution by modulating single Cu or Cd active site, consequently serving as a promising artificial enzyme with tuned catalytic selectivity for treatment of neuroinflammation in brain.

Structural properties of clusterzymes
Exceedingly different from most previously reported nanozymes, 1,4,5 the as-developed MPA-protected Au25 clusterzyme is stringently defined by its unambiguous atomic configuration and geometry structure (Figure 1a). The hydrodynamic size of Au25 is determined to be 2.0 nm by dynamic light scattering (DLS), and the zeta potentials of all clusterzymes are around -35 mV, suggesting the ultrasmall size and good colloid stability ( Figure S1). The characteristic absorption at 450 nm and 670 nm of Au25 is attributed to its unique interband transitions, 42,43 while a single-atom substitution of Cu or Cd induces a 2-3 nm minor shift, showing insignificant influence on optical properties (Figure 1b, S2). Electrospray ionization-mass spectra (ESI-MS) reveal a distinct m/z peak at ~2,271, assigned to [Au25MPA18-3H] 3- (Figure 1c). After one atom substitution by Cu and Cd, the characteristic m/z peak shifts to ~2226.6 and ~2243, respectively.
The inductively coupled plasma-mass spectrometry (ICP-MS) confirms that the ratios of Cd and Cu to the total metal are 5% and 4%, respectively, further validating the successful introduction of single atoms ( Figure S3). X-ray photoelectron spectroscopy (XPS) further confirms that Au (0) is the dominant state in all clusterzymes ( Figure S4). To identify the precise spatial atomic configuration, Extended X-ray Absorption Fine Structure (EXAFS) spectra at the Au, Cu and Cd edges were recorded (Figure 1d, e and S5-S6). The L3 edges of Au in all clusterzymes have higher white-line intensities than the bulk standard Au foil. This is ascribed to larger surface area and alloying effects from partial oxidation with more d-band vacancies from nanoscale sizes and surface molecule-like interactions (Au(I)−thiolate). The characteristic absorption edges of Au clusterzymes were found at ~11920 eV, which is assigned to the 2p→5d electronic transition of Au suggesting a reduced population of unoccupied valence d-states. The increase of intensity in MPA-protected Au24Cu1 and Au24Cd1 indicates that the density of 5d electrons of Au is decreased by the one atom substitutions of Cu and Cd through the transfer of their 4d electrons ( Figure S5). 44,45 The k-space oscillations of Au25 clusters and the Au foil are shown in Figure   S5. The k-space of the Au foil exists in typical fcc oscillation patterns which are apparently absent in all Au clusterzymes due to their small core sizes. Besides, we also investigated the XANES spectra of Cu and Cd foils as well as the corresponding atomic counterparts within clusterzymes, clearly displaying differences between single atoms and bulk metals ( Figure S6).
To further pinpoint the doping sites of Cu and Cd atoms, we performed fitting analysis on the EXAFS data of Cu and Cd. Figure 1d shows the R space of the EXAFS data of the Cu K edge in Au24Cu1. It can be seen that there is only one major peak in the range of 1.6-5.0 Å. This peak roughly corresponds to the scattering path of photoelectron waves from the X-ray absorbing Cu atom to the neighboring S atoms of different shells, and IFEFFIT program is used to fit this peak.
The EXAFS parameters obtained after fitting are shown in Table S1. The Cu-S coordination number obtained from the fitting is 1.9 ± 0.2 Å. This value is close to 2 Å, which may indicate that the replacement of Au25 by a Cu atom occurs at the oligomer site, consistent with previous work. 44 Similarly, the R space of EXAFS data on Cd L3 edge in Au24Cd1 shows a peak in the range of 1.6-5.0 Å, and the fitted Cd-S coordination number is 2.3 ± 1.7 Å, which is close to the coordination number of the bond with S at the oligomer site of Au25, indicating that Cd atom substitution may occurs at the oligomer site. (Figure 1e). 46

Antioxidant properties of clusterzymes
We tested the general antioxidative properties of all clusterzymes using the ABTS method trolox, and 7.5 and 9 times higher than anthocyanin, respectively. The reaction rates of Au24Cu1 and Au24Cd1 at 10 and 14 μM/s are 8-11 times higher than Au25, or 38 and 51 fold higher than trolox, respectively. In addtion, in a parellel comparison with other elements, substitution with exactly one atom of Cu or Cd present the foremost activity amidst all substituents ( Figure S7d).
Preceding studies have evidenced that atomically precise gold clusters, such as Au25 and Au38, are endowed with the oxidation catalytic activities, 47-50 but their antioxidant activities are rarely reported. Herein, we discovered its ultrahigh antioxidant activity with fast kinetics via atom substitution. show that the antioxidant performance is greatly improved after one atom substitution with Cu or Cd. Compared with natural antioxidants, Au24Cu1 and Au24Cd1 show 137 and 160 times higher activity than trolox, and 7.5 and 9 times higher activity than anthocyanin, respectively.

Enzyme-like properties of clusterzymes
The general catalytic profile of clusterzymes and the schematic diagram showing catalytic processes are displayed as in Figure 3a and 3b. To pinpoint catalytic selectivity of these clusterzymes, we firstly investigated the GPx-like activity of Au25, Au24Cu1 and Au24Cd1 at the concentration of 10 ng/μL. Surprisingly, Au25 shows the strongest tendency towards GPx-like activity with a maximum reation rate of 0.47 mM/min, higher than 0.34 mM/min for Au24Cu1 and 0.10 mM/min for Au24Cd1 (Figure 3c), and also significantly higher than those of previously-reported Mn3O4 nanoflowers (0.056 mM/min) 51 and Co/PMCS (0.013 mM/min). 52 The turnover frequency (TOF) value of Au25 calculated by the Michaelis-Menten equation is 320 min -1 , 4.7 times higher than Au24Cd1 ( Figure S8). This result is interesting because metals are generally considered to have low GPx-like activity, but the high GPx-like activity of Au25 can be exploited to eliminate lipid peroxides and oxidative damages. The CAT-like activity of clusterzymes were studied at the concentration of 20 ng/μL as in Figure 3d. The maximum reaction rate of Au25 is 0.074 mM/min, whereas the introduction of a Cu single atom gives rise to a 4.7-fold increase to 0.35 mM/min, suggesting its CAT-like catalytic preference. The calculated TOF value of Au24Cu1 for CAT-like activity is 116.7 min -1 (Figure S9), which is significantly higher than that of Pd octahedrons (1.51 min -1 ). 53 The SOD-like activity of pure Au25 can only inhibit 31 % of the substrate, while one Cd atom substitution considerably increases the inhibition rate to 89 %, empowering SOD-like selectivity (Figure 3e). The aforementioned results suggest enzyme-mimicking preferences of each individual clusterzyme: Au25 as GPx, Au24Cu1 and Au24Cd1 as CAT and SOD, respectively. The structures of clustezymes before and after reaction with H2O2 suggest unchanged structures of the clusterzymes (Figure S10-S11). 54,55 Previous work mainly focused on the atomic substitutions of Au25 using noble metals for catalytic reactions of hydrogen and CO/CO2. 56 (Figure 3f). However, Au24Cu1 almost completely diminishes all ESR signals (~100 %), consistent with the observed best CAT-like activity as in Figure 3d. Similarly, the scavenging of O2 •− by clusterzymes was also investigated (Figure 3g). The ESR signal stays strong for the control, and slightly decreases after addition of Au25 and Au24Cu1, with surplus remaining residues. In contrast, the ESR signal of O2 •− almost disappears in the presence of Au24Cd1 further validating its superior specialized SOD-like activity (Figure 3e). Besides, we also tested the free-radical scavenging capability of the clusterzymes towards reactive nitrogen species (RNS) including •NO, ONOO -, and DPPH•.
Au24Cd1 shows the most robust overall scavenging efficiency against DPPH• ( Figure S12). The ESR reveals that Au24Cd1 has the best scavenging capability towards •NO at a low concentration of 2.7 ng/μL, whereas Au25 presents ignorable activity ( Figure S13). Likewise, both Au24Cd1 and Au24Cu1 also manifest significantly higher scavenging efficiency towards ONOOthan Au25 ( Figure S14-S15). Au24Cd1 is more selective against RNS than Au24Cu1, while Au25 has insignificant catalytic activity. Thus, it is rational to conclude that the high selectivity for enzymes and radicals originates from the single-atom substitutions of Cu and Cd which induce redistribution of surface electrons and exert influence on electronic structures and states.

DFT calculations and the mechanism of catalytic selectivity
To reveal the catalytic mechanism, the Density Functional Theory (DFT) was employed to investigate the catalytic selectivity and quantum properties. By exploring possible structures in the literature, we adopt the gold core of the well-known Au25 clusters 60 (Table S2). Although more theoretical investigations on the surface replacements can be found in the appendix (Figure S16-S19, and Table S3), we focus on the oligomer replacement and the associated catalytic efficiency.
Unlike the surface replacement which may cause the expansion of the core, the oligomer replacement causes the oligomer bending. It is different from the normal S-Au-S chain which aligns in a (nearly) straight line ( Figure S20). The doped Cu shrinks the S-X-S chain while the doped Cd extends it. Compared with the typical bond length of S-Au at 2.3 Å, S-Cu and S-Cd bonds are 2.2 and 2.55 Å respectively, as shown in Figure 4b and Figure S21. With the bent chain, the distances between Cu/Cd atoms to the surface of the core are comparable, around 3.1 Å. The similarity between the Cu and Au atoms guarantees that the binding of S-Cu-S is so "firm" that the relative positions of Cu to S atoms can be hardly changed by the dynamics during the catalytic procedures which are discussed extensively below. In contrast, the relative position of the doped Cd atom may be significantly affected by the local environment such as the adhesion of small chemical units (Figure S22-S23).
We observed the outstanding performance of the clusterzymes in both CAT and SOD reactions with the reaction pathways summarized in Figure 4c. The CAT reaction usually refers to the catalytic degradation of hydrogen peroxide, and the decomposition mechanism of H2O2 may involve multiple chemical stages (Figure 4d, 4e). For the process of SOD, we assumed the clusters were involved in similar mechanisms to the general catalytic scheme of SOD reaction. It is worth noting that the release of oxygen completes the CAT process, while the SOD process occurs simultaneously, and the two processes are mutually permeated. The reduced cluster, Cluster(I), may also be involved in both CAT and SOD processes which depends on the concentration of different components. The SA mechanism is mainly seen in Au24Cu1. Due to the firmness of the S-Cu-S oligomer, the Cu atom is relatively rigid (Figure 4f, 4g). The SA mechanism is also seen in the first step of SOD process catalyzed by Au24Cd1. The catalytic process includes the distance change and orientation change of small units. Significant changes in the distance between the active site (doped atom) and the small units are seen in most of the SOD processes. In contrast, the orientation change is the main character in most of the CAT processes.
The MA mechanism is seen in Au24Cd1 on the 2 nd stage (Cluster(I)) of the SOD processes and most of the CAT processes. The bond modulation refers to the position change of the Cd atom which may deviate from the oligomer plane until a third S atom from another oligomer stops it. Thus, the S-Cd bonds are changed significantly (Figure 4f, 4g).  (Figure S24). During such a process, the distance between the attached oxygen atoms is slightly expanded towards the normal distance of oxygen molecules which indicates the completion of the entire catalytic procedure (Table S4). A similar procedure for the Au24Cd1 can be observed at the adhesion of OOH-at the first stage of CAT process. Such a unique process allows the doped Cd atom to be an active site that can be selfmodulated in a wide spatial range compare to the firm Cu atom. This may explain its good performance in the SOD process. To reveal the biological activity of clusterzymes, the cell toxicity for different nerve cell lines (HT22, BV2 and MA-c) were measured by the MTT assay (Figure 5a and S25), showing that Au25, Au24Cu1, and Au24Cd1 present acceptable biocompatibility. Cell survival of H2O2stimulated neuron cells was performed with the incubation of Au25, Au24Cd1 or Au24Cu1. As shown in Figure 5b, days post injury. Therefore, the decrease in SOD and GSH/GSSG levels from TBI can be well rescued by clusterzymes with prominent recoveries 7 days after treatment (Figure 5i and j).

Modulation of neuroinflammation
Comparatively, Au24Cd1 induce a better recovery in SOD than Au24Cu1, which correlates well with their in vitro SOD-like activity (Figure 3). As the byproducts of the oxidative stress, lipid peroxides and H2O2 show higher accumulations in the brain following TBI, resulting in severe oxidative damage (Figure 5k and 5l). Both Au24Cu1 and Au24Cd1 significantly inhibit the production of these harmful molecules, while Au25 barely alters the TBI-induced increase. These results are conceivable because O2 •− is known to be continuously produced by immediate injuries at the early stage, followed with subsequent production of lipid peroxides and H2O2. With regard to Au24Cd1, it can recover the diminished SOD in the first place due to its high catalytic selectivity for O2 •− , and then sustain the continuously decrease of lipid peroxides and H2O2 as the secondary catalytic options. In contrast, Au24Cu1 is primarily prone to increase the levels of lipid peroxides and H2O2 at the early stage due to its preference for CAT-like activity and •OH, but these molecules are intermediate products at relatively low concentrations afer TBI onset, and consequently it accounts for the increasing clearance capability in the long term.  (Figure 6b-6d). However, the clusterzymes treatment results in cytokines close to the normal level, indicating a better suppression effect on neuroinflammation. Further, the ELISA further validated the immunoblotting results that Au24Cd1 and Au24Cu1 are capable of decreasing the inflammatory cytokines in brain tissues such as IL-1β, IL-6, and TNFα, while Au25 does not significantly alter the inflammatory cytokine patterns (Figure 6e-6g). Au24Cd1 can eliminate IL-1β and IL-6 associated inflammatory responses, while Au24Cu1 has a better effect on reduction of TNFα, indicating their relevant selectivity towards modulation of neuroinflammation. Finally, immunofluorescence staining of cerebral cortex harvested from mice also shows that Au24Cd1 and Au24Cu1 can remarkably decrease the TBI-elevated expressions of IL-1β, IL-6, and TNFα (Figure 6h, 6i and S26-S27), therefore alleviating neuroinflammation. Colocalization studies with markers for neurons (NeuN), microglia (Iba-1) or astrocytes (GFAP) were performed in injured cortex on day 3 post injury. Figure 6h and 6i reveal that IL-1β is mainly produced by microglia after TBI, similar with IL-6 and TNFα ( Figure S26-S27). In addition, quantitative analyses of the number of positive cells show that massive microglia and astrocytes are activated and many neurons are depleted after TBI (Figure   6j). With the clusterzymes treatment, most of these nerve cells are rescued. Meanwhile, the morphology of TBI-activated astrocytes can be recovered to near normal levels after treatment with clusterzymes (Figure S28), and the neuroinflammatory responses are also prevented likewise by verified histology (Figure S29-S32). The clusterzymes can also restore the TBIinduced body weight loss ( Figure S33). Moreover, behavioral tests were studied by the Morris water maze. As shown in Figure S34a and S34b, all the mice apparently learned the task during the acquisition phase of days 13-17 and 28-31, while the distance traveled and latency to hidden platform with Au24Cu1 and Au24Cd1 treatment obviously decreased. For the probe trial on day 18 and day 32 ( Figure S34c and S34d), the percentage time in the missing platform quadrant and the number of platform crossingswere significantly reduced in the TBI group, but almost return to the normal level after Au24Cu1 and Au24Cd1 treatment. These results reveal trends in the improvements of learning ability and spatial memory with Au24Cu1 and Au24Cd1 treatment. In addition, we systematically studied the pharmacokinetics and toxicology of clusterzymes. It can be seen that the clusterzymes accumulated in major organs can be removed by the kidney (urine) and liver (feces). After 48 hours, ～80% of the total dose can be excreted, and most of it is excreted through the kidney (more than 70%) ( Figure S35). No significant changes in organs or blood chemistry or hematology are found, suggesting that renal clearable clusterzymes do not cause significant biological toxicity in vivo (Figure S36-S38). Artificial enzymes have persistently been shown to exhibit multiple enzyme-like catalytic activities with a diversified class of materials. 15 Low catalyitc activity as compared to natural enzymes, however, is one of the most noticeable disadvantages due to limited electron transfers at atomic levels. 15 The rationally-designed clusterzymes with single-atom substitutions overcome such barriers with antioxidant activity 9 times higher than that of anthocyanin which is known to be one of the most reactive antioxidant molecules in nature. Besides, unlike the structurally ambiguous traditional artificial enzymes, the definitive molecular structures of clusterzymes are accurately elucidated, allowing us to distinguish the catalytically active sites and scrutinize the electronic structures and reaction energies. [62][63][64][65] As a result, the substituting single atoms can be arranged into a specific spatial location of the clusterzyme freely, thus tuning electronic structures and affecting the catalytic activity. [66][67][68][69][70] Meanwhile, the interactions between host atoms (i.e. Au) and the introduced substituting atoms (i.e. Cu or Cd) can induce coupled electron states and in turn influence the catatlytic selectivity. 71 In our work, the GPx-, SOD-, and CAT-like catalytic selectivity were assigned to Au25, Au24Cd1, Au24Cu1, respectively via modulated bond lengths to the active center, and thus it is conceived that such a platform of clusterzymes will generate various selectivity against different molecules. By employing the three catalytically selective clusterzymes, we sucessfully established the relationship between oxidative stress and neuroinflammation, demonstrating the importance of O2 •− and long-term benefits in TBI.
Specifically, Au24Cd1 can significantly mitigate the neuroinflammation via inhibiting IL-1β and IL-6, 29,72 while Au24Cu1 differentially reduces neuroinflammation by inhibiting TNFα, showing selectivity against anti-neuroinflammation. Meanwhile, due to the innate ultrasmall size of clusterzymes, it can penetrate the kidney barriers and be exceted by renal, avoiding long-term hepatotoxicity and multi-organ injuries. Therefore, the clusterzymes are presumably influential as a biomedicine, especially in the field of neuroscience.

Conclusions
In summary, we report a systemic single-atom substitution approach to fabricate artifical enzymes on the basis of MPA-protected Au25 clusters, namely clusterzymes. The clusterzymes show the ultrahigh antioxidant activity up to 137-160 times higher than the natural trolox.
Moreover, the catalytic selectivity towards GPx, CAT, SOD, and nitrogen-related signaling molecules can be fine-tuned by single-atom substitutions. DFT calculations conclude that reaction pathways are modulated by the single active site of Au24Cd1 and Au24Cu1 at bond lengths. The biological results show that Au24Cd1 preferentially decreases IL-1β and IL-6, while Au24Cu1 tends to decrease TNFα, indicative of their different selectivity for modulating alleviation of neuroinflammation.