Catalytically Active Gold Clusters with Atomic Precision for Noninvasive Early Intervention of Neurotrauma

doi:10.21203/rs.3.rs-624731/v1

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Catalytically Active Gold Clusters with Atomic Precision for Noninvasive Early Intervention of Neurotrauma

https://doi.org/10.21203/rs.3.rs-624731/v1

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Background

Neurotrauma is a head or spine injury caused by external forces. It is associated with oxidative stress, caused by the overproduction of peroxides and superoxides, and that can induce wound infection and trigger a serial of immunological reactions. The emerging catalysts have shown great potential for the treatment of brain injury and inhabiting neurogenic inflammation, but are limited to biosafety issues and delivery efficiency.

Results

Herein, we propose the noninvasive delivery route to brain trauma by employing highly active gold clusters with enzyme-like activity for achieving the early intervention. The ultrasmall gold clusters with unique size distribution show excellent catalytic activity with 10 times decomposition rate to H₂O₂. In vitro experiment shows the gold clusters can decrease the excessive O₂^•− and H₂O₂ compared with untreated cells. In vivo experiment shows gold clusters accelerate the wound healing of brain trauma and decrease the peroxide and superoxide of brain tissue. The western blot shows noninvasive treatment can decrease the inflammatory factors via inhibiting the activation of astrocytes and microglia.

Conclusions

Present work shows noninvasive treatment is a promising route for early intervention of brain trauma.

Nanoscience

Gold nanoclusters

Catalytic activity

Traumatic brain injury

Noninvasive administration

Traumatic brain injury (TBI) is a serious public health concern leading to death and disability with high morbility globally [1–3] and causes economic burden [4–6]. Despite the primary insults, the TBI can cause secondary injury which is highly related with oxidative stress and will lead to chronic neurodegenerative diseases [1, 7–9]. Further, free radicals of reactive oxygen species (ROS) like hydroxyl radical (OH^•), superoxide anion radicals(O₂^•−), hydrogen peroxide (H₂O₂) and reactive nitrogen species (RNS) such as nitric oxide radicals (NO^•) and peroxynitrite (ONOO⁻), which can oxidatively impair the biomacromolecules and play a crucial role in TBI [1, 7, 8, 10, 11]. Thus, the elimination of reactive oxygen and nitrogen species (RONS) is vital for the treatment of TBI, and antioxidative biocatalysts that can catalytically scavenge the RONS are promising in TBI treatment. Particularly, catalytic nanoclusters acting as enzymes like catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GPx) with high catalytic performance and good stability are being developed constantly and have shown great potential for the treatment and diagnosis of TBI [12–20].

To date, several nanozymatic biocatalysts were exploited for the treatment of TBI. For instance, carbogenic and gold-based nanozymes exhibited exceeding activity against oxidative stress and neuroinflammation promising their use in TBI treatment [13, 21–24]. Nanoclusters composed of metals, metallic oxide and alloys like Pt, Pd, Cr, V and Ce possessing high enzymatic activities were developed for TBI treatment [12, 14, 15, 18, 25–27]. Especially, the ceria nanozymes exhibited enzymatic activities like SOD and CAT on scavenging RONS via redox cycle between Ce⁴⁺ and Ce³⁺ [12, 18, 25, 28]. Gold nanoclusters can simulate SOD, CAT and GPx enzymes displaying compelling effects in TBI treatment with superb antioxidant capacities [29–33]. Besides, diagnosis and monitoring methods based on nanotechnologies especially activable nanoprobes are being developed for TBI [34–36], and technologies like atom engineering, surface modification, and size modulation were employed to promote the activity and utilization of nanozymatic biocatalysts [30–32, 37–44]. To achieve rapid and painless treatment, noninvasive diagnosis and stimulation for TBI are worth considering to promote recovery and minimize disability [45, 46]. At present, multiple noninvasive methods for TBI modulation were developed and noninvasive administration of therapeutic nanozyme shows a promising prospect [12, 15, 47].

Herein, we investigated the catalytic activity and therapeutic outcome of gold nanoclusters on TBI with noninvasive intervention. Electrochemical assay unraveled their high catalytic activities toward hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), and further revealed their excellent activity toward the reduction of O₂ and H₂O₂. The good activity of the gold nanoclusters on ROS scavenging in vitro and in vivo was observed. Furthermore, noninvasive administration of the gold nanoclusters distinctly improved the wound healing on TBI mice and Morris water maze tests further confirmed significant recovery on learning ability and spatial memory with Au₂₄Cu₁ and Au₂₄Cd₁ treatment. Ex vivo assay further confirmed the ability of gold nanoclusters on mitigating oxidative stress and inhibiting neuroinflammation. Together with the results of acceptable biocompatibility, gold nanoclusters showed promising potential as noninvasive therapeutics against TBI.

Figure 1a illustrated the effects of MPA-protected Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters in brain trauma via catalytic systems. TEM images illustrated the homogenous distribution of the nanoclusters at about 2 nm (Fig. 1b, 1c), similar with L-NIBC-coated gold nanoclusters [48]. The hydrodynamic sizes for Au₂₅, Au₂₄Cd₁ and Au₂₄Cu₁ determined by dynamic light scattering (DLS) were 1.98, 1.92 and 2.58 nm respectively, which was larger than the statistical diameter of TEM. The size changed negligibly after incubation in water for 24 and 48 h, revealing the favorable stability of the nanoclusters (Fig. 1d, 1e). In addition, the zeta potentials of all nanoclusters were around − 35 mV (Fig. 1f), consistent with our previous work [29, 49]. These results indicate the ultrasmall size and good colloid stability of nanoclusters, showing the potential in biological applications.

To evaluate the electrocatalytic activities of the Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters, we employed a standard three-electrode system to conduct the electrocatalytic HER and OER activities toward HER and OER [50–52]. Figure 2a shows cyclic voltammetry (CV) curves of glassy carbon (GC) electrodes modified with as prepared Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters. Compared with a blank GC electrode, nanoclusters achieve larger negative current density toward HER (Fig. 2a), which were further verified by linear sweep voltammetry (LSV) measurement (Fig. 2b). In comparison, the current density of Au₂₄Cu₁ nanoclusters at the potential of − 0.4585 V as described in histograms (Fig. 2c) demonstrated a higher efficiency in catalysis, showing superiority on HER activity. OER shows similar results with HER that all nanoclusters improved the performance of the electrochemical reaction (Fig. 2d-2f). However, the Au₂₄Cd₁ nanocluster was more efficient than the Au₂₄Cu₁ nanocluster toward OER that the Au₂₄Cd₁ haves the lowest current onset on CV curves at 1.3 V, followed by Au₂₄Cu₁ and Au₂₅, which is a little different from the results of HER.

We also evaluated the in vitro catalytic activities of Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters in H₂O₂ reduction and oxygen reduction reaction (ORR) at the scan rate of 10 mV s^− 1 [53, 54]. Figure 3a demonstrated that all nanoclusters could enhance the electrocatalytic activity for reduction of O₂ while Au₂₄Cu₁ nanoclusters show the current density of -1.48 mA/cm^− 2 at the potential of -0.8 V, indicating the best activity of Au₂₄Cu₁ nanoclusters for ORR among all nanoclusters. The results were further verified by LSV measurement (Fig. 3b) and the current density of nanoclusters at -0.8 V was compared in Fig. 3c. For the reduction of H₂O₂, only imperceptible reduction current was observed in the presence of H₂O₂ on the GC electrode, consistent with previous works [53]. Compared with the GC electrode at -0.8 V, Au₂₄Cu₁ nanoclusters showed the current of -1.74 mA/cm^− 2, while Au₂₄Cd₁ nanoclusters showed the current of -1.56 mA/cm^− 2 (Fig. 3d, 3e), indicating excellent catalytic activities toward reduction of H₂O₂. Figure 3c and 3f demonstrated the improvement of all nanoclusters on O₂ and H₂O₂ reduction. Au₂₄Cu₁ showed the predominance toward both reactions, and Au₂₄Cd₁ and Au₂₅ followed closely toward H₂O₂ and O₂ reduction respectively. The electrochemical assessments of nanoclusters substantiated their remarkable in vitro catalytic activities in H₂O₂ and O₂ reduction reactions, inspiring us to investigate their biological responses in cells and brain-injured mice.

As the nanoclusters exhibited extraordinary catalytic activities which set the foundation for their use in ROS-related diseases, we conducted a biological inspection to evaluate their activities in vitro and in vivo. With the combination of our previous work [29], we concluded that the Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters exhibited selectivity on ROS and RNS and showed selective SOD-like and CAT-like activities, which can reduce the oxidative stress induced by brain injury and perform the therapeutic effect (Fig. 4a). Additionally, the Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters presented favorable biocompatibility and metabolic properties, which benefits their use in vivo [29].

To investigate the biological activity of nanoclusters, imaging of H₂O₂-treated neuron cells was performed with or without Au₂₅, Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters (Fig. 4b). The H₂O₂ significantly elevated the signals, indicating the presence of the excessive amount of ROS [55], especially O₂^•−. All clusterzymes can decrease the O₂^•− signals, while Au₂₄Cd₁ shows the best clearance efficiency. Meanwhile, The H₂O₂ treatment enormously increases the ROS signal, and Au₂₄Cu₁ displays better clearance capability than Au₂₅ and Au₂₄Cd₁, suggesting higher selectivity for ROS. The relative quantification of oxidative stress toward fluorescent images was shown in Fig. 4d and 4e, further confirming the remarkable biological catalytic activity and laying the groundwork for in vivo utilization. In addition, H₂O₂ induces decreases in cell viability (∼78%) due to oxidative stress and inflammation [56], while Au₂₅, Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters can rescue the decreases induced by H₂O₂ back to 90%, indicative of the good potential of nanoclusters to rescue the lethality induced by H₂O₂ (Fig. 4c). All the in vitro results manifest good catalytic activities of Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters on ROS scavenging, revealing their potential as biocatalysts and suggesting further investigation on animal models.

To investigate the usability of the nanoclusters against TBI, we performed craniotomy and Morris water maze tests as well as the assessment of oxidative stress and inflammation level in brain tissues of TBI mice. Figure 5a and 5b show the wound healing processes over time of TBI mice with or without treatment of nanoclusters. Compared with the TBI group, all nanoclusters promoted the healing progress among which the Au₂₄Cd₁ presented the best healing effect followed by Au₂₄Cu₁, demonstrating the therapeutic effect of the nanoclusters especially the Au₂₄Cd₁ and Au₂₄Cu₁. In addition, we evaluated the oxidative stress-related indicators, including SOD, GSH/GSSG, MDA, and H₂O₂ in TBI mice after nanoclusters treatment [33, 57].TBI can consume lots of SOD and GSH, leading to the decrease of SOD and GSH activity and the increase of MDA and H₂O₂ amount (Fig. 5c-f). The presence of over-excessive MDA H₂O₂ and GSSG indicates severe oxidative stress in TBI mice. After nanoclusters treatment, the activity of SOD and GSH/GSSG is increased while that of the oxidative-stress related byproducts MDA and H₂O₂ is decreased. Therefore, nanoclusters can intervene in TBI treatment by reducing oxidative stress.

The behavior tests were conducted to evaluate the spatial learning and memory abilities by Morris water maze (Fig. 6). Figure 6b showed the path of the mice searching the platform which presented the significant difference between the mice with and without administration of Au₂₄Cu₁ or Au₂₄Cd₁. Mice in the control group could quickly locate and land on the platform, while the mice in the TBI group could not locate the exact position of the platform in a short time. The mice with noninvasive administration of gold nanoclusters could find the platform after a short period and dopped gold clusters revealed better results than Au₂₅, indicating the enhancement of efficiency by Cu and Cd single-atom substitution. During 13–17 and 26–30 days after treatment, compared with the TBI group, both the total distance and the latency to the target site were reduced significantly after Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters treatment (Fig. 6c, 6d), suggesting that nanoclusters could improve the learning ability of injured mice. On days 17 and 30, the number of platform crossings was significantly increased close to the result of the control group and the average distance to the platform was reduced significantly in the groups of Au₂₄Cu₁ and Au₂₄Cd₁. All the indicators of the behavioral test demonstrated that the neuronal cognition of mice treated with Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters is improved, and the spatial learning and memory abilities of treated mice are close to healthy mice, further confirming that nanoclusters could improve the spatial learning and memory abilities.

The effects of nanoclusters on neuroinflammation were examined for further verification of the therapeutic effect. Figure 7a illustrated the TBI-associated inflammatory responses and modulation of the nanoclusters [1, 8]. The IL-1β, IL-6 and TNF-α were upregulated by TBI, revealing strong local inflammation. Au₂₄Cd₁ exhibited exceptional efficiency to downregulate all three inflammatory factors, indicating its strong anti-neuroinflammation effect in vivo. Au₂₄Cu₁ can significantly downregulate the overexpression of TNF-α, presenting superior efficacy over Au₂₄Cd₁. In comparison, Au₂₅ only showed minor downregulation toward the three inflammatory factors, as manifested in the catalytic activity induced by substitution of Cu and Cd (Fig. 7b, 7c). In addition, the ELISA further verified the immunoblot results that Au₂₄Cd₁ and Au₂₄Cu₁ are superior to Au₂₅ on inhibiting the inflammatory factors in brain tissues including IL-1β, IL-6, and TNF-α (Fig. 7d-7f). Figure 7g quantitatively analyzed the positive cell numbers in injured cortex treated with H₂O₂ with or without nanocluster and presented the significant anti-inflammation effect of Au₂₄Cd₁ and Au₂₄Cu₁. Additionally, the immunohistochemical assay illustrated the conspicuous effect of Au₂₄Cd₁ and Au₂₄Cu₁ against IL-6 (Fig. 7h). Furthermore, significant differences on the pathological slices can be seen that the TBI group exhibited swelling of nerve cells and obvious infiltration of inflammatory cells; while the groups treated with nanoclusters especially the Au₂₄Cd₁ and Au₂₄Cu₁ recovered to almost normal (Fig. 7i). These results demonstrated that the nanoclusters especially Au₂₄Cd₁ and Au₂₄Cu₁ can accelerate the wound healing of traumatically injured brain by inhibiting the inflammatory factors.

Microglia and astrocytes are the key cellular mediators of TBI and mice after TBI can activate microglia and astrocytes [1]. As shown in Fig. 8a, lots of astrocytes are produced and activated after TBI along with inflammatory cytokines, indicating strong local inflammation. However, Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters can remarkably decrease the overexpression of IL-1β and IL-6, in addition to inhibiting astrocyte activation. The relative quantification further exhibited the significant effect of Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters (Fig. 8b). Au₂₄Cd₁ can alleviate IL-1β and IL-6 associated immune response, while Au₂₄Cu₁ has a better effect on reducing TNF-α, indicating their catalytic selectivity and potential selectivity towards the treatment of neuroinflammation. The ex vivo investigation further verified the catalytic activity of Au₂₄Cd₁ and Au₂₄Cu₁ against neuroinflammation and manifested the significant effect of promoting the healing of TBI.

The present work demonstrated the high catalytic activity of Au₂₄Cd₁ and Au₂₄Cu₁ nanoclusters and their availability as TBI therapeutics. More importantly, our investigation revealed that noninvasive administration is a potent strategy for TBI treatment since it can directly and rapidly release the biocatalysts into the damaged area, eliminating the RNOS and inhibiting subsequent neuroinflammation [15, 29, 58, 59]. Necessarily, exploiting new biocatalysts with high catalytic activities and favorable biocompatibility and developing new delivery modes for the biocatalysts for the noninvasive intervention of TBI will be a constant challenge in this field [15, 45]. As more biocatalysts with distinguished catalytic activities against oxidative stress are being developed [20, 60], there will be more potential therapeutics in the use of noninvasive intervention against TBI.

We reported that noninvasive early intervention of gold nanoclusters gave a better outcome for neurotrauma and should be a powerful strategy for the cure of TBI. The nanoclusters we reported here showed high catalytic activities toward water splitting and reduction of O₂ and H₂O₂, which animated their use in oxidative stress-related diseases especially TBI. By employing the catalytical nanoclusters, we successfully demonstrated the importance of oxidative stress in the genesis and development of TBI and the exceptional effect of early intervention using enzymatic biocatalysts for the treatment. The biological results found that Au₂₄Cd₁ preferentially decreases IL-1β and IL-6, while Au₂₄Cu₁ shows the tendency to decrease TNF-α, indicating their different selectivity for alleviating neuroinflammation. Animal models illustrated their favorable effects against TBI after noninvasive administration. In conclusion, our work showed the great potential of noninvasive early intervention for the treatment of TBI with enzymatic biocatalysts.

Materials and reagents

All chemicals and reagents were purchased from commercial sources and used without further purification. Gold chloride (HAuCl₄·3H₂O) was purchased from Sigma-Aldrich. Copper nitrate (Cu(NO₃)₂), Cadmium nitrate (Cd(NO₃)₂), 3-Mercaptopropionic acid (MPA), Sodium borohydride (NaBH₄), Sodium hydroxide (NaOH), were purchased from Aladdin. Ultrapure water (18.2 MΩ*cm) was used for all the experiments. Kits and fluorescent probes were purchased from commercial sources and used as per the instructions.

Materials preparation, characterization and catalytic activities

The MPA-protected Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters were prepared as per the previous report [29, 61, 62]. Briefly, HAuCl₄ (aqueous, 20 mM, 0.25 mL) and MPA (aqueous, 5 mM, 2 mL) were added to water (2.35 mL) and stirred for 5 min at room temperature. Then, NaOH solution (aqueous, 1 M, 0.3 mL) was added to the reaction mixture, followed by the addition of 0.1 mL of NaBH₄ (43 mg of NaBH₄ powder in 10 mL of 0.2 M NaOH solution). Au₂₅MPA₁₈ was collected after the final reaction mixture stirred at room temperature for 3 hours in the dark and aged at 4 ⁰C for 12 hours. The Au₂₄Cu₁ and Au₂₄Cd₁ were synthesized based on the same method, except the Au atoms in HAuCl₄ (20 mM, 0.25 mL) were replaced by various nitrate metal ions (Cu²⁺, Cd²⁺) at a 4% molar ratio. Ultrafiltration tubes of 3 K and 10 K at 3500 rpm/min were used for ultrafiltration to remove smaller organic ligands and larger-sized clusters, and lyophilization was used to collect the purified product for further test and investigation.

A JEM-2100F electron microscope (JEOL, Japan) was employed to acquire transmission electron microscopic (TEM) images. A Malvern Zetasizer Nano ZS90 (UK) was employed to measure dynamic light scattering (DLS) to test the hydrodynamic size and determine the zeta potential of nanoclusters.

The nanoclusters were deposited onto the surface of glassy carbon (GC) electrodes for electrochemical assay [14, 42]. Briefly, as-prepared MPA-protected Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ nanoclusters (20 µL, 0.5 mg/mL) were deposited onto the surface of GC electrodes and dried naturally, then Nafion solution (3 µL) was dropped onto the surface of GC electrodes and dried, the modified electrodes were used for catalytic activity tests.

Cyclic Voltammetry (CV) and Linear Sweep Voltammetry (LSV) measurements were taken (scan rate: HER, 10 mV s^− 1; OER, 100 mV s^− 1 electrochemical analyzer, CHI660D, Shanghai) to evaluate the performance of the nanoclusters modified electrodes for HER and OER. HER was carried out in 0.5 M H₂SO₄, and a graphite rod and a saturated calomel electrode were used as the counter electrode and reference electrode, respectively. OER was carried out in 1 M KOH, and a platinum wire and an Ag/AgCl electrode were used as the counter electrode and reference electrode, respectively.

CV and LSV measurements were taken (scan rate, 50 mV s^− 1, electrochemical analyzer, CHI660D, Shanghai) to evaluate the catalytic activities for O₂ and H₂O₂ reduction. A three-electrode cell was adopted for both O₂ and H₂O₂ reduction, a platinum wire and a saturated calomel electrode were used as the counter electrode and reference electrode, respectively. Oxygen reduction reaction (ORR) was carried out in O₂-saturated in 0.01 M PBS (pH 7.4), and the H₂O₂ reduction was performed in the presence of 9.8 mM H₂O₂ in N₂-saturated 0.01 M PBS (pH 7.4).

In vitro evaluation of the catalytic activities

Mouse hippocampal neuronal cell line (HT22) was used in all the cellular experiments. Cells were seeded into the 96-well plates and grew in Dulbecco's modified Eagle's medium (DMEM) at 37°C with 5% CO₂. After being stimulated with 100 µM H₂O₂ for 6 hours, the culture medium was replaced by Au₂₅, Au₂₄Cu₁ and Au₂₄Cd₁ dissolved in the DMEM at different doses, and then cells were incubated for another 24 hours. Cell survival was analyzed using MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide, Beyotime). HT22 cells (1 x 10⁵ cells per well) were seeded in 6-well plates for 12 hours and stimulated with 100 µM H₂O₂ for 6 hours before being treated with the nanoclusters (12 ng/µL). Fluorescent staining was carried out to evaluate the intracellular oxidative stress levels with different probes such as DHE for O₂^•− and DCFH-DA for ROS, and cell images were captured by a fluorescence microscope.

In vivo treatment and behavioral experiment

TBI models: C57BL/6 mice at 21–23 g were employed to establish TBI models using an electromagnetically CCI injury device (eCCI-6.3, Custom Design & Fabrication, Inc), with an impactor of 5 m/s velocity, 0.61 mm depth, 150 ms duration, and 20º angle of dura mater on the vertical axis. The mice were divided into control, TBI, TBI + Au₂₅, TBI + Au₂₄Cu₁, and TBI + Au₂₄Cd₁ group (n = 15) randomly. All mice were anesthetized with 10 % chloral hydrate (10 mg/kg) and the scalp was cut before placed on the stereotaxic frame. The craniotomy was carried out by drilling the skull in a circle of 2 mm in diameter. The scalp was sewn together carefully, and the nanoclusters were added to the wound of TBI mice at a concentration of 50 mg/kg. The healing process was recorded photographically and the wound remaining was calculated after treatment.

Oxidative stress level: Brain tissues were taken out on days 7 and 14 post-treatment, then homogenized in 0.9 % physiological saline and analyzed for SOD, GSH/GSSG, MDA, and H₂O₂ using commercially available kits. All testing methods are carried out as per the instructions (Beyotime).

Morris water maze tests: Morris water maze (MWM) was conducted on days 13–17 and 26–30 post-treatment as described in the previously reported literature [12, 63, 64]. Briefly, the water maze was divided into four quadrants, and the platform was set in the center of quadrant I. Before spatial learning, visual discrimination learning was performed to determine whether the vision of mice was normal. In this procedure, each animal performed one trial where the platform was placed above the water to determine whether the vision of mice was normal. Animal with the visual problem would be excluded in the Morris water maze test. Each mouse was put into the pool to be trained and learned to search for the platform under the water in the order of quadrant II, III, IV and I with an inter-trial interval (ITI) of 60 min at almost the same time of each day for five days. The test was carried out without the platform on the fifth day, and each mouse was put into the pool at quadrant II and allowed for 60 seconds to track them.

Ex vivo verification

Western Blotting: The total protein in the brain tissue was extracted and the content of IL-1β, IL-6 and TNF-α was analyzed. SDS-PAGE electrophoresis was performed before transferring to the membrane. Immune responses of the specific antibodies were carried out and the images were captured with autoradiography. All antibodies were purchased from Abcam.

ELISA analysis: Inflammatory cytokines including IL-6, IL-1β and TNF-α were determined by ELISA kits (Abcam, ab100712, ab197742, ab208348, respectively), and the assays were performed as per the instructions provided by the manufacture.

Tissue staining: Brain tissues were taken out at 30 days post-injury and fixed in 4% paraformaldehyde and embedded in paraffin. Immunofluorescent staining was performed with primary antibodies including anti-GFAP, IL6, IL1β antibodies as per the instructions of (Abcam). Then the slices were incubated with Alexa Fluor 488/594-conjugated goat secondary antibody for 1-1.5 hours at room temperature under dark and counterstained with DAPI. Immunohistochemical staining for IL-6 and mice brain tissue was performed according to the instructions (Proteintech).

TBI: Traumatic brain injury; ROS:reactive oxygen species; OH^•:hydroxyl radical; O₂^•−:superoxide anion radicals; H₂O₂:hydrogen peroxide; RNS:reactive nitrogen species; NO^•:nitric oxide radicals; ONOO⁻:peroxynitrite; RONS:reactive oxygen and nitrogen species; CAT:catalase; SOD:superoxide dismutase; POD:peroxidase; GPx:glutathione peroxidase; HER:hydrogen evolution reaction; OER:oxygen evolution reaction; DLS:dynamic light scattering; GC:glassy carbon; CV:Cyclic Voltammetry; LSV:Linear Sweep Voltammetry; ORR:Oxygen reduction reaction.

Ethics approval and consent to participate

We have obtained approval of animal ethics and use committee (IRM-DWLL-2019099). All animal procedure was followed the guideline approved by the Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College.

Consent for publication

All authors gave their consent for publication.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request

Competing interests

The authors declare no conflict of interest.

Funding

This work was financially supported by the NSFC (Grant No. 91859101, 81971744, U1932107, and 81471786), the National Natural Science Foundation of Tianjin (19JCZDJC34000), the Foundation of ‘Peiyang Young Researcher’ Program of Tianjin University, and CAS Interdisciplinary Innovation Team (JCTD-2020-08).

Authors’ contributions

ZX, WH, and XJ originated the concept. ZY and SS wrote the manuscript. SS and LH worked on materials synthesis and characterizations. RQ, HW, and XQ worked on biological experiments. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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Catalytically Active Gold Clusters with Atomic Precision for Noninvasive Early Intervention of Neurotrauma

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