Anti-inflammatory and anti-oxidative stress nano-enzyme Cu5.4O-coated CNDs with cascade enzyme activity and application of nano-enzyme Cu5.4O-coated CNDs

Abstract

The invention belongs to the technical field of hepatopathy treatment drugs, and particularly relates to an anti-inflammatory and anti-oxidative stress nano-enzyme Cu5.4O atCNDs with cascade enzyme activity and application of the nano-enzyme Cu5.4O atCNDs. The nano-enzyme Cu5.4O-coated CNDs is prepared by the following steps: dissolving CuCl2 in a carbon dot aqueous solution, reacting for 10-12 minutes at the temperature of 75-80 DEG C, then adding an L-ascorbic acid aqueous solution, regulating the pH value to 7.0-8.0 by using a NaOH solution, reacting for 10-12 hours at the temperature of 75-80 DEG C, after the reaction is finished, centrifuging to remove precipitates, dialyzing supernate, and freeze-drying to obtain the Cu5.4O-coated CNDs. Research results of the invention prove that the nano-enzyme Cu5.4O-coated CNDs has the potential of treating various acute liver injury diseases, and is an intervention strategy with a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs for treating liver diseases, and particularly relates to an anti-inflammatory and antioxidant stress nanozyme Cu 5.4 O@CNDs with cascade enzyme activity and its applications. Background Art

[0002] Acute liver failure (ALF) is a clinical syndrome characterized by extensive hepatocyte necrosis, hepatic hypoplasia, and multiple organ dysfunction. The clinical symptoms of ALF usually include liver dysfunction, abnormal liver biochemical indexes, and coagulation dysfunction. Although the incidence is relatively low, up to half of the cases may develop multiple organ failure and death, with a mortality rate as high as 30%. Therefore, there is an urgent need to develop effective treatment strategies for the causes of acute liver failure.

[0003] The causes of ALF include hepatic ischemia-reperfusion injury (HIRI), viral infection, autoimmune hepatitis, and various other acute liver injury (ALI) inducing factors. Several recent studies have shown that high levels of oxidative stress and inflammatory responses in the body play a crucial role in all types of liver injury, among which reactive oxygen species (ROS) are of great importance. Hepatocytes contain a large number of mitochondria. When hepatotoxic compounds and their active metabolites, as well as other factors (such as hypoxia and reoxidation), disrupt the electron transport chain in the mitochondrial membrane, excessive ROS are generated. Due to its strong oxidative properties, ROS is an important inflammatory mediator and can cause cell damage at high concentrations. The main reactive oxygen species mainly include superoxide anion radical (·O2 -)), hydroxyl radicals (·OH) and hydrogen peroxide (H2O2). In addition, liver injury triggers inflammation, and subsequently ROS are mainly derived from activated inflammatory cells and liver sinusoidal endothelial cells (LSECs), further disrupting hepatic redox homeostasis and forming a vicious cycle of continuous stimulation. The liver is the main organ for metabolism and detoxification. Multiple enzymes with overlapping substrate specificities are expressed in the liver and are generally classified as phase I (oxidative) and phase II (conjugative) drug-metabolizing enzymes (DMEs). Approximately 90% of phase I metabolism is carried out by enzymes belonging to the cytochrome P450 (CYP) superfamily. In addition, hepatic stellate cells (HSCs) store 50%-95% of the body's vitamin A, which consists of a series of retinol compounds, including retinol, retinoic acid, and retinal. CYP450 enzymes play a crucial role in retinol metabolism, mainly involving the CYP1, CYP2C, CYP3A, and CYP26 families. Current studies have shown that the regulation of retinol metabolic homeostasis is a decisive feature of hepatic stellate cells (HSCs) in healthy and injured livers. Therefore, scavenging excessive ROS to alleviate oxidative stress, while regulating the inflammatory response, blocking continuous stimulation, and maintaining the stability of retinol metabolism, will become an effective therapeutic target for acute liver injury.

[0004] As is well known, there is a powerful antioxidant enzyme system in cells that can scavenge reactive oxygen species (ROS), such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). However, due to its lack of targeting, this system cannot effectively solve liver injury. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an anti-inflammatory and antioxidant stress nanozyme Cu 5.4 O@CNDs with cascade enzyme activity and its applications.

[0006] In a first aspect, the present invention provides an anti-inflammatory and antioxidant stress nanozyme Cu 5.4 O@CNDs with cascade enzyme activity, which is prepared by the following steps:

[0007] Dissolve CuCl2 in an aqueous solution of carbon dots, react at 75 °C to 80 °C for 10 min to 12 min, then add an aqueous solution of L-ascorbic acid, adjust the pH to 7.0 - 8.0 with NaOH solution, and react at 75 °C to 80 °C for 10 h to 12 h. In the reaction system: the concentration of CuCl2 is 15 - 20 mM, the concentration of carbon dots is 3 - 4 mg / mL, and the concentration of ascorbic acid is 300 - 400 mM. After the reaction is completed, remove the precipitate by centrifugation, and the supernatant is dialyzed and freeze-dried to obtain the anti-inflammatory and antioxidant stress nanozyme Cu 5.4 O@CNDs with cascade enzyme activity.

[0008] The nanozyme Cu of the present invention 5.4 O@CNDs has SOD and CAT enzyme activities and ·OH free radical scavenging ability, and eliminates ROS in vitro. In addition, Cu 5.4 O@CNDs shows good targeting ability in both H2O2-induced inflammatory cells and liver tissues of hepatitis mice, and has good biocompatibility in vitro and in vivo. In addition, research shows that Cu 5.4 O@CNDs improves the disruption of hepatic retinol metabolism by regulating the ROS response and liver inflammation network, effectively scavenges overloaded ROS, increases the survival rate of cells in vitro, and has an effective therapeutic effect in HIRI and LPS-ALI mouse models.

[0009] Further, the carbon dots are prepared by the following steps:

[0010] Adding activated carbon to the boiling mixed acid solution, maintaining the boiling point for 1.5 h to 2 h, cooling to room temperature, neutralizing with NaHCO3 solution, and filtering, dialyzing, concentrating, and freeze-drying the neutralized solution to obtain the carbon dots, where: the mixed acid solution is prepared by mixing equal volumes of HNO3 solution with a concentration of 8 - 10 mol / L and H2SO4 solution with a concentration of 18 - 20 mol / L, and the mass-volume ratio of activated carbon to the mixed acid solution is 0.5 g - 0.6 g:50 mL.

[0011] In a second aspect, the present invention provides the use of the nanozyme Cu 5.4 O@CNDs in the preparation of a drug for treating liver injury.

[0012] Further, the liver injury includes hepatic ischemia-reperfusion injury and acute liver injury.

[0013] Further, the drug contains the nanozyme Cu 5.4 O@CNDs, a drug carrier, and / or a drug excipient.

[0014] In a third aspect, the present invention provides a drug for treating liver injury, the drug comprising the nanozyme Cu 5.4 O@CNDs.

[0015] Further, the drug further includes a drug carrier and / or a drug excipient.

[0016] Even further, the drug carrier includes microcapsules, microspheres, nanoparticles, and / or liposomes.

[0017] Even further, the pharmaceutical excipient includes a filler, a binder, a wetting agent, a disintegrant, a lubricant, and / or a flavoring agent.

[0018] Further, the dosage form of the drug is powder, granule, capsule, tablet, pill, injection or oral liquid.

[0019] Beneficial effects:

[0020] The present invention synthesizes a nanozyme Cu 5.4 O@CNDs with cascade mimetic enzyme activity, which can protect the liver from ROS-mediated stress and inflammatory responses and improve vitamin A alcohol metabolic disorders, thereby achieving an effective therapeutic effect. Research shows that Cu 5.4 O@CNDs can improve the cell status and survival rate of THLE-2 and RAW264.7 cells under in vitro oxidative stress and inflammatory conditions. In the HIRI and LPS-induced ALI models, Cu 5.4 O@CNDs effectively scavenge ROS, reduce the expression of inflammatory cytokines, and show a significant therapeutic effect. To clarify the potential therapeutic mechanism, transcriptome sequencing results show that Cu 5.4 O@CNDs play a hepatoprotective role by regulating the ROS response and liver inflammation network, promoting the disruption of the hepatic retinol metabolic pathway, and inhibiting apoptosis. These findings confirm the potential of Cu 5.4 O@CNDs in treating various acute liver injury diseases and provide a promising intervention strategy for clinical applications. Description of the drawings

[0021] Figure 1 For the synthesis and characterization of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs, where: 5.4 O@CNDs, in which:

[0022] A is the synthesis route diagram of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs.

[0023] B is the TEM image of C-dots.

[0024] C is the TEM image of Cu 5.4 O USNPs.

[0025] D is the TEM image of Cu 5.4 O@CNDs.

[0026] E is the Zeta potential of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs.

[0027] F is C-dots, Cu 5.4 O USNPs and Cu5.4 XRD pattern of O@CNDs

[0028] G is for C-dots, Cu 5.4 O USNPs and Cu 5.4 FTIR spectrum of O@CNDs.

[0029] H is for C-dots, Cu 5.4 O USNPs and Cu 5.4 XPS graph of O@CNDs.

[0030] I is the peak fitting curve of C-1s of C-dots.

[0031] J is for Cu 5.4 Peak fitting curve of C-1s of O@CNDs.

[0032] K is for Cu 5.4 Peak fitting curve of cu2p of O USNPs.

[0033] L is for Cu 5.4 Peak fitting curve of cu2p of O@CNDs.

[0034] Figure 2 is for Cu 5.4 O USNPs, Cu 5.4 Different synthesis conditions of O@CNDs, where:

[0035] A is for different Cu 2+ : CuO USNPs synthesized under different L-AA feeding ratios (1:5, 1:10, 1:20, 1:40, 1:60) 5.4 Dissolved oxygen measurement results of the hydrogen peroxide scavenging ability of CuO USNPs.

[0036] B is for CuO@CNDs synthesized under different C-dots feeding ratios (1 - 4 mg / mL) 5.4 Dissolved oxygen measurement results of the hydrogen peroxide scavenging ability of CuO@CNDs.

[0037] C is for CuO@CNDs synthesized under different pH values (pH 6 - 7, 7 - 8, 8 - 9, 9 - 10) 5.4 Dissolved oxygen measurement results of the hydrogen peroxide scavenging ability of CuO@CNDs.

[0038] D is for different Cu 2+ concentrations for the synthesis of CuO@CNDs 5.4 Dissolved oxygen measurement of the hydrogen peroxide scavenging ability of CuO@CNDs.

[0039] Figure 3 is for C-dots, Cu 5.4 O USNPs and Cu 5.4Characterization of O@CNDs, where:

[0040] A is the particle size statistics of C-dots.

[0041] B is the particle size statistics of Cu 5.4 O USNPs.

[0042] C is the particle size statistics of Cu 5.4 O@CNDs.

[0043] Figure 4 is for the particle size statistics of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs for the test of peroxidase-like activity, where:

[0044] A is the schematic diagram of the scavenging of ·O2 - by the SOD-like enzyme.

[0045] B is the scavenging ability of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs for ·O2 - scavenging.

[0046] C is the evaluation of the SOD enzyme activity of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs by the WST-1 kit.

[0047] D is the determination of the ability of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs to scavenge ·O2 - - by the ESR method.

[0048] E is the schematic diagram of the scavenging of H2O2 by the CAT-like enzyme.

[0049] F is the ultraviolet absorption test of the ability of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs to scavenge H2O2.

[0050] G is the detection of the dissolved oxygen level of the scavenging of H2O2 by C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs.

[0051] H is the determination of the ability of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs to scavenge H2O2 by the ESR method.

[0052] I is the schematic diagram of the scavenging of ·OH and ABTS free radicals.

[0053] J is for C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs' ability to scavenge ·OH.

[0054] K is for C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs' ABTS radical scavenging ability.

[0055] Figure 5 is for Cu 5.4 O@CNDs' biocompatibility, where:

[0056] A is for Cu 5.4 O@CNDs incubated for 24 h, THLE-2 cell viability.

[0057] B is for Cu 5.4 O@CNDs incubated for 48 h, THLE-2 cell viability.

[0058] C is the hemolysis rate of each group.

[0059] D is for Cu 5.4 O@CNDs-treated normal mice, body weight change after 7 days.

[0060] E is the schematic diagram of the biocompatibility experiment.

[0061] F is for Cu 5.4 O@CNDs after intravenous administration, in vivo toxicity evaluation of major organs (heart, liver, spleen, lung, spleen) at 1 day and 7 days.

[0062] G is the serum liver function index and serum renal function index levels.

[0063] H is for normal mice (control group) and mice intravenously injected with Cu 5.4 O@CNDs, blood routine parameters. Figure 6 is for Cu 5.4 O@CNDs' in vivo therapeutic effects on HIRI and LPS-ALI mice, where:

[0064] A is the schematic diagram of the establishment and treatment plan of HIRI mice.

[0065] B is the liver tissue image.

[0066] C is the H&E staining of liver tissue.

[0067] D is the TUNEL, DCFH-DA, DHE staining of liver tissue.

[0068] E represents the levels of serum ALT and AST in HIRI mice 24 hours after different treatments.

[0069] F represents the relative expression of mRNA of cytokines IL-1β, IL-6, IL-12, and TNF-α.

[0070] G is a schematic diagram of the establishment and treatment plan of LPS-ALI mice.

[0071] H is the H&E staining of liver tissue.

[0072] I is the TUNEL, DCFH-DA, and DHE staining of liver tissue.

[0073] J represents the levels of serum ALT and AST in LPS-ALI mice 12 hours after different treatments.

[0074] K represents the relative expression of mRNA of cytokines IL-1β, IL-6, IL-12, and TNF-α. Specific implementation manners

[0075] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0076] Materials and instruments involved in the following embodiments:

[0077] Materials:

[0078] Activated carbon was purchased from Aladdin Chemical Reagent Co., Ltd. Sodium bicarbonate (NaHCO3) was acquired from Tianli Enterprise Group Co., Ltd. Sulfuric acid (H2SO4) and nitric acid (HNO3, 65% - 68%) were purchased from local suppliers. Copper chloride (CuCl2) was bought from Macklin Co., Ltd. in Shanghai, China. L-ascorbic acid (L-AA) and sodium hydroxide (NaOH) were from Sigma Aldrich Co., Ltd. in Shanghai, China. Ferrous chloride (FeCl2·4H2O) was purchased from Tianjin Bellen Biotechnology Co., Ltd. 3,3',5,5'-Tetramethylbenzidine (TMB) was bought from Shanghai Marlin Biochemical Technology Co., Ltd. Nitroblue tetrazolium chloride (NBT), L-methionine (L-met), and riboflavin were obtained from Sigma Aldrich. Hydrogen peroxide (H2O2, 30%) was purchased from Tianjin Daxiong Chemical Reagent Co., and the superoxide dismutase assay kit (S311) was bought from Dojindo Molecular Technologies Co., Ltd. The hydrogen peroxide assay kit, ABTS method total antioxidant capacity assay kit, 2′,7′-dichlorodihydrofluorescein (DCFH-DA), dihydroethidium (DHE), and animal RNA isolation kit were purchased from Beyotime Chemical Reagent Co., Ltd. 4′,6-Diamidino-2-phenylindole (DAPI) was bought from Roche Applied Science. All chemical reagents were used directly without further purification, and the detection kits were used according to the manufacturer's instructions.

[0079] Instruments:

[0080] Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai G2 F30 (FEI, USA) at an acceleration voltage of 300 kV. Powder X-ray diffraction (XRD) data were collected using a Bruker D8 ADVANCE (Germany) with a scanning rate of 6° / min. FT-IR spectra were recorded by a Thermo Fisher Nicolet 5700 (USA). X-ray photoelectron spectroscopy (XPS) was recorded using a Thermo Escalab 250Xi (USA). Electron spin resonance (ESR) spectra were recorded using a Bruker A300 - 9.5 / 12 (Switzerland) at room temperature. Flow cytometry data were collected by a FACS CaliburTM, Becton Dickinson (USA). Microplate absorbance was measured using a Tecan Spark 20m multimode microplate reader (Switzerland). Fluorescence imaging was performed using an in vivo imaging system (IVIS Lumina 3, PE, USA), and images were acquired using IVIS Living Image 3.0 software (PerkinElmer, USA). Tissue sections were imaged using a Leica DM3000 microscope (Leica, Wetzlar, Germany).

[0081] Example 1: Cu 5.4 Synthesis and Characterization of CuO@CNDs

[0082] 1. Experimental Method

[0083] (1) Synthesis of Carbon Dots (C-dots)

[0084] First, 0.5 g of activated carbon was added to a boiling solution of 50 mL of a mixed acid (equal volume mixture of 8 mol / L HNO3 solution and 18 mol / L H2SO4 solution), and the boiling point was maintained for 1.5 h. After cooling to room temperature, the sample was neutralized with NaHCO3 solution. The obtained neutralized solution was filtered through a 0.22 μm water membrane and dialyzed 4 - 5 times a day for one week. The dialyzed C-dots solution was filtered again through a 0.22 μm water membrane to remove any remaining insoluble substances. The obtained filtrate was ultrafiltered using an ultrafiltration tube with a molecular retention capacity of 100 kDa. The separated C-dots were concentrated and freeze-dried for subsequent experiments.

[0085] (2) Synthesis of CuO USNPs 5.4 10 mM of CuCl2 powder was dissolved in 50 mL of deionized water and stirred in an 80 °C magnetic stirring oil bath for 10 min. Then, an aqueous solution of L-ascorbic acid (100 mM, 50 mL) was slowly added to the above CuCl2 solution. Then, the pH of the solution was adjusted to 8.0 - 9.0 with NaOH solution (1 M) and continuously stirred at 80 °C for 12 h. After the reaction, larger aggregates were removed by centrifugation (6577×g, 15 min), and then the supernatant was dialyzed against water (Mw cut-off value: 10,000 Da) for 2 days to remove small molecules and freeze-dried for subsequent experiments.

[0086]

[0087] (3) Synthesis of CuO@CNDs 5.4

[0088] First, 15 mM of CuCl2 powder was dissolved in an aqueous solution of C-dots (3 mg / mL, 50 mL) and stirred in an 80 °C magnetic stirring oil bath for 10 min. Then, an aqueous solution of L-ascorbic acid (300 mM, 50 mL) was slowly added to the above CuCl2 solution. Then, the pH of the solution was adjusted to 7.0 - 8.0 with NaOH solution (1 M) and continuously stirred at 80 °C for 10 h. After the reaction, larger aggregates were removed by centrifugation (6577×g, 15 min). Then, the supernatant (Mw cut-off value: 3,500 Da) was dialyzed for 2 days to remove small molecules and freeze-dried for subsequent experiments and characterization.

[0089] ​​2. Experimental Results

[0090] In this example, a Cu 5.4 O@CNDs with cascade enzyme activity was synthesized. Cu 5.4 O@CNDs, C-dots, and Cu 5.4 OUSNPs were synthesized by a simple and rapid one-pot method ( Figure 1 A). To find the optimal synthesis conditions, the ratio of Cu 2+ to L-ascorbic acid (AA), the concentration of C-dots added, the pH, and the final concentration of Cu 2+ were adjusted to determine their effects on the particle size and catalytic activity of the resulting materials. The catalytic activities of the obtained Cu 5.4 O USNPs were basically the same when the feeding ratios of Cu 2+ to AA were 1:5, 1:10, 1:40, and 1:60. The best effect was obtained when the feeding ratio was 1:20; therefore, the molar ratio of Cu 2+ to AA was fixed at 1:20, the concentration of Cu 2+ was 15 mM, the concentration of C-dots was 3 mg / ml, and the pH was 7.0 - 8.0 ( Figure 2 A - D).

[0091] The transmission electron microscopy (TEM) of C-dots showed that the distribution of C-dots was uniform and monodisperse. The particle size distribution statistical chart showed that the average particle size was 2.39 ± 0.50 nm ( Figure 1 B and Figure 3 A). The TEM results of Cu 5.4 OUSNPs showed that the NPs were uniformly spherical, and the particle size distribution statistical chart showed that the average particle size was 2.06 ± 0.50 nm ( Figure 1 C and Figure 3 B). At the same time, Cu 5.4 OUSNPs and C-dots were combined to form a polymer Cu 5.4 O@CNDs. TEM showed that the NPs were uniformly spherical, and the particle size distribution statistical chart showed that the average particle size was 5.87 ± 0.94 nm ( Figure 1 D and Figure 3 C). The zeta potential of C-dots alone was -51.7 ± 0.9 mV, while after adding Cu 5.4 O USNPs, the potential of Cu 5.4 O@CNDs reached -60.47 ± 0.67 mV, indicating that C-dots and Cu 5.4 OUSNPs were successfully combined ( Figure 1 E). C-dots, Cu 5.4 O USNPs, and Cu 5.4The X-ray powder diffraction (XRD) results of O@CNDs showed that C-dots had obvious 2θ diffraction peaks at 25-30° and 42°, and Cu 5.4 O USNPs had obvious 2θ diffraction peaks at 25-30°, 42° and 50°. All appeared relative to C-dots and Cu 5.4 O USNPs, indicating that Cu 5.4 O@CNDs was successfully prepared ( Figure 1 of F). The characteristic bands of the FTIR spectrum proved that Cu 5.4 O@CNDs had the characteristic peaks of C-dots and Cu 5.4 O USNPs ( Figure 1 of G). X-ray photoelectron spectroscopy (XPS) graphs are usually used to analyze the element bonding of composite materials. The total XPS spectrum found elemental peaks of Na 1s, O 1s and C1s in C-dots, elemental peaks of Cu 2p and Cu LM in Cu 5.4 O USNPs, while elemental peaks of Na 1s, Cu 2p, Cu LM, O 1s and C1s were found in Cu 5.4 O@CNDs, proving that C-dots and Cu 5.4 O USNPs were successfully integrated ( Figure 1 of H). The C1s fractional peak fitting graphs of graphitic carbon at 284.8 eV, alcoholic carbon at 286.0 eV, carbonyl carbon at 287.8 eV and carboxyl carbon at 289.0 eV were detected in C-dots and Cu 5.4 O@CNDs, indicating that the characteristic peaks of C-dots appeared in Cu 5.4 O@CNDs ( Figure 1 of I-J). The Cu 2p peak fitting curves of Cu 5.4 OUSNPs and Cu 5.4 O@CNDs showed that there were Cu 5.4 and Cu1 0 in Cu + ( Figure 1 of K-L). The above results indicated that the synthesis and preparation of Cu 5.4 O@CNDs were successful and retained the characteristic structures and characterizations of C-dots and Cu 5.4 O USNPs respectively.

[0092] Example 2: Enzymatic characterization of C-dots, Cu 5.4 O USNPs, Cu 5.4 O@CNDs

[0093] 1. Experimental method

[0094] (1) C-dots, Cu 5.4 O USNPs, Cu 5.4 SOD-like activity of O@CNDs

[0095] To evaluate the SOD-like activity, an SOD assay kit was used, in which the reaction substrate WST reacts with ·O2 - to generate water-soluble formaldehyde, which exhibits a unique absorption peak at 450 nm. When the disproportionation of ·O2 - is blocked, indicating the presence of SOD-like activity in the sample, the enzyme activity of SOD is determined by colorimetric analysis of the WST product. The absorbance is measured at 450 nm.

[0096] (2) C-dots, Cu 5.4 O USNPs, Cu 5.4 ·O2 scavenging activity of O@CNDs

[0097] The NBT reduction method was used to evaluate the scavenging activity of ·O2 - , and this method produces blue formaldehyde in the presence of ·O2 - . When C-dots with SOD-like activity are present, they competitively scavenge ·O2 - , resulting in a lighter blue or its absence. For this purpose, samples at different concentrations (0 - 200 μg / mL) were mixed with NBT (0.05 mM), L-met (13 mM), and riboflavin (20 μM) in PBS buffer (pH 7.4) at a concentration of 25 mM, and then irradiated with LED for 5 minutes. The absorbance was measured at 560 nm.

[0098] The ESR method was used to determine its scavenging activity against ·O2 - . 100 mM DMPO, 25 μM DTPA, 0.5 mM HYP, 0.1 U / mL XOD, and 100 μg / mL Cu 5.4 O@CNDs were added to PBS (pH = 7.4), and ·O2 - was generated through the hypoxanthine / xanthine oxidase (HYP / XOD) system, and the ESR spectrum of BMPO / ·OOH was recorded after 2 min.

[0099] (3) C-dots, Cu 5.4 O USNPs, Cu 5.4 CAT-like activity of O@CNDs

[0100] The oxygen production of H2O2 scavenging by different materials was determined using the dissolved oxygen method. The dissolved oxygen meter (JPSJ-605F, RayMagnet) was used to measure the reaction system with a final volume of 15 mL, containing 60 μL of 30% H2O2 and different concentrations of different substances in ultrapure water for 15 min.

[0101] (4) C-dots, Cu 5.4 O USNPs, Cu 5.4 The activity of O@CNDs in scavenging H2O2

[0102] C-dots, Cu 5.4 O USNPs, Cu 5.4 The scavenging ability of O@CNDs for H2O2 was detected using a hydrogen peroxide detection kit (Nanjing Jiancheng Bioengineering Institute, China). H2O2 reacts with ammonium molybdate to form a stable yellow complex, showing an absorbance peak at 405 nm. Samples with different concentrations (0 - 200 μg / mL) were incubated with 2 mM H2O2 at 37 °C for 2 h. After the reaction, the remaining H2O2 concentration was measured according to the manufacturer's instructions, and the ability to scavenge H2O2 was calculated.

[0103] The scavenging activity of H2O2 was determined by the ESR method. Different 100 μg / mL materials catalyzed the degradation of H2O2 to generate O2. The ESR spin probe CTPO was used to capture O2, and the ESR spectrum of CTPO showed a proton hyperfine structure in the control nitrogen-saturated solution. As the concentration of O2 increased, the collision frequency between oxygen and the nitroxide radical also increased, resulting in the broadening of the ESR triplet spectrum and the reduction of the resolution of the proton hyperfine structure.

[0104] (5) C-dots, Cu 5.4 O USNPs, Cu 5.4 ·OH of O@CNDs - Scavenging activity

[0105] The activity of scavenging ·OH was determined by the TMB method. A 10 μM FeCl2 solution, a 50 μM H2O2 solution, and a 300 μM TMB solution were prepared with ultrapure water. Then, 100 μL of the above three solutions were uniformly mixed. Then, 10 μL of sample solutions with different concentrations (0 - 200 μg / mL) were mixed uniformly with 290 μL, and the absorbance was measured at 645 nm after reacting at room temperature for 30 min.

[0106] The scavenging activity of ·OH was evaluated by the ESR method. 100 mM DMPO, 1.0 mM FeSO4, 1.0 mM H2O2, and 100 μg / mL Cu 5.4 O@CNDs were added to ddH2O to generate ·OH. The ESR spectrum of DMPO / ·OH was recorded after 2 min.

[0107] (6) C-dots, Cu 5.4 O USNPs, Cu 5.4 ABTS radical scavenging activity of O@CNDs

[0108] According to the instructions, the T-AOC assay kit (S0119, Beyotime) was used to evaluate C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs for their ABTS radical scavenging ability. ABTS and the oxidant solution were mixed at a volume ratio of 1:1 to form a fresh working solution, which was then stored in the dark at room temperature for 12 - 16 hours before use. Subsequently, the working mixture was diluted according to the kit instructions, and the samples were analyzed at different concentrations of different substances (0 - 200 μg / mL) as specified. The absorbance was measured at 734 nm.

[0109] 2. Experimental results

[0110] ·O2 - is a common reactive oxygen species, and the SOD-like enzyme activity level of materials can be evaluated by detecting its scavenging rate ( Figure 4 of A). First, the NBT reduction method can indirectly detect C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs for their SOD-like enzyme activity. In this system, xanthine and xanthine oxidase generate ·O2 in the presence of light - , ·O2 - can make the NBT colorant produce blue formaldehyde, which has a characteristic absorption peak at 560 nm. Assuming the material has SOD activity, it can remove ·O2 - . In this case, the color development of NBT will become weaker, indirectly indicating the presence of SOD-like enzyme activity at the same time. At a series of concentration gradients, Cu 5.4 O@CNDs significantly showed better ·O2 5.4 scavenging efficiency than Cu - O USNPs, which is basically consistent with C-dots ( Figure 4 of B). At the same time, a SOD assay kit was used to quantitatively analyze the SOD-like enzyme activity of C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs. The results showed that the SOD-like enzyme activity of C-dots was 6055 U / mg, and that of Cu 5.4 O USNPs was 3063 U / mg. The SOD-like enzyme activity of Cu 5.4 O@CNDs was 8557 U / mg, which was better than the other two materials (Figure 4 C). In addition, the change in the intensity of the free radical signal was detected by ESR to evaluate the 5.4 Cu - O@CNDs' scavenging ability for ·O2 - . The results showed that there was no ESR signal for DMPO alone. When DMPO and ·O2 - coexisted, an obvious peak appeared in the signal, indicating that DMPO successfully captured ·O2 5.4 , showing a strong signal intensity. After adding Cu 5.4 O@CNDs, the peak intensity of the signal decreased significantly, indicating that Cu - O@CNDs had excellent ·O2 Figure 4 scavenging ability (

[0111] D). H2O2, as the product of SOD-catalyzed disproportionation, is also a toxic ROS. To achieve the goal of cascading ROS scavenging, H2O2 needs to be decomposed into non-toxic oxygen and water, and catalase can catalyze ( Figure 4 E). First, in H2O2 (10 mM) solution, different concentrations of C-dots, Cu 5.4 O USNPs, and Cu 5.4 O@CNDs were used. C-dots had no H2O2 scavenging ability. The absorbance values of Cu 5.4 O USNPs and Cu 5.4 O@CNDs at 240 nm decreased with increasing concentration. Cu 5.4 O@CNDs had a higher H2O2 scavenging rate than Cu 5.4 O USNPs ( Figure 4 F). Then, a dissolved oxygen meter was used to detect the dissolved oxygen concentration to measure the CAT activity of C-dots, Cu 5.4 OUSNPs, and Cu 5.4 O@CNDs. The results showed that almost no O2 was produced in the C-dots group, while the O2 produced in the Cu 5.4 O@CNDs group was twice that of the Cu 5.4 O USNPs group, indicating that C-dots had no H2O2 scavenging ability. The decomposition rate of H2O2 by Cu 5.4 O@CNDs could reach about twice that of Cu 5.4 OUSNPs. The oxygen production ability of Cu 5.4 O USNPs and Cu 5.4 O@CNDs was dose-dependent ( Figure 4G). In addition, the ESR test material was used to test the catalytic degradation of H2O2 for the generation of O2. The oxygen was captured by the ESR spin probe CTPO, and its ESR spectrum showed a proton hyperfine structure in the control nitrogen-saturated solution. As the oxygen concentration increased, the collision frequency between oxygen and the nitroxy radical increased, the ESR triplet spectrum broadened, and the resolution of the proton hyperfine structure decreased. After adding Cu 5.4 O@CNDs, the fine peaks of CTPO weakened, indicating its good hydrogen peroxide scavenging ability ( Figure 4 H).

[0112] ·OH is another important ROS with oxidative solid properties. Therefore, ·OH scavenging can also effectively protect cells from oxidative damage ( Figure 4 I). First, the TMB method was used to generate hydroxyl radicals through the Fenton reaction (Fe2 + / H2O2) and catalyze TMB to generate a soluble blue product. After adding C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs, the color changed. The scavenging rate was measured by enzyme labeling and calculated. The scavenging rate could reach over 80% at 25 μg / mL, indirectly evaluating the scavenging rate of ·OH. Cu 5.4 O@CNDs was significantly superior to the other two materials mentioned above in terms of hydroxyl radical scavenging ability ( Figure 4 J). At the same time, the ·OH scavenging ability was detected by ESR to detect the strength of the free radical signal. In this system, ·OH was generated through the Fenton reaction, and then DMPO was used to capture ·OH to form a spin adduct (DMPO / ·OH). The change in the ESR signal intensity was used to reflect the effect of scavenging ·OH. The change in the ESR signal intensity was used to respond to the influence of ·OH removal. When the Fenton reagent was mixed with DMPO, a four-line characteristic peak with an apparent signal intensity appeared, proving that a large amount of ·OH was generated in this mixed system. After adding C-dots, Cu5.4OUSNPS and Cu 5.4 O@CNDs, the intensity of the four-line intrinsic peak decreased. The intensity of the four-line intrinsic peak of Cu 5.4 O@CNDs almost weakened. The four-line state characteristic peak of Cu 5.4 O@CNDs almost disappeared, indicating that Cu 5.4 O@CNDs could effectively scavenge ·OH ( Figure 4 L).

[0113] The above experiments described the actual scavenging ability of Cu 5.4 O@CNDs when a single free radical was present. Secondly, the ABTS method was used to determine the total antioxidant capacity kit for Cu 5.4The total free radical scavenging ability of O@CNDs was evaluated. The ABTS and oxidant solutions provided in the kit were mixed at a volume ratio of 1:1 to prepare a fresh master solution, which was stored in the dark at room temperature for 12 - 16 hours before use. They diluted the mother batch according to the kit instructions and tested samples at different concentrations as required. The absorbance at 734 nm was measured using a microplate reader. The results showed that C-dots, Cu 5.4 O USNPs and Cu 5.4 O@CNDs had good total antioxidant levels, and the antioxidant ability was concentration-dependent. Among them, Cu 5.4 O@CNDs had significantly better total antioxidant ability than the other two, and the scavenging rate could reach more than 80% at 25 μg / mL ( Figure 4 K).

[0114] The above results indicated that Cu 5.4 O@CNDs not only had excellent SOD and CAT activities, but also could scavenge hydroxyl radicals and had high antioxidant ability, suggesting that Cu 5.4 O@CNDs had the potential to treat ROS-related injuries at the cellular and animal levels.

[0115] Example 4: Biosafety detection of Cu 5.4 O@CNDs

[0116] 1. Experimental method

[0117] (1) In vitro biocompatibility of Cu 5.4 O@CNDs

[0118] According to the above cell culture protocol, after co-incubation with Cu 5.4 O@CNDs nanozyme solutions at different concentrations (ranging from 0 to 100 μg / mL) for 12 hours and 24 hours, the cell viability was evaluated using the MTT method.

[0119] (4) In vivo biocompatibility of Cu 5.4 O@CNDs

[0120] To evaluate the in vivo biocompatibility of Cu 5.4 O@CNDs nanozyme, 0.5 mg / kg Cu 5.4 O@CNDs was intravenously injected into 6 - 8-week-old BALB / c mice weighing 20 - 25 g. Mice in the control group were injected with PBS. Specimens were collected at 1 day after injection and continuously at 7 days of injection. Blood samples were collected for complete blood cell analysis and serum biochemical detection. Subsequently, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were collected for H&E staining and histological examination.

[0121] 2. Experimental results

[0122] Since biocompatibility is very important for the potential clinical applications of nanomaterials, we evaluated the toxicity of Cu 5.4 O@CNDs in vitro. First, the MTT method was used to determine the cell viability of THLE-2 cells after incubation with a series of concentrations of Cu 5.4 O@CNDs for 24 h. The results showed that even at a concentration as high as 100 μg / mL, the cell viability was close to 100% ( Figure 5 A). When the incubation time reached 48 h, the cell survival rate was still higher than 80%, demonstrating good safety of Cu5.4@CNDs in vitro ( Figure 5 B). Next, a hemolysis experiment was used to detect the biocompatibility of Cu 5.4 O@CNDs. The results showed that when the concentration was 160 μg / mL, the hemolysis rate was still lower than 5%, indicating that the nanomaterials did not cause hemolysis of blood cells and intravenous injection was safe ( Figure 5 C).

[0123] In addition, all in vivo biocompatibility evaluation experiments were carried out at a concentration of 0.5 mg / kg. The effects of Cu 5.4 O@CNDs on blood chemistry and histopathology of important organs were evaluated in healthy mice to reveal its in vivo biocompatibility ( Figure 5 E). After continuous injection of Cu 5.4 O@CNDs for 7 days, the main organs (heart, liver, spleen, lung, kidney), serum, and plasma of rats were collected on the 1st and 7th days after injection, and the body weight changes of mice were followed up. The body weight record results showed that Cu 5.4 O@CNDs had no effect on the status and body weight of mice ( Figure 5 D). The H&E staining results showed that there were no obvious signs of tissue damage, indicating good biocompatibility of Cu 5.4 O@CNDs ( Figure 5 F). The results of serum biochemical analysis showed that the concentrations of serum liver function indexes (AST, ALT) and kidney function indexes (urea, CRE) in the Cu 5.4 O@CNDs group were comparable to those in the control group, indicating biocompatibility of the liver and kidney ( Figure 5 G). At the same time, the results of complete blood cell analysis of mice showed no statistically significant changes compared with the control group ( Figure 5 H). All studies consistently showed that the synthesized Cu 5.4 O@CNDs had minimal short-term and long-term toxicity in vivo.

[0124] Example 5: In vivo therapeutic effects of Cu 5.4 O@CNDs on HIRI and ALI.

[0125] 1. Experimental animals

[0126] Female BALB / c mice (6 - 8 weeks old, 20 - 25 g) were purchased from Xi'an Jiaotong University, Shaanxi Province, China. All mice were raised under standard conditions, including light, temperature, water, and food. The animal experiments were approved by the Animal Ethics Committee of Xi'an Jiaotong University.

[0127] 2. Experimental methods

[0128] (1) Establish a mouse HIRI model

[0129] Male BALB / c mice at 6 - 8 weeks of age and weighing 20 - 25 g were selected and fasted for 15 hours. Anesthesia was induced using a R500 general small animal anesthesia machine (R500IP, RWD), and then a 1.5 - cm surgical incision was made along the mid - abdominal line. After exposing the liver, the left branch of the hepatic artery, the left hepatic duct, and the portal vein were blocked using non - invasive vascular clamps. The left and middle lobes of the liver (about 70% of the total liver volume) turned white, indicating partial ischemia. After 1 hour, the clamps were removed, and the liver tissue turned red and moist, confirming the restoration of blood circulation. Subsequently, the abdominal incision was carefully sutured layer by layer. Twenty - four hours after blood flow restoration, the mice were euthanized, and blood and liver tissues were collected for analysis.

[0130] (3) Establish a mouse LPS - ALI model

[0131] Male BALB / c mice at 6 - 8 weeks of age and weighing 20 - 25 g were selected and fasted for 15 hours. They were intraperitoneally injected with 30 μg / mL LPS and 200 mg / kg D - gal, and euthanized 12 hours later. Blood and liver tissues were collected for analysis.

[0132] (4) Efficacy detection

[0133] The BALB / c mice with established HIRI models were randomly divided into sham operation (control group), HIRI, 0.1 mg / kg dose of Cu 5.4 O@CND treatment group, 0.5 mg / kg dose of Cu 5.4 O@CND treatment group, 1.0 mg / kg dose of Cu 5.4 O@CND treatment group, 2.5 mg / kg dose of Cu 5.4 O@CND treatment group, a total of 6 groups (n = 5). Twenty - four hours after administration, the body weight fluctuations of mice in each group were observed.

[0134] The BALB / c mice with established LPS - ALI models were randomly divided into three groups (n = 5): PBS (control group), LPS - ALI, 0.5 mg / kg dose of Cu 5.4 O@CND treatment group.

[0135] At 24 hours after injection, the mice were euthanized, blood samples were collected, and the levels of AST and ALT were quantitatively measured. Liver tissues were collected, and the transcriptional levels of pro-inflammatory factors, including IL-1β, IL-6, IL-12, and TNF-α, were measured. Meanwhile, part of the liver tissue was fixed with 4% paraformaldehyde and then embedded in paraffin for H&E and TUNEL staining. Another part of the liver tissue was frozen and embedded in optimal cutting temperature (O.C.T.) sample matrix, and frozen sections were made at -80 °C. The frozen liver tissue sections were stained with DAPI and DCFH / DHE at 37 °C for 30 minutes. Subsequently, the sections were rinsed three times with PBS to remove the excess dye. Then the stained sections were observed under a fluorescence microscope to qualitatively evaluate the level of ROS in the liver tissue.

[0136] 3. Experimental Results

[0137] Based on the in vitro antioxidant stress and anti-inflammatory effects of Cu 5.4 O@CNDs, the protective effect of Cu 5.4 O@CNDs on the HIRI mouse model was further studied. First, a HIRI mouse model ( Figure 6 of A) was established. Then the optimal treatment dose of Cu 5.4 O@CNDs was determined. Taking pictures of the liver tissue, visible partial tissue damage surfaces were observed. The liver status of the 0.5 mg / kg pretreatment group was better than that of the disease modeling group ( Figure 6 of B). Meanwhile, the H&E staining images of the mouse liver tissue showed that in the HIRI model group, hepatocytes showed spotty necrosis, partial cholestasis in capillaries, and inflammatory cell infiltration ( Figure 6 of C). The histopathological manifestations of the liver tissue in the pre-injected Cu 5.4 O@CNDs group were significantly improved. In the HIRI mouse model treated with 0.5 mg / kg Cu 5.4 O@CNDs, the areas of hepatocyte necrosis and cytolysis were extremely small. Subsequently, serum biochemical tests of the liver function of the mice were performed, and the results showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the HIRI model mice were higher than those in the control group, while the levels of ALT and AST in the pre-injected Cu 5.4 O@CNDs group were significantly decreased, indicating that Cu 5.4 O@CNDs had an obvious protective effect on liver function ( Figure 6 of E). When the concentration of Cu 5.4 O@CNDs was 0.5 mg / kg, the liver function index of the mice was closest to that of the control group, indicating that 0.5 mg / kg was the optimal treatment dose ( Figure 6 of E). In summary, in the HIRI mouse model, Cu 5.4 O@CNDs had a protective effect on liver function, and 0.5 mg / kg was selected as the treatment concentration for subsequent experiments.

[0138] Take frozen tissue sections for TUNEL staining to evaluate the levels of hepatocyte necrosis and apoptosis. The results showed that the green fluorescence of TUNEL in the HIRI group was significantly enhanced, and there was no significant difference between the pretreatment group and the control group, indicating that Cu 5.4 O@CNDs has good protective effects on hepatocytes and liver function ( Figure 6 of D). To evaluate the ability of Cu 5.4 O@CNDs to remove ROS in vivo, the ROS-specific fluorescent dyes DCFH-DA and DHE were used to image the in vivo ROS removal effect. After hepatic ischemia-reperfusion, the green fluorescence of DCFH-DA in liver tissues increased significantly, and the ROS level decreased in the tissues stained after pre-injection of Cu 5.4 O@CNDs ( Figure 6 of D). At the same time, DHE also enhanced the red fluorescence intensity in the modeling group and restored the stained fluorescence intensity in the pretreatment group ( Figure 6 of D). The results showed that Cu 5.4 O@CNDs can reduce liver function injury and hepatocyte apoptosis by scavenging ROS in hepatic ischemia-reperfusion injury diseases.

[0139] In addition, the liver tissues of mice were processed to extract related RNA to verify the changes in the transcriptional levels of common pro-inflammatory cytokines. After successful modeling, the levels of four common inflammatory factors (IL-1β, IL-6, IL-12, and TNF-α) were all significantly increased, and there was no significant difference in the inflammatory level between the group pre-injected with Cu 5.4 O@CNDs and healthy mice in the control group ( Figure 6 of F). The results showed that Cu 5.4 O@CNDs can effectively reduce the inflammatory response by scavenging ROS in vivo and prevent oxidative stress and inflammation in liver ischemia-reperfusion injury.

[0140] To verify that Cu 5.4 O@CNDs can be applied to other acute liver injury diseases, we established a lipopolysaccharide-induced acute liver injury model (LPS-ALI). Six hours after induction of the model with LPS + D-gal, Cu 5.4 O@CNDs (0.5 mg / kg) was injected, and the mice were sacrificed 12 hours later. Liver tissues and sera were taken for liver function verification ( Figure 6 of G). Similar to the HIRI model, the H&E staining images of mouse liver tissues showed that obvious balloon-like lesions were visible in hepatocytes of the lipopolysaccharide-induced hepatitis model, accompanied by a large number of inflammatory cell infiltrations. The pathological manifestations of liver tissues in the Cu 5.4 O@CNDs group were significantly improved, and the inflammatory symptoms were alleviated ( Figure 6 of H). Subsequently, biochemical liver function tests were performed on the sera of mice. The liver function indicators of mice with lipopolysaccharide-induced hepatitis increased, indicating severe liver function injury, while Cu5.4 In the CuO@CNDs treatment group, the liver function indicators decreased and were close to those of the control group, proving that the treatment effect was more obvious ( Figure 6 of J).

[0141] Take frozen tissue sections for TUNEL staining to evaluate the levels of hepatocyte necrosis and apoptosis. The results showed that the green fluorescence of TUNEL in the model group was significantly enhanced, and there was no significant difference between the pretreatment group and the control group, suggesting that Cu 5.4 O@CNDs has good anti-inflammatory effects ( Figure 6 of I). To evaluate the ability of Cu 5.4 O@CNDs to scavenge ROS in the liver of ALI disease, frozen sections were stained with DCFH-DA and DHE. The green fluorescence of DCFH-DA increased significantly in the liver tissue of lipopolysaccharide-induced hepatitis, and the ROS level decreased in the tissue treated with Cu 5.4 O@CNDs ( Figure 6 of I). At the same time, high-intensity red fluorescence was observed in the hepatitis tissue with DHE, and the fluorescence intensity of the tissue decreased after treatment. The results showed that Cu 5.4 O@CNDs can relieve inflammation by scavenging ROS in lipopolysaccharide-induced acute hepatitis ( Figure 6 of I).

[0142] In addition, relevant RNA was extracted by treating mouse liver tissue to verify the changes in the expression levels of common pro-inflammatory cytokines. The results showed that after successful modeling, the four common cytokines increased significantly, and the inflammation level in the Cu 5.4 O@CNDs group was not significantly different from that of healthy mice in the control group ( Figure 6 of K). It is suggested that Cu 5.4 O@CNDs can effectively improve the oxidative stress and inflammation of acute hepatitis by scavenging ROS in the body.

[0143] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent redundancy, the present invention describes preferred embodiments.

[0144] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0145] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs, characterized by: Prepared by the following steps: The CuCl2 was dissolved in the carbon dot aqueous solution, reacted at 75°C to 80°C for 10min to 12min, then L-ascorbic acid aqueous solution was added, the pH was adjusted to 7.0-8.0 with NaOH solution, and the reaction was carried out at 75°C to 80°C for 10h to 12h. In the reaction system, the CuCl2 concentration was 15-20mM, the carbon dot concentration was 3-4mg / mL, and the ascorbic acid concentration was 300-400mM. After the reaction was completed, the precipitate was removed by centrifugation, and the supernatant was dialyzed and freeze-dried to obtain the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity. 5.4 O@CNDs.

2. According to claim 1, an anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs, characterized by: The carbon dots are prepared by the following steps: The activated carbon is added to a boiling mixed acid solution, the boiling point is maintained for 1.5 h to 2 h, and after cooling to room temperature, it is neutralized with a NaHCO3 solution. The neutralized solution is filtered, dialyzed, concentrated, and freeze-dried to obtain the carbon dots, wherein the mixed acid solution is a mixture of equal volumes of an HNO3 solution with a concentration of 8 to 10 mol / L and an H2SO4 solution with a concentration of 18 to 20 mol / L, and the mass volume ratio of the activated carbon to the mixed acid solution is 0.5 g to 0.6 g: 50 mL.

3. The anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity according to any one of claims 1 to 2 5.4 Application of O@CNDs in the preparation of drugs for the treatment of liver damage.

4. The use according to claim 3, characterized in that: The liver injury includes liver ischemia-reperfusion injury and acute liver injury.

5. The use according to any one of claims 3 to 4, characterized in that: The drug contains the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity 5.4 O@CNDs, drug carriers and / or drug excipients.

6. A drug, characterized in that The drug is used to treat liver damage, and the drug comprises the anti-inflammatory and anti-oxidative stress nanozyme Cu with cascade enzyme activity according to any one of claims 1 to 2. 5.4 O@CNDs.

7. The drug according to claim 6, characterized in that The medicine also includes a drug carrier and / or a drug excipient.

8. The drug according to claim 7, characterized in that The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

9. The drug according to claim 7, characterized in that The pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents.

10. The drug according to claim 7, characterized in that The dosage form of the medicine is powder, granule, capsule, tablet, pill, injection or oral solution.

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