MXene nano-enzyme with high SOD (superoxide dismutase) activity as well as preparation method and application of MXene nano-enzyme

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an MXene nano-enzyme with high SOD (superoxide dismutase) activity as well as a preparation method and application of the MXene nano-enzyme. The MXene nano-enzyme with high SOD activity comprises Mo2CMXene loaded with a copper single atom and a polypeptide, and the polypeptide is used for modifying the copper single atom. The MXene nano-enzyme with high SOD activity has the beneficial effects that under the pathological condition of TBI, the MXene nano-enzyme with high SOD activity not only can target a brain injury part to effectively remove ROS and protect cells from death, but also has good biocompatibility and biological safety. In the mechanism, the MXene nano-enzyme with high SOD activity can regulate the redox steady state, reduce inflammatory factors and protect nerve cells. The compound is expected to become an antioxidant for treating TBI and other oxidative stress related diseases.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a MXene nanozyme with high SOD activity, a preparation method thereof, and an application thereof. Background Art

[0002] Post-traumatic escalation of reactive oxygen species (ROS) leads to secondary damage after traumatic brain injury (TBI). Effective ROS scavenging at the injury site is crucial for mitigating damage. However, conventional antioxidants are ineffective due to insufficient activity and targeting.

[0003] At present, inorganic nanozymes such as V2O5, Fe3O4, Mn3O4 and CeO2 have attracted widespread attention due to their good controllability and enzyme-like activity, and have good prospects in the treatment of TBI. However, the catalytic activity of nanozymes is not ideal, and the activity of the simulated enzyme is single. For example, V2O5 nanozymes exhibit glutathione peroxidase (GPx)-like and peroxidase (POD)-like activities, and perform poorly in simulating superoxide dismutase (SOD) and catalase (CAT). Fe3O4 nanozymes also have excellent POD-like activity, but are less efficient in SOD-like, CAT-like and Gpx-like activities. During treatment, insufficient targeting of the injury site leads to poor therapeutic effect. Summary of the Invention

[0004] The present application provides a MXene nanozyme with high SOD activity, a preparation method and application thereof, aiming to solve the problem of insufficient activity and targeting of traditional antioxidants in the treatment of traumatic brain injury (TBI).

[0005] In a first aspect, the present application provides a MXene nanozyme with high SOD activity, comprising Mo2C MXene loaded with a copper single atom and a polypeptide, wherein the polypeptide modifies the copper single atom.

[0006] According to some embodiments of the MXene nanozyme with high SOD activity described in the present application, the polypeptide includes a CAQK peptide (cysteine-alanine-glutamine-lysine).

[0007] According to some embodiments of the MXene nanozyme with high SOD activity described in the present application, the molar ratio of the copper single atom to the CAQK peptide is 1:(1-3).

[0008] The second aspect of the present application provides a method for preparing the MXene nanozyme with high SOD activity as described in the first aspect of the present application, comprising the following steps: mixing Mo2C MXene loaded with copper single atoms, a polypeptide and a solvent to obtain a MXene nanozyme with high SOD activity.

[0009] According to some embodiments of the method for preparing MXene nanozymes with high SOD activity described in this application, the solvent includes deionized water and / or ethanol.

[0010] According to some embodiments of the method for preparing MXene nanozymes with high SOD activity described in the present application, the mass ratio of the Mo2C MXene loaded with copper single atoms and the polypeptide is (20-30): (1-4).

[0011] According to some embodiments of the method for preparing MXene nanozymes with high SOD activity described in this application, the mixing temperature is 20-30°C, and the mixing time is 10-14h.

[0012] According to some embodiments of the method for preparing MXene nanozyme with high SOD activity described in the present application, the preparation method further includes centrifuging the mixed product and drying the solid product to obtain the MXene nanozyme with high SOD activity.

[0013] According to some embodiments of the method for preparing MXene nanozymes with high SOD activity described in the present application, the drying includes freeze-drying.

[0014] According to some embodiments of the method for preparing MXene nanozymes with high SOD activity described in the present application, the freeze-drying temperature is -50°C to -55°C, and the freeze-drying time is 2-3 hours.

[0015] The third aspect of the present application provides an application of the MXene nanozyme with high SOD activity described in the first aspect of the present application or the MXene nanozyme with high SOD activity obtained by the preparation method described in the second aspect of the present application in the preparation of drugs for preventing or treating traumatic brain injury.

[0016] The beneficial effects of this application include: Under the pathological conditions of TBI, the MXene nanozyme with high SOD activity described herein can not only effectively eliminate ROS at the site of brain injury and protect cells from death, but also has good biocompatibility and biosafety. Mechanistically, the MXene nanozyme with high SOD activity can regulate redox homeostasis, downregulate inflammatory factors, and protect neurons. It is expected to become an antioxidant for the treatment of TBI and other oxidative stress-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The NBT described in Example 1 and Comparative Examples 1-2 of the present application reacts with O2 in the presence of MXene nanozymes with high SOD activity. ·- Absorption spectrum of the reaction;

[0018] Figure 2 This is a fluorescence image of the ROS scavenging effect of the MXene nanozyme with high SOD activity described in Example 1 of the present application;

[0019] Figure 3 A is a Nissl-stained image of the brain injury site of TBI mice treated with the MXene nanozyme with high SOD activity described in Example 1 of the present application;

[0020] Figure 3 B is an enlarged image of hippocampal neuronal cells of TBI mice treated with MXene nanozyme with high SOD activity as described in Example 1 of the present application;

[0021] Figure 4A This is a schematic diagram of the behavioral test process after treating TBI mice with MXene nanozymes with high SOD activity as described in Example 1 of this application;

[0022] Figure 4B This is a graph showing the improvement in neurological function in TBI mice treated with MXene nanozymes with high SOD activity as described in Example 1 of the present application;

[0023] Figure 4C This is a graph showing the walking distance of TBI mice treated with MXene nanozymes with high SOD activity in the middle area as described in Example 1 of the present application;

[0024] Figure 4D This is a graph showing the time spent in the middle area by TBI mice treated with MXene nanozymes having high SOD activity as described in Example 1 of the present application;

[0025] Figure 4E This is a graph showing the average velocity results in the middle area of ​​TBI mice treated with MXene nanozymes with high SOD activity as described in Example 1 of the present application;

[0026] Figure 4F This is a graph showing the spatial memory ability test results of TBI mice treated with MXene nanozyme with high SOD activity as described in Example 1 of the present application. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. The examples of the embodiments are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0028] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0029] The present invention provides a MXene nanozyme with high SOD activity, comprising Mo2C MXene loaded with a copper single atom and a polypeptide, wherein the polypeptide modifies the copper single atom.

[0030] The MXene nanozyme with high SOD activity described in this application has ultra-high SOD-like enzyme activity, can target the site of brain injury and effectively remove ROS to achieve TBI treatment.

[0031] In some embodiments of the present application, the polypeptide includes a CAQK peptide; the CAQK peptide is composed of four amino acids (cysteine, alanine, glutamic acid and lysine), which are connected by peptide bonds to form a linear structure, cysteine-alanine-glutamine-lysine.

[0032] In some embodiments of the present application, the molar ratio of the copper atom to the CAQK peptide is 1:(1-3), for example, 1:1, 1:2, 1:2.3, 1:3, etc.

[0033] The present application also provides a method for preparing the MXene nanozyme with high SOD activity as described in the first aspect of the present application, comprising the following steps: mixing Mo2C MXene loaded with copper single atoms, a polypeptide and a solvent to obtain a MXene nanozyme with high SOD activity.

[0034] In some embodiments of the present application, a method for preparing Mo2C MXene loaded with copper single atoms comprises the following steps: dissolving Mo2C MXene and CuCl2·2H2O in a mass ratio of (10-20):(1-3) in deionized water, and then mixing them at a temperature of 20-30°C for 30-90 minutes to obtain a Cu SA / MXene nanomaterial, wherein the Mo2C MXene is a two-dimensional transition metal carbide composed of molybdenum carbide.

[0035] In some embodiments of the present application, the solvent includes deionized water and / or ethanol.

[0036] In some embodiments of the present application, the mass ratio of the Mo2C MXene loaded with copper single atoms and the polypeptide is (20-30): (1-4), for example, 20:1, 20:3, 20:4, 25:1, 25:2, 25:4, 30:1, 30:2, 30:4, etc.

[0037] In some embodiments of the present application, the mixing temperature is 20-30°C, such as 20°C, 25°C, 28°C, 30°C, etc., and the mixing time is 10-14h, such as 10h, 12h, 14h, etc.

[0038] In some embodiments of the present application, the preparation method further includes centrifuging the mixed product and drying the solid product to obtain the MXene nanozyme with high SOD activity.

[0039] In some embodiments of the present application, the drying comprises freeze-drying.

[0040] In some embodiments of the present application, the freeze-drying temperature is -50°C to -55°C, for example, -50°C, -52°C, -53°C, -55°C, etc., and the freeze-drying time is 2-3h, for example, 2h, 2.2h, 2.5h, 2.8h, 3h, etc.

[0041] The embodiments of the present application also provide an application of the MXene nanozyme with high SOD activity described in the first aspect of the present application or the MXene nanozyme with high SOD activity obtained by the preparation method described in the second aspect of the present application in the preparation of drugs for preventing or treating traumatic brain injury.

[0042] Example 1

[0043] A method for preparing a MXene nanozyme with high SOD activity comprises the following steps:

[0044] (1) 20 mg of Mo2C MXene, 3 mg of CuCl2·2H2O, and 13 ml of deionized water were mixed and stirred at 25 °C for 30 min, and then ultrasonicated with argon for 1 h to obtain a solution containing Cu SA / MXene nanomaterials;

[0045] (2) Add 1 mg of CAQK peptide to the above solution containing Cu SA / MXene nanomaterials, stir and mix at a temperature of 25°C for 12 hours to obtain a reaction solution; centrifuge the reaction solution, and freeze-dry the centrifugal precipitate at a temperature of -50°C for 3 hours to obtain the MXene nanozyme with high SOD activity.

[0046] Example 2

[0047] The preparation method of the MXene nanozyme with high SOD activity described in Example 2 is different from that in Example 1 only in that the amount of CuCl2·2H2O added is 1 mg.

[0048] The specific steps include:

[0049] (1) 20 mg of Mo2C MXene, 1 mg of CuCl2·2H2O, and 13 ml of deionized water were mixed and stirred at 25°C for 30 min, and then ultrasonicated with argon for 1 h to obtain a solution containing Cu SA / MXene nanomaterials;

[0050] (2) Add 1 mg of CAQK peptide to the above solution containing Cu SA / MXene nanomaterials, stir and mix at a temperature of 25°C for 12 hours to obtain a reaction solution; centrifuge the reaction solution, and freeze-dry the centrifugal precipitate at a temperature of -50°C for 3 hours to obtain the MXene nanozyme with high SOD activity.

[0051] Example 3

[0052] The preparation method of the MXene nanozyme with high SOD activity described in Example 3 is different from that in Example 1 only in that the amount of CuCl2·2H2O added is 4 mg.

[0053] The specific steps include:

[0054] (1) 20 mg of Mo2C MXene, 4 mg of CuCl2·2H2O, and 13 ml of deionized water were mixed and stirred at 25°C for 30 min, and then ultrasonicated with argon for 1 h to obtain a solution containing Cu SA / MXene nanomaterials;

[0055] (2) Add 1 mg of CAQK peptide to the above solution containing Cu SA / MXene nanomaterials, stir and mix at a temperature of 25°C for 12 hours to obtain a reaction solution; centrifuge the reaction solution, and freeze-dry the centrifugal precipitate at a temperature of -50°C for 3 hours to obtain the MXene nanozyme with high SOD activity.

[0056] Comparative Example 1

[0057] The only difference between the MXene nanozyme with high SOD activity described in Comparative Example 1 and Example 1 is that CAQK peptide is not used to modify copper atoms during the preparation of the MXene nanozyme with high SOD activity described in Comparative Example 1.

[0058] The specific steps include:

[0059] 20 mg of Mo2C MXene, 3 mg of CuCl2·2H2O, and 10 ml of deionized water were mixed and stirred at 25°C for 30 minutes, and then ultrasonicated with argon for 1 hour to obtain a solution containing Cu SA / MXene nanomaterials. The solution was filtered and dried to obtain Cu SA / MXene nanomaterials.

[0060] Comparative Example 2

[0061] The MXene nanozyme with high SOD activity described in Comparative Example 2 is different from that in Example 1 only in that the MXene nanozyme with high SOD activity described in Comparative Example 2 is Mo2C MXene.

[0062] 1. Study on the SOD-like activity of MXene nanozymes with high SOD activity described in Example 1 and Comparative Examples 1-2 of the present application

[0063] Test method: The SOD specific activity of the above samples was determined by nitroblue tetrazolium (NBT) method.

[0064] O2 ·- Nitroblue tetrazolium (NBT) can be reduced to blue-purple formazan, which has a maximum absorption at 560nm. O2 is detected by detecting the absorption at 560nm ·- O2 ·- The higher the content, the higher the absorption at 560nm.

[0065] First, xanthine (0.25 mM) and xanthine oxidase (0.05 U / mL) were mixed in phosphate buffer (50 mM, pH 7.4) at 37 °C to prepare O2 ·- , then NBT was added and reacted for 10 minutes (3 groups were prepared under the same conditions).

[0066] Add O2 to the above three groups respectively ·- The MXene nanozyme with high SOD activity described in Example 1 and Comparative Examples 1-2 (dosage of 100 μg / mL) was added to the solution, and then the UV-visible light absorption at 560 nm was detected, and the NBT reduction inhibition rate of each sample was calculated.

[0067] The test results are as follows Figure 1 As shown:

[0068] from Figure 1 It can be seen that the MXene nanozyme (C-Cu SA / MXene) with high SOD activity described in Example 1 of the present application retains the SOD-like activity of Cu SA / MXene and can effectively remove ROS.

[0069] 2. Study on the performance of MXene nanozymes with high SOD activity described in this application in clearing excess reactive oxygen species (ROS) produced after traumatic brain injury (TBI)

[0070] Experimental method: Fluorescent dye DCFH-DA was used to evaluate the level of intracellular ROS induced by H2O2. PC12 cells treated with H2O2 significantly expressed ROS (such as Figure 2 shown).

[0071] from Figure 2 It can be seen that after treatment with the MXene nanozyme (C-CuSA / MXene) with high SOD activity described in Example 1 of the present application, the fluorescence intensity is significantly reduced, and the effect is better than that of Cu SA / MXene, that is, the MXene nanozyme with high SOD activity described in the present application has a strong ROS scavenging property.

[0072] 3. Study on the therapeutic effect of MXene nanozymes with high SOD activity described in this application on traumatic brain injury (TBI) mice

[0073] Experimental Methods: Neuronal cell necrosis and brain tissue damage were assessed seven days after treatment in mice with traumatic brain injury (TBI).

[0074] Grouping: TBI mice were randomly divided into 5 groups: Sham group (sham operation group), TBI group (control group, no treatment after injury), CAQK group (CAQK was intravenously administered at a dose of 23 mg kg after injury), and TBI group (control group, no treatment after injury). -1 , dosage concentration 0.25 mg·mL -1 ), Cu SA / MXene group (after injury, the MXene nanozyme (Cu SA / MXene) nanodrug with high SOD activity described in Comparative Example 1 was intravenously administered at a dose of 23 mg·kg -1 , dosage concentration 5 mg·mL -1 ) and C-Cu SA / MXene group (after injury, the MXene nanozyme (C-Cu SA / MXene) nanodrug with high SOD activity described in Example 1 was intravenously administered at a dose of 23 mg·kg -1 , dosage concentration 5 mg·mL -1 ), 5 mice in each group.

[0075] 3.1. After the five groups of mice were treated for 7 days, they were deeply anesthetized by intraperitoneal injection of 1% sodium pentobarbital. 20 ml of normal saline and 20 mL of 4% paraformaldehyde were injected into the apex of the heart (left ventricle) through the catheter. The brain, heart, liver, spleen, lungs, and kidneys were dissected and fixed in a centrifuge tube filled with 4% paraformaldehyde solution. Tissue samples were Nissl stained and histological sections were observed. After data acquisition, images were analyzed using ImageJ software. The experimental results are shown in Figure 2. Figure 3 A and Figure 3 As shown in B.

[0076] from Figure 3 A and Figure 3 B shows that Nissl staining showed that the C-Cu SA / MXene group had targeted effect and significantly reduced brain tissue damage compared with the Cu SA / MXene group ( Figure 3 A). Nissl staining results showed that the survival rate of neurons in the hippocampus of TBI mice was significantly reduced compared with the sham-operated group, with only 87.33 positively stained cells detected. In contrast, C-Cu SA / MXene treatment significantly increased the survival rate of neurons in the hippocampus, with the number of positively stained cells reaching 149 ( Figure 3 B).

[0077] 3.2. Behavioral experiments were performed on days 1, 3, and 7 after treatment of mice with traumatic brain injury (TBI). The schematic diagram of the experimental timeline is shown in Figure 3. Figure 4A shown.

[0078] Experimental procedures: NSS test: The NSS test performed before injury was considered as the baseline (n = 7 per group). A 10-point task was used to analyze motor function, balance, alertness, and behavior at different time points after TBI. Failure of the task was assigned a score of 1. The total score reflects the severity of brain injury. The tasks used in the NSS included the following: (i) ability and initiative to exit a 30 cm diameter circle within 3 minutes; (ii) absence of contralateral hemiparesis; (iii) alertness, initiative, and the ability to walk upright; (iv) innate reflexes - mice should respond to loud clapping; (v) physiological behaviors indicating "interest" in the environment; (vi) ability to maintain balance on a 7 mm wide beam for at least 10 seconds; (vii) ability to maintain balance on a 5 mm diameter cylinder for at least 10 seconds; (viii) ability to cross a 3 cm wide beam to reach a shelter; (ix) the same task, but with increased difficulty on a 2 cm wide beam; (x) the same task, but with increased difficulty on a 1 cm wide beam. The assessment was performed at three different time points after injury: day 1, day 3, and day 7.

[0079] Experimental results: To evaluate the neurological recovery of mice after traumatic brain injury (TBI), the mice were scored using the Neurological Severity Score (NSS) on days 1, 3, and 7 after injury. Figure 4B shown.

[0080] from Figure 4B It can be seen that compared with the TBI group, the neurological function of mice treated with C-Cu SA / MXene was significantly improved than that of mice treated with Cu SA / MXene.

[0081] In summary, the MXene nanozyme C-Cu SA / MXene with high SOD activity described in this application can enhance the neurological function recovery of TBI mice.

[0082] 3.3. The open field test was used to evaluate the motor and anxiety behaviors of TBI mice after C-Cu SA / MXene treatment.

[0083] Specific steps: Take the treated TBI mice out of the cage and place them at the edge of the open field. For each experiment, the animals should be placed at the edge of the test box from the same position and direction to ensure consistency of experimental conditions. Immediately start the video acquisition and analysis system to record the animals' activities in the open field. The experiment usually lasts for 5-10 minutes. Observe the mice's exploration of the center. The results are as follows: Figure 4C-4E shown.

[0084] like Figure 4C-4E As shown in the figure: The movement trajectory of TBI mice is mainly concentrated in the peripheral area, while mice treated with C-Cu SA / MXene show a significant ability to explore the central area. Specifically, the walking distance in the middle area, the residence time in the central area, and the average speed increased, indicating that the movement and anxiety-related functions of TBI mice were significantly improved after treatment with the MXene nanozyme C-Cu SA / MXene with high SOD activity described in this application.

[0085] Y-maze test for assessing spatial working memory in TBI mice

[0086] The Y-maze consists of three identical arms arranged in a Y-shape with an angle of 120° between the arms. The mouse was gently placed in the center of the maze and allowed to explore freely for 10 minutes. Full entry into an arm was defined as all four paws inside. The number of spontaneous alternations (defined as the act of entering three different arms in succession) was used as an indicator of spatial working memory ability. Figure 4F shown.

[0087] from Figure 4FIt can be seen that the spatial working memory ability of mice in the TBI group decreased. Mice treated with the MXene nanozyme (C-Cu SA / MXene) with high SOD activity described in Example 1 of this application were significantly better than the Cu SA / MXene group in recovering spatial working memory. In summary, the MXene nanozyme C-Cu SA / MXene with high SOD activity described in this application can effectively restore the spatial working memory and motor ability of TBI mice, while reducing anxiety.

[0088] Although the above embodiments have been shown and described, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. Changes, modifications, substitutions and variations of the above embodiments by those skilled in the art are all within the scope of protection of the present invention.

Claims

1. A MXene nanozyme with high SOD activity, characterized in that The invention comprises Mo2CMXene loaded with copper single atoms and a polypeptide, wherein the polypeptide modifies the copper single atoms.

2. The MXene nanozyme with high SOD activity according to claim 1, characterized in that The polypeptide includes a CAQK peptide; And / or, the molar ratio of the copper atom to the CAQK peptide is 1:(1-3).

3. The method for preparing the MXene nanozyme with high SOD activity according to any one of claims 1-2, characterized in that: The following steps are involved: Mo2C MXene loaded with copper single atoms, peptides and solvents were mixed to obtain MXene nanozymes with high SOD activity.

4. The method for preparing the MXene nanozyme with high SOD activity according to claim 3, characterized in that: The solvent includes deionized water and / or ethanol.

5. The method for preparing the MXene nanozyme with high SOD activity according to claim 3, characterized in that: The mass ratio of the Mo2C MXene loaded with copper single atoms to the polypeptide is (20-30): (1-4).

6. The method for preparing the MXene nanozyme with high SOD activity according to claim 3, characterized in that: The mixing temperature is 20-30° C., and the mixing time is 10-14 h.

7. The method for preparing the MXene nanozyme with high SOD activity according to claim 3, characterized in that: The preparation method further includes centrifuging the mixed product and drying the solid product to obtain the MXene nanozyme with high SOD activity.

8. The method for preparing the MXene nanozyme with high SOD activity according to claim 7, characterized in that: The drying includes freeze-drying.

9. The method for preparing the MXene nanozyme with high SOD activity according to claim 8, characterized in that: The freeze-drying temperature is -50°C to -55°C, and the freeze-drying time is 2-3 hours.

10. Use of the MXene nanozyme with high SOD activity according to any one of claims 1-2 or the MXene nanozyme with high SOD activity obtained by the preparation method according to any one of claims 3-9 in the preparation of drugs for preventing or treating traumatic brain injury.

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