An artificial mimic enzyme for treating diabetic encephalopathy and a preparation method and application thereof

Abstract

The application belongs to the technical field of nanobiomedicine, and discloses an artificial enzyme for treating diabetic encephalopathy and a preparation method and application thereof.The application discloses an artificial enzyme, which comprises V2C quantum dot nanoscale enzyme and T7 peptide modified on the V2C quantum dot nanoscale enzyme.The artificial enzyme (i.e., V2C QDs-T7 quantum dot nanoscale enzyme) provided by the application realizes T7 peptide-mediated blood-brain barrier targeted delivery, cooperatively plays a dual simulation activity of superoxide dismutase and catalase, converts high-toxicity superoxide anion cascade catalysis into water and oxygen, significantly reduces the oxidative stress level in a diabetic encephalopathy lesion, simultaneously relieves nerve inflammation, effectively improves cognitive dysfunction, and realizes efficient targeted treatment of diabetic encephalopathy.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical technology, specifically relating to an artificial enzyme for treating diabetic encephalopathy, its preparation method, and its application. Background Technology

[0002] Diabetic encephalopathy (DE) is a common central nervous system complication of diabetes, clinically manifested primarily as cognitive decline, decreased learning and memory abilities, and neurodegenerative changes. With the continued rise in global diabetes prevalence, diabetic encephalopathy has become a serious public health problem threatening the quality of life of middle-aged and elderly populations.

[0003] Current research indicates that long-term oxidative stress and chronic neuroinflammation are the core pathological mechanisms in the development of diabetic encephalopathy. Although antioxidant and anti-inflammatory therapies are considered important intervention strategies for diabetic encephalopathy, existing treatments face two major bottlenecks. First, the existence of the blood-brain barrier. The blood-brain barrier highly selectively restricts the entry of peripheral drugs into the brain parenchyma, making it difficult for most antioxidants, anti-inflammatory drugs, or biomolecules to reach effective therapeutic concentrations in the brain. Second, traditional drugs have limited functions. Most small-molecule antioxidants can only scavenge specific types of reactive oxygen species (ROS), lack cascade catalytic capabilities, and struggle to simultaneously regulate inflammation, resulting in limited therapeutic effects.

[0004] Therefore, developing a multifunctional artificial enzyme that combines efficient ROS scavenging, inflammation regulation, and blood-brain barrier penetration is of great scientific significance and clinical translational value for the targeted treatment of diabetic encephalopathy. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide an artificial mimic enzyme for treating diabetic encephalopathy, its preparation method and application.

[0006] The first aspect of the present invention is to provide an artificial enzyme.

[0007] The second objective of this invention is to provide a method for preparing the artificially simulated enzyme of the first aspect of this invention.

[0008] The third aspect of this invention aims to provide the application of the artificially simulated enzyme of the first aspect of this invention.

[0009] The fourth aspect of this invention is to provide a product.

[0010] The fifth aspect of this invention aims to provide a method for decomposing H2O2 into oxygen.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an artificially simulated enzyme comprising a V2C quantum dot nanozyme and a T7 peptide modified on the V2C quantum dot nanozyme.

[0012] In some embodiments of the present invention, the T7 peptide is modified onto the V2C quantum dot nanozyme via electrostatic adsorption and hydrogen bonding.

[0013] In some embodiments of the present invention, the diameter of the V2C quantum dot nanozyme is 5-10 nm.

[0014] In some embodiments of the present invention, the zeta potential of the V2C quantum dot nanozyme is -0.5 mV.

[0015] In some embodiments of the present invention, the V2C quantum dot nanozyme is prepared by a method comprising the following steps: Accordion-shaped V2C powder is subjected to organic intercalation, centrifugation, washing and ultrasonic treatment in sequence to obtain a single-layer or few-layer V2C suspension. V2C quantum dot nanozymes are obtained by ultrasonically breaking down single-layer or few-layer V2C suspensions.

[0016] In some embodiments of the present invention, the step of organic intercalation of the accordion-shaped V2C powder includes: mixing the accordion-shaped V2C powder with an organic intercalating agent and reacting the mixture.

[0017] In some embodiments of the present invention, the organic intercalating agent includes at least one of tetramethylammonium hydroxide solution, tetrapropylammonium hydroxide solution, and tetrabutylammonium hydroxide solution.

[0018] In some embodiments of the present invention, the solvent of the organic intercalating agent is deionized water.

[0019] In some embodiments of the present invention, the concentration of the organic intercalating agent is 1-25 wt%, such as any value or a range formed by any two of 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 8 wt%, 10 wt%, 15 wt%, 20 wt%, or 25 wt%.

[0020] In some embodiments of the present invention, the mass-to-volume ratio of the accordion-shaped V2C powder to the organic intercalating agent is 1:(80-120), such as any ratio or range formed by any two of 1:80, 1:90, 1:100, 1:110 or 1:120.

[0021] In some embodiments of the present invention, the reaction conditions are: stirring at 20-30°C for 10-24 h.

[0022] In some embodiments of the present invention, the temperature of the reaction is 23-28°C, such as any value of 23, 24, 25, 26, 27 or 28°C or a range formed by any two of them.

[0023] In some embodiments of the present invention, the reaction time is 10-15 h, such as any value of 10, 11, 12, 13, 14 or 15 h or a range formed by any two of them.

[0024] In some embodiments of the present invention, the ultrasonic fragmentation time is 90-150 min, such as any value of 90, 100, 110, 120, 130, 140 or 150 min or a range formed by any two of them.

[0025] In some embodiments of the present invention, the ultrasonic power of the ultrasonic breaking is 20-50% of the maximum power, such as any value of 20%, 30%, 40% or 50% or a range formed by any two of them.

[0026] In some embodiments of the present invention, ultrasonic fragmentation is performed under ice-water bath conditions.

[0027] In some embodiments of the present invention, a cell ultrasonic disruptor is used for ultrasonic disruption.

[0028] In some embodiments of the present invention, the processing pulse mode of the cell ultrasonic disruptor is on / off (2-4s / 1-2s).

[0029] In some embodiments of the present invention, the centrifugation conditions are 3000-7000 r / min for 3-7 min; further, 4000-5000 r / min for 4-6 min.

[0030] In some embodiments of the present invention, the washing includes washing with deionized water as a detergent.

[0031] In some embodiments of the present invention, the duration of the ultrasonic treatment is 5-15 min, such as any value or a range formed by any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 min.

[0032] In some embodiments of the present invention, the ultrasonic power during the ultrasonic treatment is 100-500 W, such as any value of 100, 200, 300, 400 or 500 W or a range formed by any two of them.

[0033] In some embodiments of the present invention, ultrasonic treatment is performed under ice-water bath conditions.

[0034] A second aspect of the present invention provides a method for preparing an artificially simulated enzyme according to the first aspect of the present invention, comprising the following steps: mixing V2C quantum dot nanozymes and T7 peptides, reacting them to obtain the artificially simulated enzyme.

[0035] In some embodiments of the present invention, the reaction is a non-covalent coupling reaction.

[0036] In some embodiments of the present invention, the non-covalent coupling reaction is carried out under stirring at 1-6°C for 3-10 h.

[0037] In some embodiments of the present invention, the temperature of the non-covalent coupling reaction is 2-5°C, such as any value of 2, 3, 4 or 5°C or a range formed by any two of them.

[0038] In some embodiments of the present invention, the time for the non-covalent coupling reaction is 4-8 h, such as any value of 4, 5, 6, 7 or 8 h or a range formed by any two of them.

[0039] In some embodiments of the present invention, the mass ratio of the V2C quantum dot nanozyme to the T7 peptide is (8-15):1, such as any one of 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1 or any range of two of them.

[0040] A third aspect of the invention provides the application of the artificially simulated enzyme of the first aspect of the invention in (1)-(3): (1) Antioxidant stress; (2) Preparation of products with antioxidant stress (3) Preparation of therapeutic drugs.

[0041] In some embodiments of the present invention, the antioxidant stress described in (1)-(2) includes scavenging superoxide anions and scavenging reactive oxygen species.

[0042] In some embodiments of the present invention, the product includes reagents, kits, or drugs.

[0043] In some embodiments of the present invention, the therapeutic drug described in (3) includes a drug for treating at least one of the following diseases: neurodegenerative diseases, cerebrovascular diseases (such as ischemic stroke, cerebral ischemia-reperfusion injury), inflammation-related diseases, and tumors (such as liver cancer, breast cancer, lung cancer, colorectal cancer, cervical cancer, glioma, pancreas, etc.).

[0044] In some embodiments of the present invention, the inflammation-related diseases include inflammatory diseases of the central nervous system (such as diabetic encephalopathy, Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuromyelitis optica, autoimmune encephalitis, meningitis, etc.).

[0045] In some embodiments of the present invention, the inflammation-related diseases include neurodegenerative secondary inflammation, rheumatoid arthritis, ankylosing spondylitis, chronic skin inflammation, chronic hepatitis, liver fibrosis, chronic nephritis, interstitial renal inflammation, atherosclerosis, diabetic peripheral neuritis, diabetic nephropathy, diabetic retinopathy, obesity-related chronic low-grade systemic inflammation, etc.

[0046] A fourth aspect of the present invention provides a product comprising the artificially simulated enzyme of the first aspect of the present invention.

[0047] In some embodiments of the present invention, the product includes reagents, kits, or drugs.

[0048] In some embodiments of the present invention, the product can be used to combat oxidative stress or treat diseases.

[0049] In some embodiments of the present invention, the disease includes diabetic encephalopathy.

[0050] A fifth aspect of the present invention provides a method for decomposing H2O2 into oxygen, comprising the step of treating H2O2 using an artificially simulated enzyme of the first aspect of the present invention or a product of the fourth aspect of the present invention.

[0051] The beneficial effects of this invention are: The artificial enzyme (i.e., V2C QDs-T7 quantum dot nanozyme) provided by this invention is delivered to the blood-brain barrier through T7 peptide-mediated targeted delivery. It synergistically exerts dual mimicry activities of superoxide dismutase and catalase, catalyzing the conversion of highly toxic superoxide anion cascades into water and oxygen. This significantly reduces the level of oxidative stress in diabetic encephalopathy lesions, while alleviating neuroinflammation and effectively improving cognitive dysfunction, thus achieving highly efficient targeted treatment for diabetic encephalopathy.

[0052] The preparation method provided by this invention is simple and mild. It binds T7 peptide to the surface of V2C quantum dot nanozyme through a non-covalent coupling reaction. The reaction conditions are mild (room temperature aqueous phase), no complicated equipment is required, the preparation cycle is short, it is easy to scale up production, and the coupling efficiency is high, resulting in good stability of the quantum dot nanozyme.

[0053] The V2C QDs-T7 quantum dot nanozyme provided by this invention has unique dual enzyme mimicry activity and good biocompatibility, and has potential application potential in antioxidant therapy. It also has no obvious toxic side effects on normal cells and tissues, providing a safe and efficient nanoplatform for the treatment of central nervous system diseases. Attached Figure Description

[0054] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 TEM images of the V2C QDs-T7 quantum dot nanozyme (b) prepared in Example 1 and the V2C-T7 nanozyme (a) prepared in Comparative Example 3.

[0055] Figure 2 The fluorescence spectrum of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 is shown.

[0056] Figure 3 Zeta potential diagrams of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the artificial mimic enzyme V2CQDs prepared in Comparative Example 1.

[0057] Figure 4 The SOD activity test results are shown for the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the V2C-T7 nanozyme prepared in Comparative Example 3.

[0058] Figure 5 The CAT activity test results are shown for the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the V2C-T7 nanozyme prepared in Comparative Example 3; where a is the dissolved oxygen generation curve and b is the CAT activity test result.

[0059] Figure 6 The relative amount of V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the artificially simulated enzyme V2CQDs prepared in Comparative Example 1 were taken up by cells in the lower layer of the simulated blood-brain barrier system.

[0060] Figure 7 Results of ROS clearance in a diabetic encephalopathy cell model using artificial mimicry enzyme V2C QDs-T7 prepared in Example 1 and artificial mimicry enzyme V2C QDs prepared in Comparative Example 1.

[0061] Figure 8 Animal experiments using the artificial mimic enzyme V2C QDs-T7 prepared in Example 1 and the artificial mimic enzyme V2C QDs prepared in Comparative Example 1, including the water maze experiment results. Detailed Implementation

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0063] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0064] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0065] Example 1 A method for preparing an artificial enzyme for treating diabetic encephalopathy includes the following steps: (1) 0.2 g of accordion-shaped V2C powder (Xinxi Technology) was slowly added to 20 mL of tetramethylammonium hydroxide (1wt%) aqueous solution. The mixture was magnetically stirred at 25°C for 12 h, centrifuged at 5000 r / min for 5 min, washed three times with deionized water, and ultrasonically treated at 200W for 10 min to obtain a single-layer or few-layer V2C suspension.

[0066] (2) The V2C suspension prepared in step (1) was subjected to ultrasonic treatment (LICHEN Technology, LC-JY92-IIDN, power of 30% of the maximum power, pulse mode on / off (3 s / 2 s)) for 100 min in an ice-water bath to obtain a V2C QDs quantum dot suspension with a diameter of about 5-10 nm.

[0067] (3) Add T7 peptide (MeloPEG, 908001-1, the mass ratio of T7 peptide to V2C QDs quantum dots is 1:10) to the V2C QDs quantum dot suspension prepared in step (2), and react under ice-water bath conditions with magnetic stirring (400 r / min) for 6 h. Non-covalent coupling is achieved through electrostatic adsorption and hydrogen bonding, and finally artificial enzyme (referred to as V2C QDs-T7 or V2CQDs-T7 quantum dot nanozyme) is obtained.

[0068] Comparative Example 1 A method for preparing artificially mimicked enzyme V2C QDs includes the following steps: (1) 0.2 g of accordion-shaped V2C powder was slowly added to 20 mL of tetramethylammonium hydroxide (1%) aqueous solution. The mixture was magnetically stirred at 25 °C for 12 h, centrifuged at 5000 r / min for 5 min, washed three times with deionized water, and ultrasonically treated at 200 W for 10 min to obtain a single-layer or few-layer V2C suspension.

[0069] (2) The V2C suspension prepared in step (1) was subjected to ultrasonic treatment (power of 30% of the maximum power, pulse mode on / off (3 s / 2 s)) for 100 min in an ice-water bath using an ultrasonic cell disruptor to obtain a V2CQDs quantum dot suspension with a diameter of about 10 nm, namely artificial enzyme V2C QDs (denoted as V2C QDs).

[0070] Comparative Example 2 A method for preparing a single-layer or few-layer V2C suspension (denoted as V2C) includes the following steps: (1) 0.2 g of accordion-shaped V2C powder was slowly added to 20 mL of tetramethylammonium hydroxide (1%) aqueous solution. The mixture was magnetically stirred at 25 °C for 12 h, centrifuged at 5000 r / min for 5 min, washed three times with deionized water, and ultrasonically treated at 200 W for 10 min to obtain a single-layer or few-layer V2C suspension.

[0071] Comparative Example 3 A method for preparing an artificial enzyme mimicking enzyme includes the following steps: (1) 0.2 g of accordion-shaped V2C powder was slowly added to 20 mL of tetramethylammonium hydroxide (1%) aqueous solution. The mixture was magnetically stirred at 25 °C for 12 h, centrifuged at 5000 r / min for 5 min, washed three times with deionized water, and ultrasonically treated at 200 W for 10 min to obtain a single-layer or few-layer V2C suspension.

[0072] (2) Add T7 peptide (T7 peptide to V2C mass ratio of 1:10) to the monolayer or few-layer V2C suspension prepared in step (1), and react under ice-water bath conditions with magnetic stirring (400 r / min) for 6 h. Non-covalent coupling is achieved through electrostatic adsorption and hydrogen bonding, and finally artificial enzyme (referred to as V2C-T7 or V2C-T7 nanozyme) is obtained.

[0073] Effect Example 1. Microscopic morphology characterization of artificially simulated enzymes The microstructure of the artificial enzyme (V2C QDs-T7 quantum dot nanozyme) prepared in Example 1 and the artificial enzyme (V2C-T7 nanozyme) prepared in Comparative Example 3 was characterized using a transmission electron microscope (JEM-F200, TEM). The results are as follows: Figure 1As shown, V2C QDs-T7 quantum dot nanozymes are uniform in size, approximately 5-10 nm, exhibiting typical quantum confinement effects, high specific surface area, and abundant surface functional groups. These characteristics are beneficial for enhancing enzyme mimicry activity, improving T7 peptide coupling efficiency, and enhancing colloidal stability. In contrast, V2C-T7 nanozymes are larger than 2 μm and lack quantum dot properties. This suggests that the monolayer or few-layer V2C nanozymes of Comparative Example 2 and the V2C-T7 nanozymes of Comparative Example 3, due to their larger size, are not only less conducive to crossing the blood-brain barrier and achieving effective delivery to brain parenchyma, but also difficult to clear through the kidneys, increasing the risk of vascular embolism and nonspecific tissue accumulation.

[0074] 2. Determination of the fluorescence spectrum of artificially simulated enzymes The fluorescence spectrum of the artificial enzyme (V2C QDs-T7 quantum dot nanozyme) prepared in Example 1 was determined using a combined fluorescence spectrometer (Edinburgh Instruments Ltd., Edinburgh FLS 1000+LP980).

[0075] like Figure 2 The image shows the fluorescence spectrum of the artificial enzyme (V2C QDs-T7) prepared in Example 1. The results show that under UV excitation (e.g., 300-340 nm), the V2C QDs-T7 quantum dot nanozyme exhibits a significant fluorescence emission peak in the 400-600 nm range, indicating a typical quantum confinement effect and confirming that the V2C QDs-T7 quantum dot nanozyme possesses quantum dot structural characteristics.

[0076] 3. Determination of Zeta potential of artificially simulated enzymes The zeta potentials of the artificial enzyme (V2C QDs-T7 quantum dot nanozyme) prepared in Example 1, the artificial enzyme V2C QDs prepared in Comparative Example 1, and the T7 peptide were measured using a zeta potential analyzer (Malvern, Zetasizer Nano ZSE).

[0077] The results are as follows Figure 3 As shown, the zeta potential of the artificial enzyme V2C QDs is -20.5 mV, the T7 peptide is +3.5 mV, while the zeta potential of the V2C QDs-T7 quantum dot nanozyme is -0.5 mV. Compared to the artificial enzyme V2C QDs, the potential of the V2C QDs-T7 quantum dot nanozyme is significantly positively shifted, indicating that the T7 peptide has been successfully coupled to the surface of the artificial enzyme V2C QDs through electrostatic adsorption (or hydrogen bonding), confirming the successful preparation of this artificial enzyme (V2C QDs-T7 quantum dot nanozyme).

[0078] 4. SOD activity test The classic xanthine-xanthine oxidase (XOD)-WST-8 system was used to evaluate its role as a SOD mimic for scavenging superoxide anion (O2•). - The WST-8 method utilizes the ability of WST-8 to react with superoxide anions (O2•) produced by xanthine oxidase (XOD) catalysis. - The reaction produces a water-soluble formazan dye, which has a characteristic absorption peak at 450 nm. Since superoxide dismutase (SOD) catalyzes the dismutation of superoxide anions, this colorimetric process can be inhibited by SOD. The activity of SOD is negatively correlated with the amount of formazan dye produced; the enzyme activity of SOD can be calculated by colorimetric analysis of the absorbance at 450 nm.

[0079] The superoxide dismutase (SOD) simulated activity of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the V2C QDs nanozyme prepared in Comparative Example 3 was determined using the WST-8 method. The general procedure was as follows: The V2C QDs-T7 quantum dot nanozyme or V2C QDs nanozyme was diluted with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) to the corresponding concentrations, resulting in final nanozyme concentrations of 10, 20, 40, 80, and 160 μg / mL in the reaction system, with three replicates for each concentration. Following the WST-8 kit (Beyotime Biotechnology, S0101S) instructions, 20 μL of nanozyme solution of different concentrations, 160 μL of WST-8 / enzyme working solution, and 20 μL of reaction start-up working solution were added to each well sequentially. The 96-well plate was incubated at 37°C in the dark for 20 minutes, and the absorbance of each well was immediately measured at 450 nm. Simultaneously, blank and background controls were set up. The superoxide anion scavenging inhibition rate was calculated using the formula: Inhibition rate = [(A...] 空白对照1 -A 空白对照2 )-(A 样品 -A 空白对照3 )] / (A 空白对照1 -A 空白对照2 ()×100%. Among them, blank control 1 does not contain the test sample; blank control 2 does not contain the test sample and the reaction start-up working solution; blank control 3 does not contain the reaction start-up working solution.

[0080] The results are as follows Figure 4 As shown, under the same concentration gradient (10, 20, 40, 80, 160 μg / mL), the SOD activity of V2C QDs-T7 quantum dot nanozymes was significantly higher than that of V2C-T7 nanozymes, indicating that V2C QDs-T7 quantum dot nanozymes have superior superoxide dismutase mimicry activity, which is closely related to their smaller size and larger specific surface area.

[0081] 5. CAT activity test The catalase (CAT) simulated activity of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the V2C QDs nanozyme prepared in Comparative Example 3 was evaluated using dissolved oxygen assay. CAT can decompose H2O2 into H2O and O2, and the enzyme activity can be reflected by monitoring the rate of dissolved oxygen (DO) generation in the reaction system. The general detection process is as follows: V2C QDs-T7 quantum dot nanozymes or V2C QDs nanozymes were diluted to an appropriate concentration (20 μg / mL) with phosphate-buffered saline (PBS, 0.1 M, pH 7.4), with three replicates for each concentration. Measurements were performed in PBS buffer containing an appropriate amount of nanozyme solution (to a final concentration of 20 μg / mL), using H2O2 as the substrate (final concentrations of 1, 2, 5, 10, and 20 mM). The reaction chamber was quickly sealed. Dissolved oxygen concentration (mg / L) was continuously recorded using a dissolved oxygen meter (Leici, JPBJ-608) at a constant temperature of 25 °C, with data read every minute for a total of 10 minutes. A dissolved oxygen generation curve was plotted (a typical dissolved oxygen generation curve is shown below). Figure 5 As shown in Figure a), the Michaelis constant (Km) can be calculated based on the Michaelis equation.

[0082] The results are as follows Figure 5 As shown, the Km of V2C QDs-T7 quantum dot nanozymes is 5.7 mM, while the Km of V2C-T7 nanozymes is 9.4 mM. Figure 5 (As shown in b). The Km value reflects the affinity between the enzyme and the substrate; a smaller Km indicates a higher affinity. Therefore, the V2CQDs-T7 quantum dot nanozyme has a lower Km, indicating that its affinity for the substrate hydrogen peroxide (H2O2) is significantly better than that of the V2C-T7 nanozyme, enabling it to initiate a highly efficient catalytic reaction even at lower H2O2 concentrations. These results confirm that the V2C QDs-T7 quantum dot nanozyme exhibits rapid and efficient catalase-mimicking activity, capable of decomposing H2O2 into oxygen, demonstrating stronger substrate binding and catalytic capabilities.

[0083] 6. Assessment of the ability to penetrate the simulated blood-brain barrier The ability of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the artificially simulated enzyme V2C QDs prepared in Comparative Example 1 to penetrate the simulated blood-brain barrier was evaluated. The specific experiments were as follows: To simulate the blood-brain barrier, bEnd.3 cells were seeded in the upper chamber of a Transwell model (pore size 0.4 µm). The penetration ability was evaluated when the resistance reached 200 Ω·cm. 2A dense monolayer was formed, and BV2 cells were seeded in the lower chamber. Subsequently, V2C QDs-T7 or V2C QDs nanozyme (20 μg / mL, dissolved in PBS) was added to the upper chamber of the Transwell model. The cells were co-incubated at 37°C and 5% CO2 for 8 hours. After incubation, the electrical resistance was measured again to confirm BBB integrity. Finally, the cells were digested, and the vanadium (V) content in the lower chamber cells was determined by ICP-MS.

[0084] The statistical results of the relative amounts of V2C QDs-T7 quantum dot nanozymes and artificially simulated enzyme V2C QDs penetrating the lower culture dish of a simulated blood-brain barrier system are as follows: Figure 6 As shown in the figure, the relative V value in the V2C QDs group was 1.0 (normalized value), while the relative V value in the V2C QDs-T7 group significantly increased to 2.5, approximately 2.5 times that of the former. This result indicates that modification with the T7 peptide can effectively enhance the ability of V2C QDs nanozymes to cross the blood-brain barrier, promoting their entry into the brain parenchyma and uptake by nerve cells, thus providing a delivery basis for targeted therapy of diabetic encephalopathy.

[0085] 7. Assessment of the ability to clear ROS in a diabetic encephalopathy cell model The ability of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the artificially simulated enzyme V2C QDs prepared in Comparative Example 1 to scavenge ROS in a diabetic encephalopathy cell model was evaluated. Specific experiments were conducted as follows: Intracellular ROS levels were measured using a reactive oxygen species detection kit (Beyotime, S0033S). This kit utilizes the fluorescent probe DCFH-DA: DCFH-DA can freely penetrate the cell membrane and, after entering the cell, is hydrolyzed by esterases into non-fluorescent DCFH; intracellular ROS further oxidizes DCFH into green fluorescent DCF. The fluorescence intensity of DCF reflects the ROS level. Cells were first seeded into 96-well plates. After 24 hours of incubation, the old culture medium was discarded, and fresh culture medium containing V2C QDs-T7 or V2C QDs (both with a final concentration of 20 μg / mL) was added, along with H2O2 (final concentration 1000 μM), and incubation continued for 12 hours. Fresh H2O2 reagent was prepared before each experiment. Finally, the ROS levels were measured according to the kit instructions (n=3 per group).

[0086] The results of V2C QDs-T7 quantum dot nanozyme and artificially mimicked enzyme V2C QDs clearing ROS in a diabetic encephalopathy cell model are as follows: Figure 7As shown in the figure. The results showed that in hydrogen peroxide-induced microglia, the intracellular ROS level in the V2C QDs-T7 treatment group decreased to 8% of the normal level, significantly lower than that in the hydrogen peroxide-induced model group. This indicates that the V2C QDs-T7 quantum dot nanozyme exhibits excellent reactive oxygen species scavenging ability in a diabetic encephalopathy cell model.

[0087] 8. Animal water maze experiment The ability of the V2C QDs-T7 quantum dot nanozyme prepared in Example 1 and the artificially simulated enzyme V2C QDs prepared in Comparative Example 1 to improve cognitive impairment in diabetic encephalopathy mice was evaluated using an animal water maze experiment. The specific experiment was as follows: 30 minutes before each day's formal testing, the mice were transferred to the water maze room for environmental adaptation. The water temperature in the maze was maintained at 22-23℃, and an opaque titanium dioxide emulsion white background was added to the water, concealing an escape platform located 1 cm below the water surface. Training lasted for 4 days: each mouse was gently placed into the water from each of the four quadrants (facing the pool wall), and its platform-finding behavior was recorded for 90 seconds. If the mouse found the platform within 90 seconds, it was allowed to stay on the platform for 10 seconds; otherwise, it was guided to the platform and allowed to stay there for 10 seconds. After removal, the mice were dried with paper towels and placed near a heat source for rapid drying. On day 5, the platform was removed, and the mice were placed in water from the quadrant opposite to the original platform. Their movement trajectories were recorded within 90 seconds, and indicators such as the number of times they crossed the platform were analyzed to assess the mice's cognitive function.

[0088] Experimental results are as follows Figure 8 As shown, the model group mice exhibited irregular exploration paths; the V2C QDs group showed a slight improvement compared to the model group but still exhibited irregular exploration paths, indicating that the V2C QDs nanozyme itself has limited effect on improving cognitive function; while the V2CQDs-T7 group mice showed a clear platform orientation tendency. This behavioral evidence suggests that modification with the T7 peptide can significantly enhance the brain-targeted delivery capability of the V2C QDs-T7 quantum dot nanozyme, thereby effectively improving spatial cognitive dysfunction in diabetic model mice.

[0089] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An artificially simulated enzyme, comprising a V2C quantum dot nanozyme and a T7 peptide modified on the V2C quantum dot nanozyme.

2. The artificial enzyme according to claim 1, characterized in that, The T7 peptide is modified onto the V2C quantum dot nanozyme through electrostatic adsorption and hydrogen bonding.

3. The artificial enzyme according to claim 1 or 2, characterized in that, The diameter of the V2C quantum dot nanozyme is 5-10 nm.

4. The artificial enzyme according to claim 3, characterized in that, The V2C quantum dot nanozyme was prepared by the following method: Accordion-shaped V2C powder is subjected to organic intercalation, centrifugation, washing and ultrasonic treatment in sequence to obtain a single-layer or few-layer V2C suspension. V2C quantum dot nanozymes are obtained by ultrasonically breaking down single-layer or few-layer V2C suspensions.

5. The artificial enzyme according to claim 4, characterized in that, The steps for organic intercalation of the accordion-shaped V2C powder include: Mix the accordion-shaped V2C powder with an organic intercalating agent and react to obtain the product. Preferably, the organic intercalating agent comprises at least one selected from tetramethylammonium hydroxide solution, tetrapropylammonium hydroxide solution, and tetrabutylammonium hydroxide solution; Preferably, the reaction conditions are 20-30℃ for 10-24 h.

6. The artificial enzyme according to claim 4, characterized in that, The ultrasonic fragmentation time is 90-150 min; and / or the ultrasonic power of the ultrasonic fragmentation is 20-50% of the maximum power.

7. A method for preparing an artificial enzyme mimicking any one of claims 1-6, comprising the following steps: By mixing V2C quantum dot nanozymes and T7 peptides and reacting them, an artificially simulated enzyme can be obtained. Preferably, the reaction is a non-covalent coupling reaction; Preferably, the non-covalent coupling reaction is carried out under stirring at 1-6°C for 3-10 h.

8. The use of the artificially mimicked enzyme according to any one of claims 1-6 in (1)-(3): (1) Antioxidant stress; (2) Preparation of products to combat oxidative stress; (3) Preparation of therapeutic drugs; Preferably, the antioxidant stress described in (1)-(2) includes scavenging superoxide anions and scavenging reactive oxygen species; Preferably, the therapeutic drug described in (3) includes a drug for treating at least one of neurodegenerative diseases, cerebrovascular diseases, inflammation-related diseases, and tumors.

9. A product comprising any one of claims 1-6.

10. A method for decomposing H2O2 into oxygen, comprising the step of treating H2O2 with an artificially simulated enzyme according to any one of claims 1-6 or the product of claim 9.

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