Multifunctional CMPd (H) U nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a multifunctional CMPd (H) U nano-enzyme as well as a preparation method and application thereof. The multifunctional CMPd (H) U nano-enzyme is obtained by taking a metal organic framework material (MOF) and palladium as carriers and urokinase (uPA) and hydrogen as drugs, synthesizing mesoporous MOF through a template method, then anchoring palladium nanoparticles, modifying the mesoporous MOF with thrombus targeting peptide CREKA and loading the uPA and the hydrogen. The multifunctional CMPd (H) U nano-enzyme prepared by the invention is expected to solve the key problems of poor drug targeting ability, incapability of reducing reperfusion injury, adverse reaction, neuromotor function injury and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical nanotechnology, and in particular to a multifunctional CMPd(H)U nanozyme and a preparation method and application thereof. Background Art

[0002] Stroke is the second leading cause of death worldwide, including ischemic stroke and hemorrhagic stroke. It is estimated that by 2050, the annual death toll will exceed 10 million, and most survivors will be disabled. Ischemic stroke accounts for the majority, mainly caused by thromboembolism, resulting in ischemia of brain tissue and death of nerve cells.

[0003] Currently, there are limited clinical treatments for ischemic stroke, mainly including alteplase thrombolysis and surgical thrombectomy. However, the clinical treatment method is not effective and cannot reduce the reperfusion injury caused by blood flow recanalization, thereby damaging the patient's neuromotor function and causing lifelong disability. The ideal therapeutic drug should be able to target thrombus, pass through the blood-brain barrier, remove reactive oxygen, etc., with the focus on reducing the probability of disability in patients.

[0004] Therefore, it is of great significance to develop new and efficient thrombus clearance and neuroprotection treatments. Summary of the invention

[0005] The purpose of the present invention is to provide a multifunctional CMPd(H)U nanozyme and a preparation method and application thereof, aiming to solve the problems of poor drug targeting ability, inability to reduce reperfusion injury, obvious toxic and side effects, and neuromotor function damage in existing ischemic treatment methods.

[0006] In the first aspect, the present invention provides a method for preparing a multifunctional CMPd(H)U nanozyme. The multifunctional CMPd(H)U nanozyme uses a metal organic framework material (MOF) and palladium as carriers, urokinase (uPA) and hydrogen as drugs, synthesizes a mesoporous MOF by a template method, and then anchors palladium nanoparticles. The mesoporous MOF is modified with a thrombus targeting peptide CREKA and then loaded with uPA and hydrogen.

[0007] In order to construct a multifunctional drug delivery system, the present invention uses metal organic framework materials (MOF) and palladium as carriers, urokinase and hydrogen as drugs, and prepares a multifunctional CMPd (H) U nanozyme. It can not only quickly target the thrombus site, but also quickly clear the thrombus and restore blood flow with gas therapy. At the same time, the released hydrogen can remove reactive oxygen species to reduce reperfusion injury, and can also play anti-inflammatory and neuroprotective effects by regulating microglia to polarize to M2 phenotype and inhibiting neuronal pyroptosis. Ultimately, it promotes the recovery of neuromotor kinetic energy in stroke mice. Inspired by ice beer, the constructed nano drug delivery system that can quickly dissolve thrombus and release hydrogen for a long time is expected to solve key problems such as poor drug targeting ability, inability to reduce reperfusion injury, adverse reactions, and neuromotor function damage.

[0008] In some preferred embodiments, the preparation method comprises:

[0009] Mesoporous MOF nanoparticles were synthesized by reacting zirconium oxynitrate and 2-aminoterephthalic acid using Pluronic F127 as template;

[0010] The MOF solution was prepared using mesoporous MOF nanoparticles, and the sodium tetrachloropalladate solution was added to the MOF solution and stirred evenly. The sodium borohydride solution was added to 2+ Reduction to palladium nanoparticles, anchored on mesoporous MOF nanoparticles to obtain MPd nanoparticles;

[0011] CREKA peptide was linked to MPd nanoparticles using the EDC / NHS method to obtain CMPd nanoparticles;

[0012] Loading urokinase into the mesopores of CMPd nanoparticles to obtain CMPdU nanoparticles;

[0013] A hydrogen generating device was built, and hydrogen was loaded onto CMPdU nanoparticles through the hydrogen generating device to obtain CMPd(H)U nanozyme.

[0014] In some preferred embodiments, the step of synthesizing mesoporous MOF nanoparticles by reacting zirconium oxynitrate and 2-aminoterephthalic acid using Pluronic F127 as a template comprises:

[0015] Dissolve Pluronic F127 in deionized water, add acetic acid and sodium perchlorate, and continue to stir evenly;

[0016] Zirconium oxynitrate and 2-aminobenzenedicarboxylic acid were added and stirred at 40° C. for 24 h. After the reaction was completed, the mesoporous MOF nanoparticles were collected by centrifugation.

[0017] In some preferred embodiments, the MOF solution is prepared by using mesoporous MOF nanoparticles, and the sodium tetrachloropalladate solution is added to the MOF solution and stirred evenly, and the sodium borohydride solution is added to Pd 2+ The steps of reducing to palladium nanoparticles and anchoring to mesoporous MOF nanoparticles to obtain MPd nanoparticles include:

[0018] Mesoporous MOF nanoparticles were added to a mixed solution of ethanol and deionized water, and after ultrasonic dispersion, an aqueous solution of sodium tetrachloropalladate was added. After magnetic stirring for 3.5 h, the system was placed in an ice bath, and then an ice-cold aqueous solution of sodium borohydride was added. The system was stirred in the ice bath for 1 h, and then washed three times with water after centrifugation to obtain black MPd nanoparticles.

[0019] In some preferred embodiments, the step of connecting CREKA peptide to MPd nanoparticles using the EDC / NHS method to obtain CMPd nanoparticles comprises:

[0020] MPd nanoparticles were added to deionized water and then sonicated. EDC and NHS were added after sonication, stirred for 1 h in an ice bath, and then centrifuged.

[0021] After centrifugation, deionized water and CREKA were added and stirred at room temperature for 12 h;

[0022] The product was washed three times with deionized water to remove free polypeptides, and CMPd nanoparticles were collected by centrifugation.

[0023] In some preferred embodiments, the step of loading urokinase into the mesopores of CMPd nanoparticles to obtain CMPdU nanoparticles comprises:

[0024] Deionized water was added to the CMPd nanoparticles to prepare a CMPd aqueous solution, and uPA was added. The mixture was stirred at room temperature in the dark, and then washed twice with water. The mixture was collected by centrifugation to obtain CMPdU nanoparticles.

[0025] In some preferred embodiments, the steps of constructing a hydrogen generator and loading hydrogen onto CMPdU nanoparticles through the hydrogen generator to obtain CMPd(H)U nanozyme include:

[0026] Take two vials, defined as vial A and vial B;

[0027] The CMPdU nanoparticles were dissolved in deionized water and ultrasonically dispersed to prepare a CMPdU solution;

[0028] The prepared CMPdU solution was injected into bottle A and sealed with a rubber stopper, sodium borohydride was added into bottle B and sealed with a rubber stopper, and bottles A and B were connected by a hose, and then a needle was inserted into bottle A to connect to the atmosphere;

[0029] Dilute sulfuric acid was injected into bottle B to generate hydrogen gas, and the hydrogen gas was pumped into the CMPdU solution for 30 min to obtain the CMPd(H)U nanozyme solution.

[0030] In a second aspect, the present invention provides a multifunctional CMPd(H)U nanozyme, which is prepared according to the above-mentioned method for preparing the multifunctional CMPd(H)U nanozyme.

[0031] In a third aspect, the present invention provides a use of the above-mentioned multifunctional CMPd(H)U nanozyme in a drug for treating ischemic stroke.

[0032] Compared with the prior art, the present invention also has the following advantages:

[0033] 1. Rapid targeting of thrombus: The CMPd(H)U nanozyme of the present invention can accurately target thrombus;

[0034] 2. Synergistic thrombolytic effect: CMPd(H)U nanozyme combined with NIR irradiation can enhance the release of urokinase (uPA) and release reducing hydrogen bubbles at the same time, achieving the synergistic effect of uPA, photothermal and hydrogen bubbles, and improving the thrombolytic efficiency.

[0035] 3. Long-term hydrogen therapy: The released hydrogen can not only remove reactive oxygen species and reduce reperfusion injury, but also inhibit the M1 polarization of microglia, promote M2 polarization, and exert anti-inflammatory effects, which provides a new idea for long-term treatment after stroke.

[0036] 4. Neuroprotective effect: CMPd(H)U nanozyme protects nerves by inhibiting neuronal pyroptosis, thus helping to restore neuromotor function after stroke, which is of great significance for reducing disability caused by stroke and improving the quality of life of patients.

[0037] 5. Improve drug delivery efficiency: Since hydrogen therapy can enhance the delivery efficiency of nanodrugs into the brain, the strategy of the present invention improves the efficacy, especially in the presence of the blood-brain barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 (a1) is a SEM image of the MOF synthesized in one embodiment of the present invention; Figure 1 (a2) is a SEM image of MPd nanoparticles synthesized in one embodiment of the present invention; Figure 1 (a3) is a SEM image of CMPd nanoparticles synthesized in one embodiment of the present invention; Figure 1 (a4) is a SEM image of CMPdU nanoparticles synthesized in one embodiment of the present invention;

[0039] Figure 2 X-ray diffraction (XRD) patterns of MOF nanoparticles and CMPd(H)U nanozymes synthesized in one embodiment of the present invention;

[0040] Figure 3 This is a graph showing the uPA release ability test results of the CMPd(H)U nanozyme synthesized in one embodiment of the present invention;

[0041] Figure 4 This is a graph showing the hydrogen release ability test results of the CMPd(H)U nanozyme synthesized in one embodiment of the present invention;

[0042] Figure 5 (a) is one of the detection results of CD86 in microglia cells according to an embodiment of the present invention; Figure 5(b) is the second figure of the detection result of CD86 in microglia cells according to an embodiment of the present invention; Figure 5 (c) is one of the detection result graphs of CD206 in microglia cells according to one embodiment of the present invention; Figure 5 (d) is the second figure of the detection result of CD206 in microglia cells according to an embodiment of the present invention;

[0043] Figure 6 (a) is a graph showing the inhibitory effect of the CMPd(H)U nanozyme synthesized in one embodiment of the present invention on neuronal pyroptosis; Figure 6 (b) is the second test result of the inhibitory effect of the CMPd(H)U nanozyme synthesized in one embodiment of the present invention on neuronal pyroptosis;

[0044] Figure 7 In vitro fluorescence images of the distribution of MPd(H)U and CMPd(H)U nanozymes synthesized in one embodiment of the present invention in the brain;

[0045] Figure 8 (a) Schematic diagram of the rotarod test apparatus; Figure 8 (b) is one of the test results of the CMPd(H)U nanozyme synthesized in one embodiment of the present invention on mouse behavior; Figure 8 (c) is a schematic diagram of the apparatus for the cylinder experiment; Figure 8 (d) is the second figure of the test results of the CMPd(H)U nanozyme synthesized in one embodiment of the present invention on mouse behavior.

[0046] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be understood by people with general skills in the field to which the present invention belongs. "Including" and similar words used in this article mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0048] Example 1

[0049] 1. Synthesis of MOF nanoparticles:

[0050] Dissolve Pluronic F127 (50 mg) in 3 mL of deionized water, add acetic acid (0.4 mL) and sodium perchlorate (150 mg) and continue stirring. Add zirconium nitrate (115.6 mg) and 2-aminobenzenedicarboxylic acid (50 mg) and stir at 40 ° C for 24 hours. After the reaction is completed, collect the solid by centrifugation. Wash twice with deionized water and DMF respectively, soak the synthesized sample in anhydrous ethanol for 2 days, and replace fresh ethanol once during the period. Finally, the product is vacuum dried at 60 ° C for 12 hours and then used.

[0051] 2. Synthesis of MPd nanoparticles:

[0052] 20 mg of MOF nanoparticles were added to a mixed solution of 2 mL of ethanol and 2 mL of deionized water. After ultrasonic dispersion, 500 μL of an aqueous solution of sodium chloropalladate (3.6 mg) was added. After magnetic stirring for 3.5 h, the system was placed in an ice bath, and 2.4 mL of an ice-cold aqueous solution of sodium borohydride (4.5 mg) was added. The stirring in the ice bath was continued for 1 h. After centrifugation, the system was washed three times with water to obtain black MPd nanoparticles.

[0053] 3. Synthesis of CMPd nanoparticles:

[0054] The EDC / NHS method was used to connect the CREKA peptide. Take 20 mg of MPd nanoparticles, add 5 mL of deionized water, and then sonicate for 10 minutes. Add EDC (0.1 mmol) and NHS (0.5 mmol), and stir for 1 hour in an ice bath. After centrifugation, add 5 mL of deionized water and CREKA (5 mg), and stir at room temperature for 12 hours. Wash the product three times with deionized water to remove free peptides. Collect by centrifugation to obtain CMPd nanoparticles.

[0055] 4. Synthesis of CMPdU Nanoparticles:

[0056] Deionized water was added to the CMPd nanoparticles to make a solution of 1 mg mL -1 Add 4.5 mg uPA to the CMPd aqueous solution, stir overnight at room temperature in the dark, wash twice with water, collect by centrifugation to obtain CMPdU nanoparticles, freeze-dry in vacuum and store in a refrigerator at 4°C for later use.

[0057] 5. Synthesis of CMPd(H)U nanozyme:

[0058] 10 mg of CMPdU nanoparticles were dispersed in 10 mL of deionized water and ultrasonically dispersed to prepare a CMPdU solution. Then, a hydrogen generation device was constructed: two 10 mL vials (bottle A and bottle B), 2 mL of CMPdU solution was injected into bottle A and sealed with a rubber stopper, and 1 g of sodium borohydride was added to bottle B and sealed with a rubber stopper. The two vials were connected by a hose. A needle was inserted into bottle A to connect to the atmosphere. Then, 1 mL of dilute sulfuric acid with a pH of 3 was injected into bottle B to generate hydrogen, and hydrogen was pumped into the CMPdU solution for 30 minutes to obtain 1 mg mL -1 The CMPd(H)U nanozyme solution was sealed and stored in a dark place at 4°C for future use. "The CMPd(H)U nanozyme solution should be prepared and used immediately", and the CMPd(H)U nanozyme solution was centrifuged and freeze-dried to obtain CMPd(H)U nanozyme powder.

[0059] Test Example 1

[0060] Characterization of CMPd(H)U by SEM and XRD

[0061] like Figure 1 As shown, it can be directly observed from the SEM images that the surfaces of (a1) MOF nanoparticles, (a2) MPd nanoparticles and (a3) ​​CMPd nanoparticles prepared in Example 1 contain a large number of uniformly open pores, and the pores on the surface of (a4) CMPdU nanoparticles have disappeared, indicating that uPA has been successfully loaded into the mesopores of CMPd; the prepared MOF and CMPd(H)U nanozymes have obvious characteristic peaks at 2θ=7.43°, 8.55°, and 25.76°, as shown in FIG. Figure 2 As shown, it is in good agreement with the standard card of MOF (CCDC1405751), indicating that the framework structure of MOF is still well maintained after a series of modifications.

[0062] Test Example 2

[0063] uPA release ability of CMPd(H)U nanozymes

[0064] 5 mg of CMPd(H)U prepared in Example 1 was dispersed in 5 mL of PBS buffer at pH 7.4 and the mixture was stirred at 808 nm at 1.5 W cm -2 Irradiate under NIR or place at 37° C. Collect the supernatant every 20 min, and measure the release amount of uPA in the supernatant using BCA protein quantitative detection kit (Biyuntian).

[0065] Results: This test case studied the uPA release behavior of CMPd(H)U nanozyme under NIR irradiation (With NIR) and at 37°C (Without NIR). Figure 3As shown, under NIR irradiation (808 nm, 1.5 W / cm 2 ), the release of uPA in 2 h was about 78.80%, while it was only 8.51% at 37°C. The uPA release rate under NIR was about 9 times that without light.

[0066] Test Example 3

[0067] Hydrogen release ability of CMPd(H)U nanozymes

[0068] Methylene blue (MB) was used as a probe to detect the release of reduced hydrogen in CMPd(H)U nanozymes. First, the standard curve of MB absorbance at 664 nm was determined for quantitative analysis of the amount of reduced hydrogen released. 150 μL CMPd(H)U (1 mg mL -1 ) nanozyme solution was added to 2.85 mL MB (10 μg mL -1 ) solution, and then the absorption curve of MB was measured in real time using an ultraviolet spectrophotometer, and the NIR group was irradiated with near-infrared laser.

[0069] Results: Figure 4 As shown in the figure, the process of releasing reduced hydrogen in CMPd(H)U nanozyme can last for about 60 hours, and the first 8 hours is a rapid release stage (about 50% of hydrogen can be released). This shows that NIR enhances the release of reduced hydrogen in CMPd(H)U nanozyme, shortening the release process to 12 hours.

[0070] Test Example 4

[0071] Anti-inflammatory effect of CMPd(H)U nanozyme

[0072] Set up G1: Control, G2: LPS, G3: LPS + CMPdU + NIR, G4: LPS + CMPd(H)U, G5: LPS + CMPd(H)U + NIR, a total of five groups of experiments, including:

[0073] G1: Normal cultured microglia.

[0074] G2: Add LPS (1 μg mL -1 ) to induce an inflammatory model. After incubation for 24 h, the cells were collected and resuspended, and then antibodies CD11b and CD86 or CD206 were added. After mixing, they were stained at room temperature in the dark for 30 min. After washing with PBS twice, the expression of CD206 and CD86 in microglia was detected by flow cytometry.

[0075] G3: Add LPS (1 μg mL -1) to induce an inflammatory model, and then mixed with 100 μg mL -1 After 4 h of incubation, the NIR group was treated with 808 nm NIR (1.5 W cm -2 ) irradiated the cells for 10 min, incubated for another 20 h, and then collected and resuspended. Antibodies CD11b and CD86 or CD206 were then added, mixed, and stained at room temperature in the dark for 30 min. After washing with PBS twice, the expression of CD206 and CD86 in microglia was detected by flow cytometry.

[0076] G4: Add LPS (1 μg mL -1 ) to induce an inflammatory model, and then mixed with 100 μg mL -1 After 4 hours of incubation, the cells were collected and resuspended for another 20 hours. Antibodies CD11b and CD86 or CD206 were then added, mixed and stained at room temperature in the dark for 30 minutes. After washing with PBS twice, the expression of CD206 and CD86 in microglia was detected by flow cytometry.

[0077] G5: Add LPS (1 μg mL -1 ) to induce an inflammatory model, and then mixed with 100 μg mL -1 After 4 h of incubation, the NIR group was treated with 808 nm NIR (1.5 W cm -2 ) irradiated the cells for 10 min, incubated for another 20 h, and then collected and resuspended. Antibodies CD11b and CD86 or CD206 were then added, mixed, and stained at room temperature in the dark for 30 min. After washing with PBS twice, the expression of CD206 and CD86 in microglia was detected by flow cytometry.

[0078] It should be noted that CD11b is a marker for microglia, CD86 is a marker for M1 (pro-inflammatory) microglia, and CD206 is a marker for M2 (anti-inflammatory) microglia.

[0079] Results: Figure 5As shown, flow cytometry was used to detect the expression of CD206 and CD86 in microglia after different treatments. The number of CD206-positive cells in the LPS+CMPd(H)U+NIR group was approximately 6.7 times that of the LPS group, approximately 2.2 times that of the LPS+CMPdU+NIR group, and approximately 1.3 times that of the LPS+CMPd(H)U group; the number of CD86-positive cells in the LPS+CMPdU+NIR group was approximately 73.8% of the LPS group, the number of CD86-positive cells in the LPS+CMPd(H)U group was approximately 54.5% of the LPS group, and the number of CD86-positive cells in the LPS+CMPd(H)U+NIR group was approximately 42.2% of the LPS group.

[0080] Test Example 5

[0081] Inhibitory effect of CMPd(H)U nanozyme on neuronal pyroptosis

[0082] Five groups of experiments were set up: G1: Control, G2: OGD / R, G3: OGD / R+CMPdU+NIR, G4: OGD / R+CMPd(H)U, G5: OGD / R+CMPd(H)U+NIR. After the cells were cultured according to the predetermined plan, they were lysed and proteins were collected for detection by western blotting, among which:

[0083] G1: Normal cultured mouse hippocampal neuronal cells (HT22).

[0084] G2: The DMEM complete medium (with glucose) of HT22 cells was replaced with the sugar-free DMEM medium (without glucose), and the cells were cultured in a sealed chamber filled with an anaerobic bag with 5% CO2. After 4 hours, the cells were taken out of the chamber and replaced with the DMEM complete medium, and cultured for another 24 hours.

[0085] G3: The HT22 DMEM complete medium (with glucose) was replaced with sugar-free DMEM medium (without glucose), and the cells were cultured in a sealed chamber filled with an anaerobic bag containing 5% CO2. After 4 h, the cells were taken out of the chamber and replaced with 100 μg mL -1 The DMEM medium containing CMPdU was irradiated with 808 nm NIR (1.5 W cm -2 ) Irradiate the cells for 10 min and continue culturing for 24 h.

[0086] G4: The HT22 DMEM complete medium (with glucose) was replaced with sugar-free DMEM medium (without glucose), and the cells were cultured in a sealed chamber filled with an anaerobic bag containing 5% CO2. After 4 h, the cells were taken out of the chamber and replaced with 100 μg mL -1The cells were cultured in complete DMEM medium containing CMPd(H)U and the culture was continued for 24 h.

[0087] G5: The HT22 DMEM complete medium (with glucose) was replaced with sugar-free DMEM medium (without glucose), and the cells were cultured in a sealed chamber filled with an anaerobic bag containing 5% CO2. After 4 h, the cells were taken out of the chamber and replaced with 100 μg mL -1 The CMPd(H)U in DMEM complete medium was irradiated with 808 nm NIR (1.5 W cm -2 ) Irradiate the cells for 10 min and continue culturing for 24 h.

[0088] Results: GSDMD-N is a marker of cell pyroptosis. This test case induced pyroptosis of HT22 cells by the oxygen-glucose deprivation / reoxygenation model (OGD / R). The effect of nanoparticles on inhibiting pyroptosis of HT22 cells induced by oxygen-glucose deprivation / reoxygenation (OGD / R) was analyzed by using protein immunoblotting. Figure 6 The results showed that compared with the OGD / R group, the expression level of GSDMD-N protein in the OGD / R+CMPdU+NIR group decreased by approximately 5.5%, in the OGD / R+CMPd(H)U group decreased by approximately 63.6%, and in the OGD / R+CMPd(H)U+NIR group decreased by approximately 68.1%.

[0089] Test Example 6

[0090] Distribution of CMPd(H)U in mouse brain

[0091] Cy5-labeled MPd(H)U (non-targeted group) and CMPd(H)U (targeted group) were injected into the tail vein, and the mice were euthanized at the preset time points, and the brains were collected for in vitro imaging. Figure 7 As shown, the fluorescence signal observed in the right (stroke side) brain 1 h after injection of Cy5-CMPd(H)U was approximately 1.7 times that of the left (normal side) brain, indicating that CMPd(H)U nanozyme can rapidly target thrombi at the site of cerebral ischemia.

[0092] Test Example 7

[0093] Evaluation of the therapeutic effect of CMPd(H)U on neuromotor function after stroke in mice

[0094] 1. Construction of mouse stroke model

[0095] After the mice were anesthetized with 2% isoflurane, the hair of the scalp was shaved, the scalp was cut and the periosteum was removed, and the mice were placed in a stereotaxic apparatus and anesthetized with 1% isoflurane. Then 1% (w / w) Bengal rose red solution (30 mg / kg) was injected intraperitoneally. Five minutes later, a 532nm green laser (power of 100mW) was used to irradiate the sensorimotor cortex of the right brain of the mice for 10 minutes to form an infarct lesion. After the laser irradiation, the scalp was sutured, iodine was applied for disinfection, and the mice were placed on a 37°C warming blanket until they woke up, and then raised normally. Among them, the sham operation group was only injected with rose red solution without green laser irradiation.

[0096] 2. Grouping and Treatment of Mice

[0097] After the mouse ischemic stroke disease model was established, the mice were divided into 5 groups for treatment, with 8 mice in each group. G1: sham operation group; G2: uPA treatment group; G3: CMPd(H)+NIR treatment group; G4: CMPdU+NIR treatment group; G5: CMPd(H)U+NIR treatment group. The injection volume of the G2 group was 1.6 mg / kg, and the injection volumes of G3-G5 were all 5 mg / kg. The amount of uPA in the G2 group was the same as that in the G5 group, and the NIR was 808 nm (1.5 W / cm 2 ), the irradiation time was 20 minutes, and 1 mg / kg means that 1 mg of uPA or CMPd(H) or CMPdU or CMPd(H)U needs to be injected per kg of mouse.

[0098] In addition, the preparation process of CMPd (H) solution is as follows: 10 mg of CMPd nanoparticles are dispersed in 10 mL of physiological saline, and the CMPd solution is uniformly prepared by ultrasonic dispersion. Then a hydrogen generating device is constructed: two 10 mL vials (bottle A and bottle B, respectively), 2 mL of CMPd solution is injected into bottle A and sealed with a rubber stopper, 1 g of sodium borohydride is added to bottle B and sealed with a rubber stopper. The two vials are connected by a hose. Insert a needle into bottle A and connect to the atmosphere. Then, 1 mL of dilute sulfuric acid is injected into bottle B to generate hydrogen, and the hydrogen is pumped into the CMPd solution for 30 minutes to obtain a CMPd (H) solution.

[0099] 3. Mouse behavioral testing

[0100] The rotarod test and cylinder test were used to evaluate the neuromotor kinetic recovery of stroke mice after CMPd(H)U nanozyme treatment.

[0101] Rotarod test: Three days before modeling, mice were placed on the rotarod for training, 3 times a day, 5 minutes each time, 30 minutes apart. Tests were performed on the 1st, 3rd, and 7th days after treatment. The speed was accelerated from 4 rpm to 40 rpm within 2 minutes and continued for 5 minutes until the animal fell. The average time on the rod was recorded as the fall latency. Clean the instrument with medical alcohol after training or testing.

[0102] Cylinder test: Place the mouse in a 500 mL transparent beaker and place a mirror behind the beaker to record the mouse's forelimb movements from all directions. Use a video camera to record the contact between the forepaw and the wall for 5 minutes.

[0103] Calculate the forelimb use asymmetry score according to the formula.

[0104]

[0105] Among them, R, L and B refer to the number of times the right front paw alone, the left front paw alone and the two front paws were used simultaneously when contacting the cylinder wall.

[0106] Results: The apparatus for the rotating rod test and the cylinder test were as follows: Figure 8 a, 8c. The motor coordination ability of mice after stroke is very poor, as shown in Figure 8 As shown in b, the motor coordination ability of mice treated with CMPd(H)U+NIR was greatly improved, and they could stay on the rotating rod for a longer time (about 2.3 times that of the uPA treatment group, 2.2 times that of the CMPd(H)+NIR treatment group, and 1.6 times that of the CMPdU+NIR treatment group). The shape of the cylinder can stimulate mice to use their forelimbs to vertically explore the cylinder wall. The nerve damage caused by stroke makes stroke mice tend to use the unaffected forelimbs, so stroke mice show obvious asymmetry in the use of forelimbs. Figure 8 As shown, compared with the uPA-treated group, the forelimb asymmetry score of stroke mice treated with CMPd(H)+NIR in the cylinder test 7 days after treatment was not significantly decreased; the CMPdU+NIR-treated group decreased by approximately 42.1%, and the CMPd(H)U+NIR group decreased by approximately 71.7%.

[0107] In summary, the present invention uses metal organic framework materials (MOF) and palladium as carriers, and urokinase and hydrogen as drugs to prepare a multifunctional CMPd(H)U nanozyme. It can not only quickly target the thrombus site, but also cooperate with gas therapy to quickly clear the thrombus and restore blood flow. At the same time, the released hydrogen can remove reactive oxygen species to reduce reperfusion injury, and can also regulate microglia to polarize to M2 phenotype and inhibit neuronal pyroptosis to play anti-inflammatory and neuroprotective effects. Ultimately, it promotes the recovery of neuromotor kinetic energy in stroke mice. Inspired by ice beer, the constructed nano drug delivery system that can quickly dissolve thrombus and release hydrogen for a long time is expected to solve key problems such as poor drug targeting ability, inability to reduce reperfusion injury, adverse reactions, and neuromotor function damage.

[0108] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A method for preparing a multifunctional CMPd(H)U nanozyme, characterized in that: The multifunctional CMPd(H)U nanozyme is prepared by using a metal organic framework material and palladium as carriers, urokinase and hydrogen as drugs, synthesizing a mesoporous MOF by a template method, then anchoring palladium nanoparticles, modifying the mesoporous MOF with a thrombus targeting peptide CREKA, and then loading uPA and hydrogen.

2. The method for preparing the multifunctional CMPd(H)U nanozyme according to claim 1, characterized in that: The preparation method comprises: Mesoporous MOF nanoparticles were synthesized by reacting zirconium oxynitrate and 2-aminoterephthalic acid using Pluronic F127 as template; The MOF solution was prepared using mesoporous MOF nanoparticles, and the sodium tetrachloropalladate solution was added to the MOF solution and stirred evenly. The sodium borohydride solution was added to 2+ Reduction to palladium nanoparticles, anchored on mesoporous MOF nanoparticles to obtain MPd nanoparticles; CREKA peptide was linked to MPd nanoparticles using the EDC / NHS method to obtain CMPd nanoparticles; Loading urokinase into the mesopores of CMPd nanoparticles to obtain CMPdU nanoparticles; A hydrogen generating device was built, and hydrogen was loaded onto CMPdU nanoparticles through the hydrogen generating device to obtain CMPd(H)U nanozyme.

3. The method for preparing the multifunctional CMPd(H)U nanozyme according to claim 2, characterized in that: The steps of synthesizing mesoporous MOF nanoparticles by using Pluronic F127 as a template and reacting zirconium oxynitrate and 2-aminoterephthalic acid include: Dissolve Pluronic F127 in deionized water, add acetic acid and sodium perchlorate, and continue to stir evenly; Zirconium oxynitrate and 2-aminobenzenedicarboxylic acid were added and stirred at 40° C. for 24 h. After the reaction was completed, the mesoporous MOF nanoparticles were collected by centrifugation.

4. The method for preparing the multifunctional CMPd(H)U nanozyme according to claim 2, characterized in that: The MOF solution is prepared by using mesoporous MOF nanoparticles, and sodium tetrachloropalladate solution is added to the MOF solution and stirred evenly, and sodium borohydride solution is added to Pd 2+ The steps of reducing palladium nanoparticles and anchoring them on mesoporous MOF nanoparticles to obtain MPd nanoparticles include: Mesoporous MOF nanoparticles were added to a mixed solution of ethanol and deionized water, and after ultrasonic dispersion, an aqueous solution of sodium tetrachloropalladate was added. After magnetic stirring for 3.5 h, the system was placed in an ice bath, and then an ice-cold aqueous solution of sodium borohydride was added. The system was stirred in the ice bath for 1 h, and then washed three times with water after centrifugation to obtain black MPd nanoparticles.

5. The method for preparing the multifunctional CMPd(H)U nanozyme according to claim 2, characterized in that: The step of connecting the CREKA peptide to the MPd nanoparticles using the EDC / NHS method to obtain the CMPd nanoparticles comprises: MPd nanoparticles were added to deionized water and then sonicated. EDC and NHS were added after sonication, stirred for 1 h in an ice bath, and then centrifuged. After centrifugation, deionized water and CREKA were added and stirred at room temperature for 12 h; The product was washed three times with deionized water to remove free polypeptides, and CMPd nanoparticles were collected by centrifugation.

6. The method for preparing the multifunctional CMPd(H)U nanozyme according to claim 2, characterized in that: The step of loading urokinase into the mesopores of CMPd nanoparticles to obtain CMPdU nanoparticles comprises: Deionized water was added to the CMPd nanoparticles to prepare a CMPd aqueous solution, and uPA was added. The mixture was stirred at room temperature in the dark, and then washed twice with water. The mixture was collected by centrifugation to obtain CMPdU nanoparticles.

7. The method for preparing the multifunctional CMPd(H)U nanozyme according to claim 2, characterized in that: The steps of constructing a hydrogen generating device and loading hydrogen onto CMPdU nanoparticles through the hydrogen generating device to obtain CMPd(H)U nanozyme include: Take two vials, defined as vial A and vial B; The CMPdU nanoparticles were dispersed in deionized water and ultrasonically dispersed to uniformly prepare a CMPdU solution; The prepared CMPdU solution was injected into bottle A and sealed with a rubber stopper, sodium borohydride was added into bottle B and sealed with a rubber stopper, and bottles A and B were connected by a hose, and then a needle was inserted into bottle A to connect to the atmosphere; Dilute sulfuric acid was injected into bottle B to generate hydrogen gas, and the hydrogen gas was pumped into the CMPdU solution for 30 min to obtain the CMPd(H)U nanozyme solution.

8. A multifunctional CMPd(H)U nanozyme, characterized in that: The multifunctional CMPd(H)U nanozyme is prepared according to the preparation method of the multifunctional CMPd(H)U nanozyme according to any one of claims 1-7.

9. Use of the multifunctional CMPd(H)U nanozyme as claimed in claim 8 in a drug for treating ischemic stroke.

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