A metal-organic framework nanomaterial, its preparation method and application

Abstract

A metal-organic framework (MOF) nanomaterial, its preparation method, and its application belong to the field of bionanomaterials technology. It addresses the technical problem of inhibiting atherosclerosis using biomimetic nanoenzymes derived from metal-organic frameworks. The solution involves the composition and proportion of the MOF nanomaterial as follows: [The remaining text appears to be incomplete and requires further context for accurate translation.] 4+ The molar ratio of ions to fumaric acid ligand is 5:1. The preparation method is as follows: a mixed solution is prepared by measuring FA regulator and water; then fumaric acid and cerium ammonium nitrate are added dropwise to the mixed solution; the mixture is stirred at room temperature and centrifuged; the solution is washed repeatedly with alternating water and ethanol; and finally, it is dried overnight in a vacuum oven. This invention provides a simple, efficient, and convenient method for synthesizing MOF nanomaterials. The synthesized MOF nanomaterials possess excellent CEH-like enzyme activity and intrinsic antioxidant activity, and can be applied to increase lipid efflux and reduce lipid uptake, decrease ox-LDL-induced foam cell formation, and thus alleviate atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of bionanomaterials technology, specifically relating to a metal-organic framework nanomaterial, its preparation method, and its applications. Background Art

[0002] Atherosclerosis (AS), an inflammatory, immune, and lipid metabolism disorder, is a leading cause of death and disability worldwide due to cardiovascular and cerebrovascular diseases such as coronary heart disease, stroke, and myocardial infarction. Therefore, the treatment of AS plays a crucial role in combating cardiovascular disease. A hallmark of AS progression is the formation and accumulation of cholesterol-rich foam cells in the subendothelial layer of affected arteries. Foam cell formation is primarily due to an imbalance in lipid uptake, lipid processing, and reverse cholesterol transport. During the plaque stage and later progression of AS, large amounts of unlipidated free cholesterol accumulate extracellularly and are primarily transported by high-density lipoprotein (HDL) via reverse cholesterol transport (RCT). Numerous studies have shown that damage to macrophage RCT accelerates AS progression, while enhancing macrophage RCT can prevent or even reverse AS. Since cholesterol exists primarily in foam cells formed by macrophages as a bound form of cholesterol esters, studies have explored the use of natural cholesterol ester hydrolases (CEHs) to improve atherosclerosis. However, the preparation of natural enzymes is complex, unstable, expensive, and has low recycling rates, making this approach very challenging. Furthermore, the efficacy of improving atherosclerosis solely through increasing lipid efflux—a single lipid downregulation mechanism—is limited because macrophages continue to uptake lipids to form foam cells; therefore, it is necessary to simultaneously reduce lipid uptake.

[0003] Macrophages are the main source of foam cells, and oxidized low-density lipoprotein (ox-LDL), a modified low-density lipoprotein, is considered to be the main apolipoprotein accumulated during foam cell formation. Numerous studies have shown that ox-LDL levels in plaques are closely related to oxidative stress induced by high levels of reactive oxygen species (ROS). Generally, intracellular ROS-modified LDL produces ox-LDL, suggesting that reducing ROS production in plaques to inhibit ox-LDL formation may attenuate atherosclerosis. In recent years, nanoparticles with natural enzyme-mimicking catalytic activity have attracted widespread attention due to their high stability, low cost, and ease of preparation. Most importantly, nanozymes, compared to natural enzymes and traditional enzyme mimics, can rationally integrate multiple enzyme activities.

[0004] Metal-organic frameworks (MOFs) are a class of novel porous inorganic-organic hybrid materials composed of metal ions / clusters and organic ligands. Due to their diverse metal ion and ligand structures, high specific surface area, efficient drug loading, and high safety, MOF-based nanozymes have shown broad application prospects and have been widely used to scavenge free radicals and treat ROS-mediated ischemic stroke, rheumatoid arthritis, radiation damage, and other diseases. However, nanozymes with CEH-like enzyme activity have rarely been discovered; moreover, little attention has been paid to the therapeutic effects of nanozymes themselves on atherosclerosis. Therefore, it is necessary to develop new MOF materials that simultaneously possess CEH-like enzyme activity and intrinsic antioxidant activity to increase lipid efflux and reduce lipid uptake, thereby reducing ox-LDL-induced foam cell formation and alleviating atherosclerosis. Summary of the Invention

[0005] The main objective of this invention is to overcome the shortcomings of the prior art and solve the technical problem of inhibiting atherosclerosis with metal-organic framework biomimetic nanoenzymes. This invention provides a metal-organic framework nanomaterial, its preparation method, and its application.

[0006] This invention is achieved through the following technical solution:

[0007] A metal-organic framework nanomaterial, characterized in that: the composition and ratio of the metal-organic framework nanomaterial are as follows: the metal ion is Ce. 4+ The ion, with fumaric acid as the ligand, Ce 4+ The molar ratio of ions to fumaric acid is 5:1.

[0008] Furthermore, the metal-organic framework nanomaterials possess cholesterol ester hydrolase activity and intrinsic antioxidant activity.

[0009] A method for preparing metal-organic framework nanomaterials includes the following steps:

[0010] S1. Measure 1.132mL-18.68mL of formic acid regulator and water to prepare a 30mL mixed solution. Mix the formic acid regulator and water evenly.

[0011] S2. Add 1.74g fumaric acid and 1.6447g cerium ammonium nitrate dropwise to the mixed solution prepared in step S1, stir at room temperature for 10 minutes, then centrifuge at 4000rpm for 5 minutes, and finally wash repeatedly with water and ethanol three times to obtain solid material.

[0012] S3. The solid material prepared in step S2 is dried overnight in a vacuum oven at 60°C to obtain metal-organic framework nanomaterials.

[0013] Furthermore, in step S3, the size range of the metal-organic framework nanomaterials obtained is 50-200 nm, and the more formic acid regulator is added in step S1, the larger the size of the metal-organic framework nanomaterials obtained.

[0014] An application of the metal-organic framework nanomaterials described above includes:

[0015] Its application in the preparation of drugs that alleviate atherosclerosis;

[0016] Applications in the preparation of drugs that increase lipid outflow and reduce lipid upflow;

[0017] Application in the preparation of drugs that reduce macrophages and foam cells in endothelial cells.

[0018] The beneficial effects of the present invention are:

[0019] This invention provides a simple, efficient, and convenient method for synthesizing MOF nanomaterials. The synthesized MOF nanomaterials have excellent CEH enzyme-mimicking activity and intrinsic antioxidant activity, increase lipid efflux and reduce lipid uptake, and reduce ox-LDL-induced foam cell formation, thereby alleviating atherosclerosis. Attached Figure Description

[0020] Figure 1 Transmission electron microscope image of the MOF nanomaterials prepared in Example 1;

[0021] Figure 2 High-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of the MOF nanomaterials prepared in Example 1;

[0022] Figure 3 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the MOF nanomaterials prepared in Example 1.

[0023] Figure 4 The image shows the CEH enzyme-mimicking activity test result of the MOF nanomaterials prepared in Example 1.

[0024] Figure 5 The image shows the SOD activity of the MOF nanomaterials prepared in Example 1.

[0025] Figure 6 The image shows the CAT activity test results of the MOF nanomaterials prepared in Example 1.

[0026] Figure 7 This is a biosafety test diagram of the MOF nanomaterials prepared in Example 1;

[0027] Figure 8The graph shows the antioxidant properties of the MOF nanomaterials prepared in Example 1.

[0028] Figure 9 This is a test image showing the MOF nanomaterials prepared in Example 1 clearing excess ROS generated in cells;

[0029] Figure 10 This is a test image showing the inhibition of foam cell formation by the MOF nanomaterials prepared in Example 1.

[0030] Figure 11 Transmission electron microscope image of the MOF nanomaterials prepared in Example 2;

[0031] Figure 12 Transmission electron microscope image of the MOF nanomaterials prepared in Example 3. DETAILED DESCRIPTION

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Example 1

[0033] A method for preparing metal-organic framework nanomaterials includes the following steps:

[0034] S1. Measure 2.264 mL of formic acid (FA) regulator and 27.736 mL of water to prepare a 30 mL mixed solution. Mix the FA regulator and water thoroughly.

[0035] S2. Add 1.74 g (15 mmol) fumaric acid (FMA) and 1.6447 g (15 mmol) cerium ammonium nitrate Ce(NH4)2(NO3)2 dropwise to the mixed solution prepared in step S1, stir at room temperature for 10 minutes, then centrifuge at 4000 rpm for 5 minutes, and finally wash repeatedly with water and ethanol three times to obtain solid material.

[0036] S3. The solid material prepared in step S2 is dried overnight in a vacuum oven at 60°C to obtain metal-organic framework (MOF) nanomaterials.

[0037] The composition and proportions of the MOF nanomaterials prepared in Example 1 are as follows: the metal ion is Ce. 4+ Ions, with ligands FMA and Ce 4+ The molar ratio of ions to FMA is 5:1.

[0038] Figure 1 Transmission electron microscope images of the MOF nanomaterials prepared in Example 1, by Figure 1 It can be seen that MOF nanomaterials were successfully synthesized, with a particle size of 100 nm and slight aggregation.

[0039] Figure 2 Here are high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images of the MOF nanomaterials prepared in Example 1, produced by... Figure 2 It can be known that its main elements are Ce, C, and O.

[0040] The application of MOF nanomaterials in the preparation of drugs to alleviate atherosclerosis: Example 1 specifically describes the preparation of drugs to alleviate apolipoprotein E deficiency (ApoE). - / - The application of MOF nanomaterials in drugs for treating atherosclerosis in mice is due to their excellent CEH activity and intrinsic antioxidant activity. Therefore, they can be used to increase lipid efflux and reduce lipid uptake, thereby reducing foam cells (ox-LDL-induced foam cells) in macrophages and endothelial cells, as detailed below.

[0041] I. CEH Enzyme-Mimicking Activity Assay of MOF Nanomaterials

[0042] 1) Experimental principle: Cholesterol esters are first hydrolyzed under the catalysis of cholesterol ester hydrolase to generate free cholesterol and fatty acids. The free cholesterol esters are further oxidized by cholesterol oxidase with oxygen to generate H2O2 and cholesterol ketones. The content of free cholesterol is determined by detecting the absorbance of the reaction product of H2O2 and Amplex Red, halogenated ester. Natural cholesterol ester hydrolase is used as a positive control.

[0043] 2) Prepare a cholesterol benzoate solution (50 μmol) and add cholesterol ester hydrolase or MOF nanomaterial solutions of different concentrations (0, 50, 100 and 200 μg / mL);

[0044] 3) Add cholesterol detection liquid, incubate at 37°C in the dark for 30 minutes, and measure the absorbance at 570nm.

[0045] II. Superoxide Dismutase (SOD) Activity Assay of MOF Nanomaterials

[0046] 1) Using xanthine (1 mM) and xanthine oxidase (0.1 U / mL) as O2• - Generation system;

[0047] 2) MOF nanomaterial solutions of different concentrations (0, 50, 100 and 200 μg / mL) were reacted with the above system at 37°C for 30 minutes;

[0048] 3) Detect residual O2• - Calculate the concentration of O2• - Its ability to clear debris.

[0049] III. Catalase (CAT) Activity Assay in MOF Nanomaterials

[0050] 1) CAT activity, namely the hydroxyl radical scavenging ability of MOF nanomaterials, is obtained by measuring the degree of inhibition of 2-hydroxyterephthalic acid;

[0051] 2) Mix 0.5 mM terephthalic acid, 10 mM H2O2 and MOF nanomaterial solutions of different concentrations (0, 50, 100 and 200 μg / mL) with 25 mM phosphate buffer solution at pH 7.4 and react at 37°C for 24 hours.

[0052] 3) Since terephthalic acid itself has no fluorescence, it can capture hydroxyl radicals to form 2-hydroxyterephthalic acid, which has strong fluorescence. This oxidation product is excited at 320 nm and emits at 425 nm. Therefore, the fluorescence spectrum at 425 nm under the excitation wavelength of 320 nm is collected.

[0053] IV. Biocompatibility Testing of MOF Nanomaterials

[0054] 1) Human umbilical vein endothelial cells (HUVECs) were injected at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of / wells into 96-well plates and incubated at 37°C. o Overnight growth under C and 5% CO2 atmosphere;

[0055] 2) Remove the upper culture medium and add 100 μL of fresh culture medium containing MOF nanomaterials at concentrations of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 and 200 μg / mL, respectively. Incubate together for 24 hours.

[0056] 3) Add 10 µL of CCK-8 solution to each well, incubate for 2 hours, and then measure the absorbance at 450 nm to calculate cell viability.

[0057] V. Antioxidant Performance Testing of MOF Nanomaterials

[0058] 1) To detect the scavenging of excess reactive oxygen species (ROS) generated intracellularly by MOF nanomaterials, HUVECs were subjected to a concentration of 4 × 10⁻⁶. 4 Cells were seeded at a density of / wells into 24-well plates and incubated at 37°C. o Overnight growth under C and 5% CO2 atmosphere;

[0059] 2) Remove the upper culture medium, add culture medium containing different concentrations (0, 50, 100 and 200 μg / mL) of MOF nanomaterials and co-culture for 4 hours. Then add serum-free culture medium with a H2O2 concentration of 60 μM to induce oxidative damage.

[0060] 3) After 20 hours, add 10 µL of CCK-8 solution to each well, incubate for 2 hours, and then measure the absorbance at 450 nm to calculate cell viability;

[0061] 4) If used for taking fluorescence images, after step 2, gently wash the cells twice with PBS to remove the old solution, add freshly prepared 2',7'-dichlorofluorescein (DCF, 10µM) dye to each well and incubate for 2 hours, then take fluorescence images of the cells using an inverted fluorescence microscope.

[0062] VI. MOF Nanomaterials Inhibit Foam Cell Formation Test

[0063] 1) HUVEC at 2×10 4 Cells were seeded at a density of / wells into 12-well plates and incubated at 37°C. o Overnight growth under C and 5% CO2 atmosphere;

[0064] 2) After removing the upper culture medium, stimulate with lipopolysaccharide (LPS, 1 µg / mL) for 24 hours, add culture medium containing different concentrations (0, 50, 100 and 200 μg / mL) of MOF nanomaterials for co-culture.

[0065] 3) After 2 hours, incubate with 50 µg / mL ox-LDL for 48 hours. The normal control group is treated with fresh culture medium, while the model group is stimulated with ox-LDL only.

[0066] 4) After 48 hours, stain the cells with Oil Red O staining (ORO) kit and observe them with an optical microscope.

[0067] The results of the above six MOF nanomaterial performance tests are analyzed as follows:

[0068] Figure 3 The X-ray photoelectron spectroscopy (XPS) spectrum of the MOF nanomaterial prepared in Example 1 shows the Ce2 content. Peak segmentation calculations yielded the Ce2 content. 4+ The atomic percentage is 86.8%, Ce 3+ The atomic percentage is 13.2%.

[0069] Figure 4 The image shows the CEH enzyme-mimicking activity test result of the MOF nanomaterials prepared in Example 1. Figure 4 It is known that MOF nanomaterials can effectively remove cholesterol esters in a concentration-dependent manner.

[0070] Figure 5 This is a graph showing the SOD activity of the MOF nanomaterials prepared in Example 1. Figure 5 It is known that MOF nanomaterials can effectively remove ROS in a concentration-dependent manner.

[0071] Figure 6 The image shows the CAT activity test results of the MOF nanomaterials prepared in Example 1. Figure 6 It is known that MOF nanomaterials can effectively remove H2O2 in a concentration-dependent manner.

[0072] Figure 7 This is a biosafety test diagram of the MOF nanomaterials prepared in Example 1, by... Figure 7 It can be seen that after co-incubating MOF nanomaterials with HUVECs cells for 24 hours, the cell survival rate was above 80%, indicating that MOF nanomaterials have good biosafety.

[0073] Figure 8 The graph shows the antioxidant properties of the MOF nanomaterials prepared in Example 1. Figure 8 It can be seen that the activity of HUVECs cells decreased after H2O2 treatment; however, the addition of MOF nanomaterials can effectively resist the damage caused by H2O2 to the cells.

[0074] Figure 9 This is a test image showing the MOF nanomaterials prepared in Example 1 clearing excess ROS generated intracellularly. (See image for details.) Figure 9 As shown, HUVECs cells exhibited the highest fluorescence intensity after H2O2 treatment, indicating the generation of excessive ROS. In contrast, MOF nanomaterial treatment significantly reduced the fluorescence intensity within HUVECs cells, demonstrating that cellular oxidation could be effectively inhibited. This indicates that MOF nanomaterials can effectively suppress ROS generation, thereby protecting cells from oxidative stress.

[0075] Figure 10 This is a test image showing the inhibition of foam cell formation by the MOF nanomaterials prepared in Example 1. Figure 10 As shown, ox-LDL stimulation causes cells to produce many lipid droplets, inducing the formation of foam cells. However, MOF nanomaterial treatment can effectively reduce the formation of foam cells. Example 2

[0076] A method for preparing metal-organic framework nanomaterials includes the following steps:

[0077] S1. Measure 1.132 mL of FA regulator and 29.887 mL of water to prepare a 30 mL mixed solution. Mix the FA regulator and water thoroughly.

[0078] S2. Add 1.74 g (15 mmol) FMA and 1.6447 g (15 mmol) Ce(NH4)2(NO3)2 dropwise to the mixed solution prepared in step S1, stir at room temperature for 10 minutes, then centrifuge at 4000 rpm for 5 minutes, and finally wash repeatedly with water and ethanol three times to obtain solid material.

[0079] S3. The solid material prepared in step S2 is dried overnight in a vacuum oven at 60°C to obtain MOF nanomaterials.

[0080] The composition and ratio of the MOF nanomaterials prepared in Example 2 are as follows: the metal ion is Ce. 4+ Ions, with ligands FMA and Ce 4+ The molar ratio of ions to FMA is 5:1.

[0081] Figure 11 Transmission electron microscope images of the MOF nanomaterials prepared in Example 2, by Figure 11 It can be seen that its particle size is 50nm and it has slight aggregation.

[0082] The MOF nanomaterials prepared in Example 2 have the same cholesterol ester hydrolase activity and intrinsic antioxidant activity as the MOF nanomaterials prepared in Example 1. They can increase lipid efflux and reduce lipid uptake, thereby reducing foam cell formation in macrophages and endothelial cells. Example 3

[0083] A method for preparing metal-organic framework nanomaterials includes the following steps:

[0084] S1. Measure 18.68 mL of FA regulator and 11.32 mL of water to prepare a 30 mL mixed solution. Mix the FA regulator and water thoroughly.

[0085] S2. Add 1.74 g (15 mmol) FMA and 1.6447 g (15 mmol) Ce(NH4)2(NO3)2 dropwise to the mixed solution prepared in step S1, stir at room temperature for 10 minutes, then centrifuge at 4000 rpm for 5 minutes, and finally wash repeatedly with water and ethanol three times to obtain solid material.

[0086] S3. The solid material prepared in step S2 is dried overnight in a vacuum oven at 60°C to obtain MOF nanomaterials.

[0087] The composition and ratio of the MOF nanomaterials prepared in Example 3 are as follows: the metal ion is Ce. 4+ Ions, with ligands FMA and Ce 4+ The molar ratio of ions to FMA is 5:1.

[0088] Figure 12 Transmission electron microscope images of the MOF nanomaterials prepared in Example 3, by Figure 12 It is known that its particle size is 200nm and it has slight aggregation.

[0089] The MOF nanomaterials prepared in Example 3 have the same cholesterol ester hydrolase activity and intrinsic antioxidant activity as the MOF nanomaterials prepared in Examples 1 and 2. They can increase lipid efflux and reduce lipid uptake, thereby reducing foam cell formation in macrophages and endothelial cells.

[0090] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of a metal-organic framework nanomaterial in the preparation of drugs to alleviate atherosclerosis, characterized in that: The composition and proportions of the metal-organic framework nanomaterial are as follows: the metal ion is Ce. 4+ The ion, with fumaric acid as the ligand, specifically consists of 1.74 g fumaric acid and 1.6447 g cerium ammonium nitrate.

2. The application of the metal-organic framework nanomaterial according to claim 1 in the preparation of drugs to alleviate atherosclerosis, characterized in that: The metal-organic framework nanomaterials possess cholesterol ester hydrolase activity and intrinsic antioxidant activity.

3. The application of the metal-organic framework nanomaterial as described in claim 1 in the preparation of a drug for alleviating atherosclerosis, characterized in that, The preparation method of metal-organic framework nanomaterials includes the following steps: S1. Measure 1.132-18.68 mL of formic acid regulator and water to prepare a 30 mL mixed solution. Mix the formic acid regulator and water thoroughly. S2. Add 1.74g fumaric acid and 1.6447g cerium ammonium nitrate dropwise to the mixed solution prepared in step S1, stir at room temperature for 10 minutes, then centrifuge at 4000rpm for 5 minutes, and finally wash repeatedly with water and ethanol three times to obtain solid material. S3. The solid material prepared in step S2 is dried overnight in a vacuum oven at 60°C to obtain metal-organic framework nanomaterials. In step S3, the size range of the metal-organic framework nanomaterials obtained is 50-200 nm, and the more formic acid regulator is added in step S1, the larger the size of the metal-organic framework nanomaterials obtained.

4. The application of the metal-organic framework nanomaterial as described in claim 1 in the preparation of a drug for alleviating atherosclerosis, characterized in that: The application of the metal-organic framework nanomaterials in the preparation of drugs that increase lipid efflux and reduce lipid uptake.

5. The application of the metal-organic framework nanomaterial as described in claim 1 in the preparation of a drug for alleviating atherosclerosis, characterized in that: The application of the metal-organic framework nanomaterials in the preparation of drugs that reduce macrophages and foam cells in endothelial cells.

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