阅读说明：本技术 金属有机骨架材料-酶复合物及其制备方法和应用 (Metal organic framework material-enzyme compound and preparation method and application thereof ) 是由 戈钧 刘雨 于 2021-09-13 设计创作，主要内容包括：本发明涉及固定化酶技术领域,具体而言,涉及一种金属有机骨架材料-酶复合物及其制备方法和应用。金属有机骨架材料-酶复合物的制备方法包括以下步骤：将抗氧化酶、过渡金属离子及有机配体溶于溶剂中进行共沉淀反应,抗氧化酶选自超氧化物歧化酶与过氧化氢酶、谷胱甘肽过氧化物酶或辣根过氧化物酶的组合酶,或谷胱甘肽还原酶与谷胱甘肽过氧化物酶、葡萄糖脱氢酶或乙醇脱氢酶的组合酶,过渡金属离子来源于可溶性金属盐,有机配体为咪唑类化合物。所述金属有机骨架材料-酶复合物具有强活性氧清除能力。(The invention relates to the technical field of immobilized enzymes, in particular to a metal organic framework material-enzyme compound and a preparation method and application thereof. The preparation method of the metal organic framework material-enzyme complex comprises the following steps: dissolving antioxidant enzyme, transition metal ions and organic ligand in a solvent for coprecipitation reaction, wherein the antioxidant enzyme is selected from the group consisting of combined enzyme of superoxide dismutase and catalase, glutathione peroxidase or horseradish peroxidase, or the combined enzyme of glutathione reductase and glutathione peroxidase, glucose dehydrogenase or alcohol dehydrogenase, the transition metal ions are derived from soluble metal salt, and the organic ligand is imidazole compound. The metal organic framework material-enzyme complex has strong active oxygen scavenging capacity.)

1. A preparation method of a metal organic framework material-enzyme complex is characterized by comprising the following steps:

dissolving antioxidase, transition metal ions and organic ligands in a solvent for coprecipitation reaction;

the antioxidant enzyme is selected from a combined enzyme of superoxide dismutase and catalase, glutathione peroxidase or horseradish peroxidase, or a combined enzyme of glutathione reductase and glutathione peroxidase, glucose dehydrogenase or alcohol dehydrogenase;

the transition metal ions are derived from soluble metal salts, and the organic ligand is an imidazole compound.

2. The method of claim 1, further comprising the steps of adding an active protectant and a synergist after the co-precipitation reaction, mixing, and drying;

the activity protective agent is one or more of glucose, fructose, sucrose, mannose, trehalose, arginine, glycine, glycerol, polyethylene glycol, chitosan and human serum albumin, and the synergistic agent is one or more of vitamin C, vitamin C palmitate, vitamin C succinate, vitamin C phosphate, vitamin E succinate, glutathione, nicotinamide mononucleotide, nicotinamide adenine dinucleotide phosphate, coenzyme Q10, resveratrol, lipoic acid, ferulic acid and phloretin.

3. The method of claim 2, wherein the active protectant and the synergist are independently selected from 0.1% to 30% by weight.

4. The method for preparing a metal-organic framework material-enzyme complex according to claim 1, wherein the mass ratio of the superoxide dismutase to the catalase, the glutathione peroxidase or the horseradish peroxidase is 1: (0.001 to 1000);

the mass ratio of the glutathione reductase to the glutathione peroxidase, the glucose dehydrogenase or the alcohol dehydrogenase is 1: (0.001-1000).

5. The method for preparing the metal-organic framework material-enzyme complex according to claim 1, wherein the transition metal ions are zinc ions, copper ions or ferrous ions, and the mass ratio of the antioxidant enzyme to the transition metal ions is (0.00001-1): 1.

6. the method of claim 1, wherein the organic ligand is one or more of 2-methylimidazole, 2-imidazolecarboxaldehyde, imidazole, and benzimidazole.

7. The method of claim 1, wherein the molar ratio of the transition metal ion to the organic ligand is 1: (0.01-200).

8. The method according to any one of claims 1 to 7, wherein the solvent is one or more of water, methanol, ethanol, dimethyl sulfoxide, acetonitrile, acetone, and N, N-dimethylformamide.

9. The metal-organic framework material-enzyme complex prepared by the preparation method of any one of claims 1 to 8.

10. A daily chemical product, food, medical material or pharmaceutical product comprising the metal-organic framework material-enzyme complex according to claim 9.

Technical Field

The invention relates to the technical field of immobilized enzymes, in particular to a metal organic framework material-enzyme compound and a preparation method and application thereof.

Background

Reactive Oxygen Species (ROS) are normal products of the metabolic cycle in vivo, including electron reduction products of oxygen such as superoxide radical ions, hydrogen peroxide, hydroxyl radicals, singlet oxygen, and the like, and play an important role in the processes of metabolism, immunity, and signal transduction. A complex antioxidant symbiotic ring exists in a human body to maintain the active oxygen level under normal physiological conditions, and comprises enzyme substances such as superoxide dismutase (SOD), Catalase (CAT), glutathione peroxidase (GPx), Glutathione Reductase (GR) and the like, and non-enzyme small molecules such as vitamin C, vitamin E, coenzyme Q, Glutathione (GSH), flavonoids and the like. However, when the organism is exposed to a specific environment or toxic substances, excessive active oxygen is generated, which brings about the harm of oxidative stress on biological macromolecules such as lipid, DNA, protein and the like, destroys the normal structure and function of cells, and even causes various diseases such as Parkinson's disease, cancer and the like.

Compared with the sacrificial small molecules, the enzyme antioxidants are used as catalysts rather than reactants to participate in the process, and are not theoretically consumed in the reaction process. Enzymes have relatively higher safety and biocompatibility than metal particles or metal oxides, and are preferred antioxidants. However, free enzymes are susceptible to various physical and chemical degradations in vivo and in vitro environments to be inactivated, and negatively charged proteins are difficult to enter cytoplasm wrapped by phospholipid bilayers to function.

Disclosure of Invention

Based on the metal organic framework material-enzyme compound, the invention provides a metal organic framework material-enzyme compound with strong active oxygen scavenging capacity, and a preparation method and application thereof.

In one aspect of the present invention, a method for preparing a metal organic framework material-enzyme complex is provided, which comprises the following steps:

dissolving antioxidase, transition metal ions and organic ligands in a solvent for coprecipitation reaction;

the antioxidant enzyme is selected from a combined enzyme of superoxide dismutase and catalase, glutathione peroxidase or horseradish peroxidase, or a combined enzyme of glutathione reductase and glutathione peroxidase, glucose dehydrogenase or alcohol dehydrogenase;

the transition metal ions are soluble metal salts, and the organic ligand is an imidazole compound.

On one hand, the invention also provides the metal organic framework material-enzyme compound prepared by the preparation method.

In another aspect of the present invention, there is provided a daily chemical product, food, medical material or pharmaceutical product comprising the metal-organic framework material-enzyme complex.

The metal organic framework material-enzyme complex provided by the invention has a protein release mechanism depending on pH, and also depends on a proton sponge effect, so that the complex can effectively escape from a slightly acidic endosome after being taken into cells, and the antioxidase can play a role in clearing active oxygen in cytoplasm. And the metal organic framework material-enzyme compound has better stability and dispersibility, can be recycled after simple separation, and can still keep higher catalytic activity after repeated use.

In addition, the coprecipitation method used in the invention is convenient and simple, greatly reduces the cost of experimental synthesis, and can be prepared in batch.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a scanning electron microscope image of a metal organic framework material-enzyme complex prepared in example 1 of the present invention;

FIG. 2 is a thermogravimetric analysis curve of the metal-organic framework material-enzyme complex prepared in example 1 of the present invention;

FIG. 3 is a graph showing protein release curves of the metal-organic framework material-enzyme complex prepared in example 1 under different pH conditions;

FIG. 4 is a scanning electron microscope image of the metal organic framework material-enzyme complex prepared in example 2 of the present invention;

FIG. 5 is a scanning electron microscope image of the metal organic framework material-enzyme complex prepared in example 3 of the present invention;

FIG. 6 is a scanning electron microscope image of the metal organic framework material-enzyme complex prepared in example 4 of the present invention;

FIG. 7 is an X-ray diffraction pattern of the metal-organic framework material-enzyme complex prepared in examples 1 to 4 of the present invention;

FIG. 8 is a scanning electron micrograph of a metal organic framework material-enzyme complex prepared in example 9 of the present invention;

FIG. 9 is a scanning electron micrograph of a metal organic framework material-enzyme complex prepared in example 10 of the present invention;

FIG. 10 is a scanning electron micrograph of a metal organic framework material-enzyme complex prepared in example 11 of the present invention;

FIG. 11 is a graph showing cytotoxicity test results of the metal-organic framework material-enzyme complexes prepared in examples 1 to 3 of the present invention and comparative examples 1 to 3;

FIG. 12 is a graph showing the protection of the organometallic framework material-enzyme complexes prepared in example 1 of the present invention and comparative example 1 against paraquat-induced oxidative stress cytotoxicity.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one aspect of the present invention, a method for preparing a metal organic framework material-enzyme complex is provided, which comprises the following steps:

dissolving antioxidase, transition metal ions and organic ligands in a solvent for coprecipitation reaction;

wherein the antioxidant enzyme is selected from the combined enzyme of superoxide dismutase and catalase, glutathione peroxidase or horseradish peroxidase, or the combined enzyme of glutathione reductase and glutathione peroxidase, glucose dehydrogenase or alcohol dehydrogenase;

the transition metal ions are derived from soluble metal salts, and the organic ligand is an imidazole compound.

The metal organic framework material-enzyme complex has a pH-dependent protein release mechanism and also depends on a proton sponge effect, and can effectively escape from a slightly acidic inclusion body after being taken into cells, so that the metal organic framework material-enzyme complex can be stably stored under physiological conditions, and can effectively escape from the slightly acidic inclusion body after being taken into the cells, so that the antioxidase plays a role in clearing active oxygen in cytoplasm. Specifically, for example, under the condition of pH 5.0, about 80% of the loaded protein is rapidly released in 1h from the metal-organic framework material-enzyme complex, and under the condition of pH 7.4, only 16% of the protein is released after 28 h. And the metal organic framework material-enzyme compound has better stability and dispersibility, can be recycled after simple separation, and can still keep higher catalytic activity after repeated use. Specifically, after 10 times of repeated use, the enzyme activity can still be kept above 80%. In addition, the coprecipitation method is convenient and simple, greatly reduces the cost of experimental synthesis, and can be prepared in batch.

In some embodiments, the mass ratio of superoxide dismutase (SOD) to Catalase (CAT), glutathione peroxidase (GPx), or horseradish peroxidase (HRP) is 1: (0.001-1000), and may be 1:0.01, 1:0.1, 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, or the like.

In some embodiments, the mass ratio of Glutathione Reductase (GR) to glutathione peroxidase, Glucose Dehydrogenase (GDH), or Alcohol Dehydrogenase (ADH) is 1: (0.001-1000), and may be 1: (0.001-1000), and may be 1:0.01, 1:0.1, 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, or the like. By adjusting the proportion of the cascade enzyme, the antioxidant efficiency can be improved, and the secondary harm caused by the accumulation of toxic and harmful intermediate products can be eliminated.

In some embodiments, the preparation method further comprises the steps of adding an active protective agent and a synergistic agent after the coprecipitation reaction is carried out, mixing, and drying;

the activity protective agent can be one or more of saccharides, amino acids, alcohols, proteins and the like. The saccharide includes, but is not limited to, glucose, fructose, sucrose, mannose, trehalose, chitosan, etc., the amino acid may be arginine, glycine, etc., the alcohol may be selected from glycerol, polyethylene glycol, etc., and the protein may be human serum albumin. The synergistic agent can be selected from one or a mixture of vitamins and derivatives thereof, vitamin analogs, polypeptides, nucleotides, alcohols, acids, ketones and the like. The vitamins and derivatives thereof comprise vitamin C, vitamin C palmitate, vitamin C succinate, vitamin C phosphate, vitamin E and vitamin E succinate, the polypeptide is preferably glutathione, the nucleotide can be nicotinamide mononucleotide, nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, the vitamin analogue is preferably coenzyme Q10, the alcohol is preferably resveratrol, the acid can be lipoic acid, ferulic acid and the like, and the ketone is preferably phloretin.

In some embodiments, the content of the active protective agent and the synergistic agent is independently selected from 0.1% to 30% by mass, and may be 0.5%, 1%, 5%, 10%, 12%, 15%, 20%, 25% by mass, or the like.

In some embodiments, the drying may be any manner known to those skilled in the art, and may be at least one of vacuum drying, freeze drying and natural air drying, and the drying time is 10h to 120 h.

In some embodiments, the transition metal ion may be zinc ion, copper ion or ferrous ion, and the mass ratio of the antioxidant enzyme to the transition metal ion is (0.00001-1): 1, 0.00005:1, 0.0001:1, 0.0008:1, 0.001:1, 0.006:1, 0.01:1, 0.05:1, 0.08:1, 0.1:1, 0.4:1, 0.8:1, etc. may also be used. Preferably, the transition metal ion is a zinc ion.

In some embodiments, the organic ligand may be one or more of 2-methylimidazole, 2-imidazolecarboxaldehyde, imidazole, and benzimidazole. Preferably, the organic ligand is 2-methylimidazole.

In some embodiments, the molar ratio of transition metal ion to organic ligand is 1: (0.01-200), and may be 1:0.1, 1:1, 1:10, 1:20, 1:40, 1:50, 1:60, 1:70, 1:80, 1:100, 1:110, 1:120, 1:150, 1:180, and the like. The particle size, enzyme loading capacity, enzyme activity and the like of the metal organic framework material-enzyme compound can be regulated and controlled by regulating the mass ratio of the transition metal ions to the organic ligand, so that the compound is suitable for any scene.

In some embodiments, the solvent may be one or more of water, methanol, ethanol, dimethyl sulfoxide, acetonitrile, acetone, and N, N-dimethylformamide.

In some embodiments, the method further comprises the step of centrifuging and washing the obtained substance after the coprecipitation reaction and before the active protective agent and the synergist are added.

In some embodiments, the speed of centrifugation is 4000rpm to 15000rpm and the time of centrifugation is 2min to 20 min.

In some embodiments, the solvent used for washing is a solvent commonly used in the art, preferably the same solvent as the solvent in which the antioxidant enzyme, the transition metal ion, and the organic ligand are dissolved.

In some embodiments, in order to completely dissolve the solute in the solvent, the solubilization treatment may be performed by stirring, ultrasonic treatment, or the like, preferably stirring at a speed of 10rpm to 1000 rpm.

On one hand, the invention also provides the metal organic framework material-enzyme compound prepared by the preparation method.

In another aspect of the present invention, there is provided a daily chemical product, food, medical material or pharmaceutical product comprising the metal-organic framework material-enzyme complex.

In some embodiments, the daily chemical product may be a skin care product, which may be a sunscreen cream, a cream, an emulsion, a lotion, a essence, a lyophilized powder, a facial cleanser, a bath lotion, a mask, or the like.

In some embodiments, the food may be a health product, which may be an anti-glycation capsule, an anti-oxidation capsule, or the like, or a snack food, which may be a jelly, a beverage, a confection, or the like.

In some embodiments, the medical material may be gauze, a band-aid, a mask, a wound dressing, a dental filling material, a dental implant material, or the like, and the drug may be a plaster.

The metal organic framework material-enzyme complex of the present invention, the preparation method thereof, and the use thereof will be described in further detail below with reference to specific examples and comparative examples.

Example 1

(1) Respectively preparing 1.25 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 2.5mg/mL SOD solution and 7.0mg/mL CAT solution, and performing ultrasonic treatment for 10min at room temperature until complete dissolution is achieved, wherein the molar ratio of zinc ions to 2-methylimidazole is 1: 40;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of SOD solution and 100 mu L of CAT solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) pre-freezing the precipitate obtained in the step (3) at-20 ℃ for 12h, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound which is recorded as SC @ ZIF-8.

The scanning electron microscope of the metal organic framework material-enzyme complex prepared in this example is shown in fig. 1. As can be seen from FIG. 1, the main body of the metal-organic framework material-enzyme complex is approximately spherical, and the particle size of the particles is 400 nm-700 nm. The thermogravimetric curve of the metal-organic framework material-enzyme complex is shown in FIG. 2. As can be seen from FIG. 2, the metal organic framework material-enzyme complex starts to decompose at about 250 ℃, while the ZIF-8 crystal is degraded at about 450 ℃, and the mass fraction of the antioxidant enzyme in the complex is about 4.5 wt% according to curve difference. The protein release profiles of the metal-organic framework material-enzyme complexes at different pH conditions are shown in FIG. 3. As can be seen from fig. 3, about 80% of the loaded protein was released rapidly within 1 hour at pH 5.0, while only 16% of the protein was released after 28 hours at pH 7.4. The pH-dependent protein release mechanism of the metal organic framework material-enzyme complex is shown, so that the stable storage of the metal organic framework material-enzyme complex under physiological conditions can be ensured, the complex can effectively escape from a slightly acidic endosome after being taken into cells, and the antioxidase can play a role in scavenging active oxygen in cytoplasm.

Example 2

This example is prepared substantially identically to example 1, except that: the concentration of the 2-methylimidazole aqueous solution is 1.875 mol/L. The method comprises the following specific steps:

(1) respectively preparing 1.875 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 2.5mg/mL SOD solution and 7.0mg/mL CAT solution, and performing ultrasonic treatment for 10min at room temperature until complete dissolution is achieved, wherein the molar ratio of zinc ions to 2-methylimidazole is 1: 60;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of SOD solution and 100 mu L of CAT solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) pre-freezing the precipitate obtained in the step (3) at-20 ℃ for 12h, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound which is recorded as SC @ ZIF-8.

The scanning electron microscope of the metal organic framework material-enzyme complex prepared in this example is shown in fig. 4. As can be seen from FIG. 4, the main body of the metal-organic framework material-enzyme complex is approximately spherical, and the particle size of the particles is between 200nm and 400 nm.

Example 3

This example is prepared substantially identically to example 1, except that: the concentration of the 2-methylimidazole aqueous solution was 2.5 mol/L. The method comprises the following specific steps:

(1) respectively preparing 2.5 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 2.5mg/mL SOD solution and 7.0mg/mL CAT solution, and performing ultrasonic treatment for 10min at room temperature until complete dissolution is achieved, wherein the molar ratio of zinc ions to 2-methylimidazole is 1: 80;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of SOD solution and 100 mu L of CAT solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) pre-freezing the precipitate obtained in the step (3) at-20 ℃ for 12h, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound which is recorded as SC @ ZIF-8.

The scanning electron microscope of the metal organic framework material-enzyme complex prepared in this example is shown in fig. 5. As can be seen from FIG. 5, the main body of the metal-organic framework material-enzyme complex is approximately spherical, and the particle size of the particles is between 80nm and 200 nm.

Example 4

This example is prepared substantially identically to example 1, except that: the concentration of the 2-methylimidazole aqueous solution was 3.125 mol/L. The method comprises the following specific steps:

(1) respectively preparing 3.125 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 2.5mg/mL SOD solution and 7.0mg/mL CAT solution, and performing ultrasonic treatment for 10min at room temperature until complete dissolution is achieved, wherein the molar ratio of zinc ions to 2-methylimidazole is 1: 100;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of SOD solution and 100 mu L of CAT solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) pre-freezing the precipitate obtained in the step (3) at-20 ℃ for 12h, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound which is recorded as SC @ ZIF-8.

The scanning electron microscope of the metal organic framework material-enzyme complex prepared in this example is shown in fig. 6. As can be seen from FIG. 6, the main body of the metal-organic framework material-enzyme complex is approximately spherical, and the particle size of the particles is between 60nm and 160 nm.

The X-ray diffraction pattern of the metal-organic framework material-enzyme complex prepared in examples 1-4 is shown in FIG. 7. As can be seen from fig. 7, the metal organic framework material-enzyme complex exhibited the crystal structure and good crystallinity of the metal organic framework compound (ZIF-8).

Example 5

This example is prepared substantially identically to example 1, except that: the volume of CAT solution in step (2) was 50. mu.L.

Example 6

This example is prepared substantially identically to example 1, except that: the volume of CAT solution in step (2) was 200. mu.L.

And (3) performing enzyme activity determination on the metal organic framework material-enzyme compound prepared in the embodiment 1-6:

SOD enzyme activity determination: the WST-8 method is used for measuring the enzymatic activity of the SOD, and comprises the following specific steps: WST-8/enzyme working solution and reaction initiation working solution were prepared, and sample wells and control wells were set according to Table 1. After adding the reaction initiation medium, incubation was carried out at 37 ℃ for 30min, and then absorbance at 450nm was measured. The SOD enzyme activity was defined as: when the inhibition percentage in the coupling reaction system is 50%, the SOD has 1 enzyme activity unit (1U).

Percent inhibition ═ a [ ("a_B1-A_B2)-(A_S-A_B3)]/(A_B1-A_B2)×100％

SOD activity as inhibition percentage/(1-inhibition percentage) (U)

TABLE 1 SOD enzyme activity determination sample well and control well arrangement table

And (3) CAT enzyme activity determination: a50 mM phosphate buffer solution (pH 7.0) was prepared, and 30% hydrogen peroxide was added thereto to give a concentration of 20 mM. 980. mu.L of hydrogen peroxide solution was added to 20. mu.L of enzyme solution, and the change in absorbance at 240nm was measured using an ultraviolet-visible spectrophotometer. CAT enzyme activity unit (1U) is defined as: the amount of enzyme required to raise the substrate absorbance value by 0.001 per minute at 25 ℃.

The calculation formula of relative enzyme activity and enzyme activity yield is as follows:

relative enzyme activity ═ A/A_f×100％

Wherein A is_fThe activity of the free enzyme at the corresponding protein concentration, and A is the apparent total activity of the metal-organic framework material-enzyme complex.

The yield of enzyme activity is A/A_pre×100％

Wherein A is the apparent total activity of the metal organic framework material-enzyme complex, A_preIs the total activity of the free enzyme added at the time of dosing.

The test results are shown in tables 2 and 3:

TABLE 2 relative enzyme activities of different metal organic framework material-enzyme complexes

Group of	Relative enzyme activity of SOD (%)	CAT relative enzyme activity (%)
			Example 1	12.7	81.8
Example 2	10.0	72.7
			Example 3	12.0	33.0
Example 4	20.6	21.6
			Example 5	26.1	85.4
Example 6	18.6	94.1

TABLE 3 enzymatic Activity Retention of different Metal organic framework Material-enzyme complexes

Group of	SOD enzyme activity retention (%)	CAT enzyme activity retention (%)
			Example 1	11.4	41.6
Example 2	13.3	71.1
			Example 3	20.3	33.0
Example 4	34.2	21.2
			Example 5	8.4	17.0
Example 6	6.0	20.0

Example 7

This example is prepared substantially identically to example 1, except that: the active protective agent and the synergistic agent are added, and the volume of the components is different. The method comprises the following specific steps:

(1) respectively preparing 1.25 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 2.5mg/mL SOD solution and 7.0mg/mL CAT solution, and performing ultrasonic treatment at room temperature for 10min until the 2-methylimidazole aqueous solution, the zinc nitrate aqueous solution, the SOD solution and the CAT solution are completely dissolved;

(2) adding 16mL of zinc nitrate aqueous solution, 5mL of SOD solution and 10mL of CAT solution into 160mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) and (3) after the precipitate obtained in the step (3) is resuspended in 10mL of deionized water, adding 8 wt% of a mixture of sucrose and glycine (the mass ratio is 60:40) and 5 wt% of a mixture of vitamin C, glutathione and nicotinamide mononucleotide (the mass ratio is 50:30:20), pre-freezing for 12h at-20 ℃, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound.

Example 8

This example is prepared substantially identically to example 1, except that: the added antioxidant enzyme is the combination of superoxide dismutase and glutathione peroxidase. The method comprises the following specific steps:

(1) respectively preparing 1.875 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 2.5mg/mL SOD solution and 1.0mg/mL GPx solution, and performing ultrasonic treatment at room temperature for 10min until the 2-methylimidazole aqueous solution, the zinc nitrate aqueous solution and the GPx solution are completely dissolved;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of SOD solution and 100 mu L of GPx solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) and (3) resuspending the precipitate obtained in the step (3) with 0.1mL of deionized water, adding 8 wt% of glutathione, pre-freezing for 12h at-20 ℃, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound, which is recorded as SOD-GPx @ ZIF-8.

Example 9

This example is prepared substantially identically to example 1, except that: the added antioxidant enzyme is the combination of glutathione reductase and glutathione peroxidase. The method comprises the following specific steps:

(1) respectively preparing 1.875 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 1.0mg/mL GR solution and 1.0mg/mL GPx solution, and performing ultrasonic treatment for 10min at room temperature until complete dissolution;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of GR solution and 50 mu L of GPx solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) and (3) resuspending the precipitate obtained in the step (3) with 0.1mL of deionized water, adding a mixture of 8 wt% of glutathione and nicotinamide adenine dinucleotide (the molar ratio is 1:10), pre-freezing for 12h at-20 ℃, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound, which is marked as GR-GPx @ ZIF-8, wherein the measured scanning electron microscope image is shown in figure 8.

Example 10

This example is prepared substantially identically to example 1, except that: the added antioxidant enzyme is the combination of glutathione reductase and alcohol dehydrogenase. The method comprises the following specific steps:

(1) respectively preparing 1.875 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 1.0mg/mL GR solution and 6.6mg/mL ADH solution, and performing ultrasonic treatment for 10min at room temperature until the solutions are completely dissolved;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of GR solution and 50 mu L of ADH solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) and (3) resuspending the precipitate obtained in the step (3) with 0.1mL of deionized water, adding a mixture of 1 wt% oxidized glutathione and nicotinamide adenine dinucleotide (the molar ratio is 1:2), pre-freezing for 12h at-20 ℃, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound, namely GR-ADH @ ZIF-8, wherein the measured scanning electron microscope image is shown in figure 9.

Example 11

This example is prepared substantially identically to example 1, except that: the antioxidant enzyme is a combination of glutathione reductase and glucose dehydrogenase. The method comprises the following specific steps:

(1) respectively preparing 1.875 mol/L2-methylimidazole aqueous solution, 0.31mol/L zinc nitrate aqueous solution, 1.0mg/mL GR solution and 7.5mg/mL GDH solution, and performing ultrasonic treatment for 10min at room temperature until the solutions are completely dissolved;

(2) adding 160 mu L of zinc nitrate aqueous solution, 50 mu L of GR solution and 50 mu L of GDH solution into 1.6mL of 2-methylimidazole aqueous solution at the same time, and stirring for 30min at room temperature by using a magnetic stirrer with the rotating speed of 600 rpm;

(3) centrifuging the solution obtained in the step (2) at the speed of 15000rpm for 5min, removing the supernatant, then resuspending the solution with deionized water, and repeatedly washing the solution for 2 times;

(4) and (3) resuspending the precipitate obtained in the step (3) with 0.1mL of deionized water, adding 8 wt% of a mixture of oxidized glutathione, nicotinamide adenine dinucleotide and glucose (the molar ratio is 1:2:5), pre-freezing for 12h at-20 ℃, and freeze-drying for 24h to obtain the metal organic framework material-enzyme compound, namely GR-GDH @ ZIF-8, wherein the detected scanning electron microscope image is shown in figure 10.

Comparative example 1

This comparative example was prepared substantially the same as example 1, except that: no antioxidant enzyme was added.

Comparative example 2

This comparative example was prepared substantially the same as example 2, except that: no antioxidant enzyme was added.

Comparative example 3

This comparative example was prepared substantially the same as example 3, except that: no antioxidant enzyme was added.

Comparative example 4

This comparative example was prepared substantially the same as example 1, except that: no CAT was added. The metal organic framework material-enzyme complex is marked as SOD @ ZIF-8.

Comparative example 5

This comparative example was prepared substantially the same as example 1, except that: the antioxidant enzymes are SOD and GR. The metal organic framework material-enzyme complex is marked as SOD-GR @ ZIF-8.

Comparative example 6

This comparative example was prepared substantially the same as example 1, except that: the antioxidant enzyme is SOD and GDH. The metal organic framework material-enzyme complex was designated as SOD-GDH @ ZIF-8.

The cytotoxicity of the metal organic framework material-enzyme complexes prepared in examples 1 to 3 and comparative examples 1 to 3 was examined by the MTT method. The method comprises the following specific steps: HeLa cells were seeded at 8000 cells per well in a 96-well plate, and DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was incubated at 37 ℃ with 5% CO₂The culture box of (1) was cultured overnight. The medium was removed, washed 1 time with PBS, and 200. mu.L of medium containing different concentrations (0, 10, 20, 30, 40, 50. mu.g/. mu.L) of the metal-organic framework material-enzyme complex or metal-organic framework compound was added and incubated for 24 h. The medium was removed, washed 2 times with PBS, and 100. mu.L of DMEM medium containing 0.5mg/mL MTT was added and incubated at 37 ℃ for 4 h. 10 μ LFormazan lysate was added, incubated at 37 ℃ for 4h and the absorbance value was measured at 560 nm. The cell viability was calculated according to the following formula, using the sample wells without cells as blank (blank), the sample wells without drug as control (control), and the drug-treated sample wells as sample groups (sample): cell survival rate ═ a_s-A_b)/(A_c-A_b) X 100%. The test results are shown in fig. 11. As can be seen from FIG. 11, the incubation concentration of 50. mu.g/. mu.L resulted in only about 20% decrease in cell viability, indicating that the metal-organic framework material-enzyme complex and the metal-organic framework compound (ZIF-8) had good biocompatibility.

In vitro antioxidant activity test of metal organic framework material-enzyme complex:

methyl viologen (paraquat, PQ) is used as a redox poison, and the protection effect of a metal organic framework material-enzyme complex on cells is observed. The specific operation is as follows: inoculating HeLa cells to a 96-well plate at a density of 10000 cells per well, culturing overnight at 37 ℃, removing the culture medium, and washing 1 time with PBS; adding 200 μ L of metal organic framework material-enzyme complex or metal organic framework compound containing 30 μ g/μ L, and incubating at 37 deg.C for 10 hr to complete endocytosis and release process of cells. The medium was removed, washed 2 times with PBS, 200. mu.L of medium containing 6mM PQ was added, and incubated at 37 ℃ for 12 h. The medium was removed, 100. mu.L of MTT reagent-containing medium was added and incubated at 37 ℃ for 4 hours, 100. mu.L of Formazan solution was added and incubated at 37 ℃ for 4 hours, and the absorbance at 560nm was measured and the cell viability was analyzed as above. The protection of example 1 and comparative example 1 against paraquat-induced oxidative stress cytotoxicity is shown in fig. 12. As can be seen from FIG. 12, the cell survival rate was significantly reduced to 36% (control group) by 6mM PQ treatment, whereas the cell survival rate was improved by 20% by pre-incubation with the metal-organic framework material-enzyme complex prepared in example 1. Meanwhile, it was observed that the free antioxidase, the metal organic framework compound (ZIF-8) did not show a significant protective effect.

The catalytic activity of SOD in the metal organic framework material-enzyme complexes prepared in example 1 and comparative examples 4 to 6 was measured by the WST-8 method, and the test results are shown in table 4. As can be seen from table 4, the SOD activity of the antioxidant enzyme combination used in example 1 was significantly higher than that of the metal-organic framework material-enzyme complex prepared from SOD single enzyme and other enzymes.

TABLE 4 SOD enzyme activity of different metal organic framework material-enzyme complexes

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.