CN113549611A

CN113549611A - Cascade nanoenzyme and preparation method and application thereof

Info

Publication number: CN113549611A
Application number: CN202110649536.6A
Authority: CN
Inventors: 穆婧; 黄鹏
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-06-10
Filing date: 2021-06-10
Publication date: 2021-10-26
Anticipated expiration: 2041-06-10
Also published as: CN113549611B

Abstract

The invention discloses a cascade nanoenzyme and a preparation method and application thereof. The cascade nanoenzyme belongs to nanometer size, has natural advantage of passive accumulation in liver compared with small molecular drug, can increase enrichment of drug in liver, and has higher targeting property. The platinum nanoparticles have excellent active oxygen (ROS) scavenging capacity as a nano enzyme, can catalyze the active oxygen to generate oxygen, and the generated oxygen reacts with arginine under the catalysis of nitric oxide synthase (iNOS) to generate NO in situ, so that the bioavailability of the NO is improved. Therefore, the cascade nanoenzyme improves the targeting property and the bioavailability of NO, and can simultaneously realize the elimination of ROS and the regulation of NO, thereby realizing the effective intervention on the injury caused by liver ischemia-reperfusion.

Description

Cascade nanoenzyme and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to cascade nanoenzyme and a preparation method and application thereof.

Background

The rapid development of liver surgery has not left the surgical anatomy and has also benefited from the use of new materials and techniques. The most common methods for treating liver diseases such as benign and malignant liver tumors, intrahepatic bile duct stones, liver trauma and the like in clinic are liver resection and liver transplantation. Liver surgery generally requires the blocking of the Hepatic portal to control blood flow and reduce blood loss, the liver is partially damaged during Ischemia, and when blood flow is restored, hypoxic organ damage is exacerbated, resulting in Hepatic Ischemia-Reperfusion Injury (HIRI). When HIRI occurs, a large amount of Reactive Oxygen Species (ROS) and cytokines are produced, causing an acute inflammatory immune response, resulting in severe liver function damage and even multiple organ failure and death. The pathophysiological processes of HIRI are complex and thus NO complete treatment is available, both endogenous and exogenous Nitric Oxide (NO) can relieve the damage caused by HIRI during generation of HIRI, NO-based therapies have shown great application prospects in the field of regenerative medicine, NO gas inhalation or NO donor drug use has shown a positive protective effect on HIRI, however, studies in this field are still in an early stage and many potential problems remain unsolved: (1) the half-life period of NO gas is short, the bioavailability is low, and the treatment effect is not ideal; (2) NO donor small molecule drugs such as nitrite and sodium nitroprusside have short circulation time in vivo, have NO tissue specificity, and potential toxic and side effects caused by interaction of multiple sites and multiple ways, which hinder clinical application of the NO donor small molecule drugs. Therefore, improving the targeting and bioavailability of NO formulations to further provide safer and feasible HIRI protective measures is one of the research trends in the field of liver diseases in the future.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a cascade nanoenzyme, which aims to solve the problem of low targeting and bioavailability of the existing NO preparations when NO is used for treating HIRI.

The technical scheme of the invention is as follows:

in a first aspect of the present invention, there is provided a cascade nanoenzyme, wherein the cascade nanoenzyme comprises a metal-organic framework support, a nitric oxide synthase bound to the metal-organic framework support, and a platinum nanoparticle bound to the metal-organic framework support.

Optionally, the nitric oxide synthase is bound inside the metal-organic framework support, and the platinum nanoparticles are bound inside the metal-organic framework support and on the surface of the metal-organic framework support.

Optionally, the particle size of the cascade nanoenzyme is 50-100 nm.

Optionally, the platinum nanoparticles have a particle size of 1-10 nm.

Optionally, the mass of the nitric oxide synthase accounts for 1% -10% of the mass sum of the nitric oxide synthase and the metal-organic framework carrier.

Optionally, the ratio of the mass of the metal in the metal-organic framework to the mass of the platinum nanoparticles is (1-10): 1.

optionally, the surface of the platinum nanoparticle contains a first ligand selected from one or more of polyvinylpyrrolidone, polyethylene glycol, and polyoxyethylene polyoxypropylene ether block copolymer.

In a second aspect of the present invention, there is provided a method for preparing the cascade nanoenzyme, comprising the steps of:

providing an aqueous solution of platinum nanoparticles;

mixing a second ligand with the platinum nanoparticle aqueous solution;

and adding a mixed aqueous solution of metal salt and nitric oxide synthetase, and reacting to obtain the cascade nanoenzyme.

Optionally, the second ligand is selected from one or two of 2-methylimidazole and 2-aldehydic imidazole, and/or the metal salt is selected from one or more of zinc salt, cobalt salt, zirconium salt and copper salt.

In a third aspect of the invention, an application of the cascade nanoenzyme of the invention in preparation of a medicament for treating hepatic ischemia-reperfusion injury is provided.

Has the advantages that: the invention provides a cascade nanoenzyme and a preparation method and application thereof, wherein the cascade nanoenzyme comprises a metal organic framework carrier, nitric oxide synthase combined with the metal organic framework carrier and platinum nanoparticles combined with the metal organic framework carrier, belongs to a nanometer size, has the natural advantage of passive accumulation in liver compared with small molecular drugs, can increase the enrichment in liver and has higher targeting property. The platinum nanoparticles have excellent active oxygen (ROS) scavenging capacity as a nano enzyme, can catalyze the active oxygen to generate oxygen, and the generated oxygen reacts with arginine under the catalysis of nitric oxide synthase (iNOS) to generate NO in situ, so that the bioavailability of the NO is improved. Because the specific surface area of the metal organic framework carrier is large and the pores are adjustable, the metal organic framework carrier not only can provide effective protection for iNOS combined in the metal organic framework carrier to prevent the iNOS from being inactivated and degraded in vivo, but also can promote the diffusion between molecules and the reaction, because the synthesis of NO has the characteristic of oxygen dependence, reactants (such as oxygen generated by the catalytic ROS of platinum nanoparticles) and a catalyst are limited in the metal organic framework carrier, the content of the oxygen in the metal organic framework carrier can be increased, the probability of the reaction is greatly increased, and the catalytic efficiency is improved. Therefore, the cascade nanoenzyme improves the targeting property and the bioavailability of NO, and can simultaneously realize the removal of ROS and the regulation of NO, thereby realizing the effective intervention on the injury caused by liver ischemia-reperfusion.

Drawings

FIG. 1 is a synthesis scheme of a cascade of nanoenzymes in an example of the present invention.

FIG. 2 is a TEM image of ZIF @ Pt @ NOS in example 1 of the present invention.

FIG. 3 is an XRD pattern of ZIF @ Pt @ NOS in example 1 of the present invention and ZIF @ Pt in comparative example 2.

FIG. 4 is a graph showing the clearance of Pt NPs from hydrogen peroxide in comparative example 4 of the present invention.

FIG. 5 is a graph showing the clearance of superoxide radicals by Pt NPs in comparative example 4 of the present invention.

FIG. 6 is a graph showing the results of NO production tests for ZIF @ Pt @ NOS in example 1 of the present invention, ZIF @ Pt in comparative example 2, and ZIF @ NOS in comparative example 3.

FIG. 7 is a graph showing the results of the ZIF @ Pt @ NOS antioxidant activity test in example 1 of the present invention.

FIG. 8 is a graphical representation of fluorescence imaging of the reactive oxygen species of parenchymal hepatocytes after incubation with ZIF @ Pt @ NOS in example 1 of the present invention, ZIF in comparative example 1, ZIF @ Pt in comparative example 2, and with hydrogen peroxide stimulation.

FIG. 9 is a graph showing the effect of hydrogen peroxide scavenging by ZIF @ Pt @ NOS in example 1 of the present invention after incubation with parenchymal hepatocytes and stimulation with hydrogen peroxide.

FIG. 10 is a graph showing the survival rate of parenchymal hepatocytes after treatment with ZIF @ Pt @ NOS in example 1 of the present invention in the presence of hydrogen peroxide.

FIG. 11 is a graph showing the profile of ZIF @ Pt @ NOS in example 1 of the present invention in major organs after 24 hours of tail vein injection into mice.

FIG. 12 is a graph showing fluorescence images of ZIF @ Pt @ NOS in example 1 of the present invention injected into mice at different times through the tail vein.

FIG. 13 is a graph showing the level of alanine Aminotransferase (ALT) in the serum of mice after tail vein injection of ZIF @ Pt @ NOS in example 1 of the present invention, ZIFs in comparative example 1, and ZIFs in comparative example 2 into mice injured by hepatic ischemia reperfusion.

FIG. 14 is a graph showing the serum glutamic-oxaloacetic transaminase (AST) content of mice after tail vein injection of ZIF @ Pt @ NOS in example 1 of the present invention, ZIF in comparative example 1, and ZIF @ Pt in comparative example 2 into bodies of mice injured by hepatic ischemia reperfusion.

FIG. 15 is a graph showing the results of liver function evaluation of ZIF @ Pt @ NOS in example 1 of the present invention in normal mice for 7 days.

FIG. 16 is a graph showing the results of renal function assessment of ZIF @ Pt @ NOS in example 1 of the present invention in normal mice over 7 days.

FIG. 17 is a graph showing the results of a hemolysis experiment using ZIF @ Pt @ NOS in example 1 of the present invention.

Detailed Description

The invention provides a cascade nanoenzyme and a preparation method and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the HIRI generation process, both endogenous and exogenous Nitric Oxide (NO) can relieve the damage caused by HIRI, NO-based therapy has shown a huge application prospect in the field of regenerative medicine, but the existing NO preparation has low targeting property and low bioavailability. In addition, oxidative stress in the liver from ischemia-reperfusion can cause Reactive Oxygen Species (ROS) such as H₂O₂、O^2·-、^·Due to the over-expression of OH, excessive ROS can cause damage and death of cells, so that the key for protecting the cells from damage is to eliminate ROS and reduce oxidative stress, and the important significance is to provide a medicament capable of realizing the double protection effects of ROS elimination and NO regulation.

Based on this, embodiments of the present invention provide a cascade nanoenzyme, wherein the cascade nanoenzyme includes a metal-organic framework support, a nitric oxide synthase bound to the metal-organic framework support, and a platinum nanoparticle bound to the metal-organic framework support.

The nano enzyme is economical and stable, can keep good reaction activity in vivo, is limited in the type of catalytic reaction and has low selectivity on a substrate. In contrast, the natural enzyme has high substrate selectivity, good biocompatibility and wider catalytic reaction selection range, but the natural enzyme has poor stability. Therefore, the inventor deeply researches the characteristics of the nanoenzyme and the natural enzyme to integrate and complement the advantages, and because the specific surface area of the metal organic framework material is large and the pores are adjustable, the method not only can provide effective protection for the natural protease to prevent the natural protease from inactivation and degradation in vivo, but also can promote the diffusion between molecules and the reaction.

The cascade nanoenzyme in the embodiment belongs to a nanometer size, has the natural advantage of passive accumulation in the liver compared with small molecular drugs, can increase the enrichment of drugs in the liver, and has higher targeting property. The platinum nanoparticles have excellent active oxygen (ROS) scavenging capacity as a nano enzyme, can catalyze the active oxygen to generate oxygen, the generated oxygen reacts with arginine under the catalysis of nitric oxide synthase (iNOS, a natural enzyme) to generate NO in situ, and the NO is generated in situ, so that the bioavailability of the NO is improved. Because the specific surface area of the metal organic framework carrier is large and the pores are adjustable, the metal organic framework carrier not only can provide effective protection for iNOS combined in the metal organic framework carrier to prevent the iNOS from being inactivated and degraded in vivo, but also can promote the diffusion between molecules and the reaction, because the synthesis of NO has the characteristic of oxygen dependence, reactants (such as oxygen generated by the ROS catalyzed by platinum nanoparticles) and a catalyst are limited in the metal organic framework carrier, the content of the oxygen in the metal organic framework carrier can be increased, the probability of the reaction is greatly increased, and the catalytic efficiency is improved. Therefore, the cascade nanoenzyme not only improves the targeting property and the bioavailability of NO, but also can simultaneously realize the elimination of ROS and the regulation of NO, thereby realizing the effective intervention on the injury caused by the liver ischemia-reperfusion.

The inventor utilizes the effective enrichment effect of the nano material in liver tissues through deep understanding of a disease damage mechanism, takes ROS generated when HIRI occurs as a reactant, namely ROS as a stimulus, triggers a catalytic reaction, platinum nanoparticles catalyze ROS to react to generate oxygen, the generated oxygen can further react with arginine under the catalysis of iNOS to generate NO in situ, the whole cascade reaction is completed, the platinum nanoparticles catalyze ROS to react to generate oxygen in the process of the cascade reaction, the ROS is effectively eliminated, the generated oxygen can further react with arginine under the catalysis of iNOS to generate NO in situ, and the bioavailability of NO is improved. The cascade reaction process can reduce oxidative stress injury, inhibit apoptosis and inflammatory factor expression, and finally realize effective intervention on HIRI. The cascade nanoenzyme activated by active oxygen not only improves the targeting property and bioavailability of NO, but also triggers cascade reaction by the enrichment effect at the liver part, realizes the dual protection effects of ROS elimination and NO regulation, and provides a new idea and clinical application prospect for developing safe and effective HIRI treatment medicines.

In one embodiment, the nitric oxide synthase is bound inside the metal-organic framework support, and the platinum nanoparticles are bound inside the metal-organic framework support and on the surface of the metal-organic framework support.

In this embodiment, the metal-organic framework support comprises metal ions and organic ligands, the amino group of the nitric oxide synthase forms a coordination with the metal ions in the metal-organic framework support, the nitric oxide synthase and the organic ligands and metal ions in the metal-organic framework support have charge interaction, that is, the nitric oxide synthase is specifically bound in the metal-organic framework through the coordination between the amino group of the nitric oxide synthase and the metal ions in the metal-organic framework support and the interaction between the nitric oxide synthase and the charges of the organic ligands and metal ions in the metal-organic framework support, and the platinum nanoparticles are specifically bound in the metal-organic framework support and on the surface of the metal-organic framework support through physical adsorption. Because the platinum nano-particles are very small in size, the platinum nano-particles can be combined in the metal-organic framework carrier and on the surface of the metal-organic framework carrier through a physical adsorption mode. The nitric oxide synthetase is combined in the metal organic framework carrier, so that the nitric oxide synthetase can provide effective protection for iNOS, and can prevent the iNOS from being inactivated and degraded in vivo.

In one embodiment, the particle size of the cascade of nanoenzymes is between 50 and 100 nm. The cascade nanoenzyme with the particle size range has good dispersibility, can ensure effective aggregation at liver parts, improves the targeting property of subsequently generated NO, and simultaneously, the metal organic framework carrier in the cascade nanoenzyme can be slowly degraded in vivo, so that the cascade nanoenzyme can be effectively removed in vivo while the catalytic reaction is ensured.

In one embodiment, the platinum nanoparticles have a particle size of 1 to 10 nm. The platinum nanoparticles are small in size, large in comparison area and multiple in active sites, and are more favorable for catalyzing ROS to generate oxygen and water as nanoenzyme, so that ROS can be removed more favorably, therefore, the platinum nanoparticles in the particle size range have good enzyme simulation characteristics, and in addition, the platinum nanoparticles in the particle size range have renal removal performance.

In one embodiment, the mass of the nitric oxide synthase is 1% to 10% of the mass of the sum of the nitric oxide synthase and the metal-organic framework support. The proportion can ensure that the loading of the nitric oxide synthetase forms stable cascade nano enzyme particles, and simultaneously, the overlarge particle size of the cascade nano enzyme is avoided. In addition, the increase of the content of nitric oxide synthase easily causes aggregation of the formed composite particles, and the dispersibility is poor.

In one embodiment, the ratio of the mass of the metal in the metal-organic framework to the mass of the platinum nanoparticles is (1-10): 1. the cascade nanoenzyme obtained in the proportion range has good dispersibility and stability, and can simultaneously ensure the activity of iNOS and platinum nanoparticles.

In one embodiment, the metal organic framework support is selected from one of ZIF-8 and ZIF-90, but is not limited thereto. For example, the metal skeleton support in the present embodiment may be selected from metal organic skeleton materials obtained by performing a coordination reaction using one of zinc ions, cobalt ions, zirconium ions, copper ions, and the like as a metal ion, and using one of 2-methylimidazole, 2-aldehydic imidazole, and the like as an organic ligand.

In one embodiment, the surface of the platinum nanoparticle contains a first ligand selected from one or more of polyvinylpyrrolidone, polyethylene glycol, and polyoxyethylene polyoxypropylene ether block copolymer, but is not limited thereto. The ligand on the surface of the platinum nano-particle can effectively stabilize the platinum nano-particle, and the platinum nano-particle is controlled to have a very small size. The platinum nanoparticles are small in size, large in specific surface area and multiple in active sites, and are more beneficial to catalyzing ROS to generate oxygen and water as nanoenzyme, so that the scavenging of ROS is more facilitated.

In one embodiment, the mass ratio of the ligand contained on the surface of the platinum nanoparticles to the platinum nanoparticles is (10-60): 1. This ratio can allow the surface ligands to more effectively stabilize the platinum nanoparticles, controlling the platinum nanoparticles to have a very small size.

The embodiment of the invention also provides a preparation method of the cascade nanoenzyme, which comprises the following steps:

s1, providing a platinum nanoparticle aqueous solution;

s2, mixing a second ligand and the platinum nano-particle aqueous solution;

and S3, adding a mixed aqueous solution of metal salt and nitric oxide synthetase, and reacting to obtain the cascade nanoenzyme.

In the embodiment, the mixed aqueous solution of the metal salt and the nitric oxide synthase is prepared by adding the metal salt and the nitric oxide synthase into water and uniformly mixing, in the process, the amino functional group and the metal ion in the nitric oxide synthase have the mutual attraction of positive and negative charges, then when the mixed aqueous solution of the metal salt and the nitric oxide synthase is added into the mixed solution of the second ligand and the platinum nanoparticle aqueous solution, the metal ion in the metal salt aqueous solution and the second ligand generate the coordination reaction to form the metal organic framework carrier, and simultaneously, the nitric oxide synthase and the platinum nanoparticle can be wrapped in the metal organic framework carrier in situ, namely, the nitric oxide synthase is combined in the metal organic framework by the coordination of the amino group and the metal ion and the interaction between the amino group and the second ligand in the metal organic framework carrier and the charge of the metal ion, the platinum nanoparticles are combined with the metal organic framework carrier in a physical adsorption mode, and not only can be combined in the metal organic framework carrier, but also can be combined on the surface of the metal organic framework. In this embodiment, a co-precipitation method is used to prepare a metal organic framework carrier-based nitric oxide synthase and platinum nanoparticle complex, i.e., the cascaded nanoenzyme. The coprecipitation method has advantages in that the size of the protein (e.g., iNOS) is not limited, the conditions of the metal organic framework support in the synthesis are mild, macromolecules such as the protein can be coated therein without denaturing the protein, and the molecular weight of the protein is not limited by the pore size of the metal organic framework support.

In step S1, the specific steps of providing the platinum nanoparticle aqueous solution include: the platinum nano-particles are prepared by adopting a sodium borohydride reduction method, and then the platinum nano-particles are prepared into an aqueous solution containing the platinum nano-particles, namely the aqueous solution of the platinum nano-particles. The present invention is not limited to the above-described method for producing platinum nanoparticles, and other production methods capable of producing platinum nanoparticles are also applicable to the present invention, and examples thereof include an ethanol reduction method.

In step S2, a second ligand is mixed with the platinum nanoparticles, and the platinum particles are wrapped and adsorbed inside and on the surface of the metal-organic framework carrier during the formation of the metal-organic framework carrier.

In one embodiment, the second ligand is selected from one or two of 2-methylimidazole and 2-aldehydic imidazole, but is not limited thereto.

In step S3, the reaction temperature is 20-30 ℃, and the reaction time is 10 min-2 h. The preparation method in the embodiment can be completed at normal temperature, and is energy-saving and environment-friendly.

In one embodiment, the molar ratio of the metal salt to the second ligand is 1 (20-100).

In one embodiment, the metal salt is selected from one or more of zinc salt, cobalt salt, zirconium salt, copper salt, but is not limited thereto. For example, zinc nitrate, cobalt sulfate, zirconium sulfate, copper sulfate, etc. may be mentioned.

The metal-organic framework support in the present embodiment is prepared by a coordination reaction between one of zinc ions, cobalt ions, zirconium ions, and copper ions and one of 2-methylimidazole and 2-aldehydiimidazole, and may be prepared, for example, by preparing one of ZIF-8 and ZIF-90, but is not limited thereto.

In one embodiment, the method further comprises the following steps after the metal salt aqueous solution is added for reaction and before the cascade nanoenzyme is obtained:

centrifuging the solution after reaction to obtain a precipitate;

and washing the precipitate with deionized water, and drying to obtain the cascade nanoenzyme.

In this embodiment, since iNOS is water-soluble, and iNOS adsorbed on the surface of the metal-organic framework support can be removed by washing with deionized water, in the obtained cascade nanoenzyme, iNOS is bound inside the metal-organic framework by the coordination of the amino group thereof with the metal ion and the interaction between the amino group thereof and the charge of the second ligand and the metal ion, and is not adsorbed on the surface of the metal-organic framework.

Taking zinc ions and 2-methylimidazole as metal ions and organic ligands of a metal-organic framework carrier respectively as an example, a synthetic route of the cascade nanoenzyme is described, as shown in fig. 1, mixing the zinc ions, the 2-methylimidazole, platinum nanoparticles (Pt NPs) and iNOS uniformly by using a coprecipitation method, and reacting to obtain the cascade nanoenzyme ZIF @ Pt @ NOS, so that in-situ wrapping of the platinum nanoparticles (Pt NPs) and the iNOS is realized.

The embodiment of the invention also provides application of the cascade nanoenzyme in preparation of a medicine for treating hepatic ischemia-reperfusion injury.

In the embodiment, the cascade nanoenzyme belongs to a nanometer size, has the natural advantage of passive accumulation in the liver compared with small molecular drugs, can increase the enrichment of the drugs in the liver, and has higher targeting property. The platinum nanoparticles have excellent active oxygen (ROS) scavenging capacity as a nano enzyme, can catalyze the active oxygen to generate oxygen, and the generated oxygen reacts with arginine under the catalysis of nitric oxide synthase (iNOS) to generate NO in situ, so that the bioavailability of the NO is improved. Because the specific surface area of the metal organic framework carrier is large and the pores are adjustable, the metal organic framework carrier not only can provide effective protection for iNOS combined in the metal organic framework carrier to prevent the iNOS from being inactivated and degraded in vivo, but also can promote the diffusion between molecules and the reaction, because the synthesis of NO has the characteristic of oxygen dependence, reactants (such as oxygen generated by the catalytic ROS of platinum nanoparticles) and a catalyst are limited in the metal organic framework carrier, the content of the oxygen in the metal organic framework carrier can be increased, the probability of the reaction is greatly increased, and the catalytic efficiency is improved. Therefore, the cascade nanoenzyme not only improves the targeting property and bioavailability of NO, but also triggers cascade reaction by the enrichment effect of the nanoenzyme in the liver part, and can simultaneously realize the removal of ROS and the regulation of NO when the cascade nanoenzyme is used as a medicament for relieving the liver ischemia-reperfusion injury, thereby realizing the effective intervention on the injury caused by the liver ischemia-reperfusion. Specifically, ROS generated when HIRI occurs is used as a reactant, namely ROS is used as stimulation to trigger catalytic reaction, platinum nanoparticles catalyze ROS to react to generate oxygen, the generated oxygen can further react with arginine to generate NO in situ under the catalysis of iNOS, the whole cascade reaction is completed, the platinum nanoparticles catalyze ROS to react to generate oxygen in the process of the cascade reaction, the ROS is effectively eliminated, the generated oxygen can further react with arginine to generate NO in situ under the catalysis of iNOS, and the bioavailability of NO is improved. The cascade reaction process can reduce oxidative stress injury, inhibit apoptosis and inflammatory factor expression, and finally realize effective intervention on HIRI.

The invention is further illustrated by the following specific examples.

The following examples and tests are given with no indication of the particular experimental conditions and methods by following the conventional conditions such as: the conditions and methods described in the guidelines for laboratory animal care and use, cell protocols, etc., of the national institutes of health, or as recommended by the instrument manufacturer.

Example 1

Adding 111.1mg of polyvinylpyrrolidone (PVP) into 10mL of 1mM chloroplatinic acid solution, stirring for 15 minutes, adding newly prepared 200uL of 100mM sodium borohydride solution, stirring for 12 hours to obtain a dark brown solution, filtering and washing to obtain platinum nanoparticles, and mixing the platinum nanoparticles with a proper amount of water to prepare an aqueous solution of the platinum nanoparticles with the mass concentration of 1 mg/mL.

2-methylimidazole (0.26g,3.15mmol) and the platinum nanoparticle aqueous solution (0.1mL) are added into 0.9mL of water and mixed uniformly, then 100 μ L of a 0.5M mixed aqueous solution (the molar number of zinc nitrate is 0.05mmol) of zinc nitrate and nitric oxide synthase (0.6mg) is added, after reaction, centrifugal filtration and washing are carried out, and the cascade nanoenzyme, namely ZIF @ Pt @ NOS, is obtained, wherein a TEM image of the cascade nanoenzyme is shown in FIG. 2, the particle size of the ZIF @ Pt @ NOS is less than 100nm, and uniformly distributed Pt nanoparticles can be observed.

Comparative example 1

2-methylimidazole (0.26g,3.15mmol) was added to 0.9mL of water and mixed well, and then 100. mu.L of 0.5M aqueous zinc nitrate solution (zinc nitrate mole number 0.05mmol) was added to react to obtain a metal organic framework support, designated as ZIF.

Comparative example 2

2-methylimidazole (0.26g,3.15mmol) was added to 0.9mL of water and mixed well, then 0.1mL of the above platinum nanoparticle aqueous solution was added and mixed well, and then 100. mu.L of 0.5M zinc nitrate aqueous solution (zinc nitrate mole number 0.05mmol) was added, and the product obtained after the reaction was designated ZIF @ Pt.

Comparative example 3

2-methylimidazole (0.26g,3.15mmol) was added to 0.9mL of water and mixed well, and then 100. mu.L of a 0.5M aqueous solution (0.05 mmol of zinc nitrate in moles) of a mixed solution of zinc nitrate and nitric oxide synthase (0.6mg) was added thereto, and the product obtained after the reaction was designated as ZIF @ NOS. FIG. 3 is an XRD pattern of ZIF in comparative example 1 and ZIF @ NOS in comparative example 3. As can be seen in fig. 3, loading of iNOS does not destroy the crystal structure properties of the metal organic framework.

Comparative example 4

To 10mL of a 1mM solution of chloroplatinic acid was added 111.1mg of polyvinylpyrrolidone (PVP), and after stirring for 15 minutes, 200uL of a 100mM solution of sodium borohydride was added in a fresh state, and after stirring for 12 hours, the solution was dark brown, and was filtered and washed to obtain platinum nanoparticles, which were designated as Pt NPs.

Tests were conducted on ZIF @ Pt @ NOS in example 1, ZIF in comparative example 1, ZIF @ Pt in comparative example 2, and Pt NPs in comparative example 4.

Test 1

Capacity test of Pt NPs for scavenging active oxygen

(1) Test for scavenging Hydrogen peroxide

The efficiency test of the Pt NPs in the comparative example 4 for removing hydrogen peroxide is carried out by preparing aqueous solutions with different concentrations of 0.5 mu g/mL, 1 mu g/mL, 2 mu g/mL and 4 mu g/mL by a hydrogen peroxide probe kit (Abcam, USA), and carrying out the efficiency test of the Pt NPs with different concentrations for removing hydrogen peroxide according to the scheme provided by a manufacturer of the hydrogen peroxide probe kit, and the result is shown in FIG. 4, and as can be seen from FIG. 4, the Pt NPs can effectively remove hydrogen peroxide and have the characteristic of concentration dependence, and the inhibition rate of hydrogen peroxide is higher when the concentration is higher.

(2) Test for scavenging superoxide radical

The efficiency of eliminating superoxide radical by Pt NPs was tested by SOD detection kit (Sigma-Aldrich, USA), the Pt NPS in comparative example 4 was prepared into different concentrations of aqueous solutions of 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, respectively, and the efficiency of eliminating superoxide radical by Pt NPs was tested according to the protocol provided by the manufacturer of SOD detection kit (Sigma-Aldrich, USA), and the results are shown in FIG. 5, from which it can be seen that Pt NPs can effectively eliminate superoxide radical and have a concentration-dependent characteristic, the higher the concentration of superoxide radical inhibition rate.

Test 2

Test of NO generation capability of ZIF @ Pt @ NOS cascade enzyme

The efficiency of NO production by ZIF @ Pt @ NOS in example 1, ZIF @ Pt in comparative example 2, and ZIF @ NOS in comparative example 3 was tested by the Griess kit method (Promega, USA). Arginine (5mg/mL) and hydrogen peroxide (10mM) were added to aqueous ZIF @ Pt solution, aqueous ZIF @ NOS solution, and aqueous ZIF @ Pt @ NOS solution (the amount of Pt added in example 1 was changed so that the Pt contents were 0.58%, 1.15%, and 2.3%, respectively) at the same concentrations for 24 hours. The NO production test was performed according to the protocol provided by the nitric oxide kit manufacturer, and it can be seen from FIG. 6 that the NO production was proportional to the Pt loading, and the NO production by ZIF @ Pt @ NOS was superior to that by ZIF @ Pt and ZIF @ NOS.

Test 3

Antioxidant Capacity testing of ZIF @ Pt @ NOS in example 1

To evaluate the overall antioxidant capacity of the prepared ZIF @ Pt @ NOS nano-platform, 2' -Azino-bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) assay was used. As shown in FIG. 7, blue ABTS in the presence of ZIF @ Pt @ NOS^·+The free radicals show a significant decrease in absorbance and discoloration. In addition, the radical scavenging efficiency is positively correlated with the nanoparticle concentration. These results clearly indicate that ZIF @ Pt @ NOS has good antioxidant ability to scavenge ATBS free radicals.

Test 4

ZIF @ Pt @ NOS ability to scavenge active oxygen at cellular level and effect on survival of liver parenchymal cells (FL38B cells)

(1) Ability test of ZIF @ Pt @ NOS to scavenge active oxygen at cellular level

After incubating Phosphate Buffered Saline (PBS), ZIF in comparative example 1, ZIF @ Pt in comparative example 2, and ZIF @ Pt @ NOS in example 1 with FL38B cells for a period of time (the concentrations of Phosphate Buffered Saline (PBS), ZIF @ Pt, and ZIF @ Pt @ NOS in the cell mixture were the same), H was added to each of the cells₂O₂Simulating the oxidative stress reaction in the cells, continuously incubating for 4H, and adding a 2',7' -Dichlorofluorescin diacetate (DCFH-DA) fluorescent probe to detect the H in the cells₂O₂Content, in particular analysis of cells by confocal laser microscopy and flow cytometryInner H₂O₂Variation of the contents with provision for not adding H₂O₂The control group of (1). The results are shown in fig. 8, in which DAPI (4', 6-diamidino-2-phenylindole) was used to stain the nucleus and DCF (2',7' -dichlorofluorescein) was used as the reactive oxygen indicator, and it can be seen from fig. 8 that the DCF fluorescence was significantly reduced in the cells of ZIF @ Pt @ NOS group and close to the cells of the control group after hydrogen peroxide stimulation compared with the cells of the control group (cells not stimulated with hydrogen peroxide, control group in fig. 8), which indicates that ZIF @ Pt @ NOS could effectively scavenge the reactive oxygen in FL38B cells. ZIF @ Pt @ NOS was formulated into aqueous solutions of various concentrations (1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, respectively), and after co-incubation with FL38B cells for a period of time, H was added₂O₂The intracellular oxidative stress was simulated and then the hydrogen peroxide content was tested, and the results are shown in figure 9, with increasing concentrations of ZIF @ Pt @ NOS, the reactive oxygen species level in FL38B cells decreased significantly.

(2) Influence of ZIF @ Pt @ NOS on survival of liver parenchymal cells (FL38B cells) was tested

FL38B cells were seeded at 1X 104 density per well in 96-well plates and placed at 37 ℃ with 5% CO₂Incubating for 12h under the condition, adding ZIF @ Pt @ NOS aqueous solutions with different concentrations (1, 2, 4 and 8 mug/mL respectively) for incubation for 2h, and adding 250 mug M H₂O₂And after incubation is continued for 24h, evaluating the toxicity of ZIF @ Pt @ NOS on hepatocytes by adopting an MTT method, specifically, adding 100 mu L of MTT culture medium solution (0.8mg/mL) into each well, continuing to culture for 4h, sucking out residual culture medium in a 96-well plate, adding 150 mu L of DMSO solution into each well, after slight shaking, detecting the OD value (the detection wavelength is 570nm) of each well on a microplate reader, and calculating the cell survival rate by using the following formula. Cell viability (percent) (%) (OD 570 value of sample/blank OD570 value) × 100%. The results are shown in FIG. 10, where the survival rate of FL38 cells 38B cells was significantly higher when treated with hydrogen peroxide after incubation of ZIF @ Pt @ NOS with FL38B cells than when treated with FL38B cells without incubation of ZIF @ Pt @ NOS, indicating that ZIF @ Pt @ NOS can protect parenchymal hepatocytes from reactive oxygen species.

Test 5

Biodistribution test and liver accumulation evaluation of ZIF @ Pt @ NOS in mice

ZIF @ Pt @ NOS was injected into mice C57BL/6(6-8 weeks) in vivo via tail vein, and distribution of ZIF @ Pt @ NOS in major organs and accumulation of liver tissues of the mice at different time points of 1h, 4h, 12h, 24h, 48h and the like were measured by inductively coupled plasma atomic emission spectroscopy (ICP-OES) and live imaging of the mice, respectively.

As shown in FIG. 11, after 24H of tail vein injection, the accumulation of liver still reaches 22.8% ID/g, and the accumulation of other major organs such as heart (corresponding to H in the figure), spleen (corresponding to Sp in the figure), lung (corresponding to Lu in the figure) and kidney (corresponding to K in the figure) is less than 10% ID/g, as found by inductively coupled plasma atomic emission spectroscopy. As shown in fig. 12, the accumulation of ZIF @ Pt @ NOS in liver tissues at different time points of 1h, 4h, 10h, 24h, 48h, etc. was observed by fluorescence observation of the small animal living body, indicating that 4 hours to 10 hours had good liver accumulation effect.

Test 6

Treatment efficacy and biological safety evaluation of ZIF @ Pt @ NOS on mice with liver ischemia-reperfusion injury

(1) Evaluation of treatment efficacy of ZIF @ Pt @ NOS on mice with liver ischemia-reperfusion injury

A C57BL/6(6-8 weeks) mouse liver ischemia-reperfusion injury model is constructed, and tail vein injection administration (divided into PBS, Pt NPs, ZIF @ Pt and ZIF @ Pt @ NOS) is carried out to form HIRI positive groups, namely PBS group, Pt NPs group, ZIF @ Pt and ZIF @ Pt @ NOS group. Selecting the optimal time to perform the operation, and the specific operation is as follows: mice were opened along the mid-abdominal incision, exposing the hepatic portal area, and the hepatic pedicles including portal vein, hepatic artery and hepatic duct were clamped using a non-invasive vascular clamp. After 1h of liver ischemia, the arterial clamp was released to cause reperfusion. And a blank control group which is not injected with any medicine and is not subjected to liver ischemia reperfusion injury is set. Blood is collected for 12 hours after the operation of different groups of mice respectively, and the liver and kidney function indexes are analyzed by measuring alanine Aminotransferase (ALT), glutamic-oxaloacetic transaminase (AST), Blood Urea Nitrogen (BUN) and Creatinine (CREA) in the blood serum of the mice by a full-automatic biochemical analyzer.

As shown in fig. 13 and 14, ALT and AST levels were significantly increased after hepatic ischemia-reperfusion injury in PBS group compared to blank control group. Liver damage in Pt NPs and ZIF injected mice was not slowed. In contrast, the group ZIF @ Pt @ NOS showed the best therapeutic effect. Therefore, ZIF @ Pt @ NOS can effectively relieve and treat hepatic ischemia reperfusion injury.

(2) Safety evaluation-liver and kidney function test

Tail veins of ZIF @ Pt @ NOS were injected into mice C57BL/6(6-8 weeks), and a blank control group without ZIF @ Pt @ NOS was set, and after 7 days, ALT, AST, CRE, BUN levels of the mice were tested.

As can be seen from fig. 15 and 16, after 7 days, the levels of ALT, AST, CRE, and BUN in the ZIF @ Pt @ NOS treated group were substantially indistinguishable from the placebo zone, indicating that ZIF @ Pt @ NOS did not impair the hepatic and renal functions of mice (ALT, AST in fig. 15 are indicators reflecting hepatic function, and CRE, BUN in fig. 16 are indicators reflecting renal function), further indicating that ZIF @ Pt @ NOS has good biocompatibility and biosafety.

(3) Safety evaluation-hemolytic test

300 mu L of PBS (phosphate buffer solution) of ZIF @ Pt @ NOS in different mass concentrations (0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mu g/mL respectively) and 1.2mL of erythrocyte suspension (the erythrocyte concentration is 4%) are mixed, incubated at 37 ℃ for 4h, centrifuged at 5000r/min for 5min, 150 mu L of supernatant is taken in a 96-well plate, an ultraviolet absorbance (A) value is measured at 540nm by a microplate reader, each concentration is 3 parts in parallel, PBS is used as a negative control, and distilled water is used as a positive control. The hemolysis rate was calculated. Hemolysis rate (a value of experimental group-a value of negative control group)/(a value of positive control group-a value of negative control group). As shown in fig. 17, it can be seen that the hemolyzing rate of erythrocytes is very low after PBS and other isotonic solutions of ZIF @ Pt @ NOS with different mass concentrations are mixed with the erythrocyte suspension, which indicates that ZIF @ Pt @ NOS does not cause hemolysis of cells, and has biocompatibility and biosafety.

In conclusion, the invention provides the cascade nanoenzyme, the preparation method and the application thereof, the cascade nanoenzyme belongs to nanometer size, has the natural advantage of passive accumulation in liver compared with small molecular drugs, can increase the enrichment of drugs in liver, and has higher targeting property. The platinum nanoparticles have excellent active oxygen (ROS) scavenging capacity as a nano enzyme, can catalyze the active oxygen to generate oxygen, and the generated oxygen reacts with arginine under the catalysis of nitric oxide synthase (iNOS) to generate NO in situ, so that the bioavailability of the NO is improved. Because the specific surface area of the metal organic framework carrier is large and the pores are adjustable, the metal organic framework carrier not only can provide effective protection for iNOS combined in the metal organic framework carrier to prevent the iNOS from being inactivated and degraded in vivo, but also can promote the diffusion between molecules and the reaction, because the synthesis of NO has the characteristic of oxygen dependence, reactants (such as oxygen generated by the catalytic ROS of platinum nanoparticles) and a catalyst are limited in the metal organic framework carrier, the content of the oxygen in the metal organic framework carrier can be increased, the probability of the reaction is greatly increased, and the catalytic efficiency is improved. Therefore, the cascade nanoenzyme not only improves the targeting property and bioavailability of NO, but also triggers a cascade reaction by the enrichment effect of the cascade nanoenzyme at the liver part, and can simultaneously realize the removal of ROS and the regulation of NO, thereby realizing the effective intervention on the injury caused by liver ischemia-reperfusion. Excessive ROS in HIRI is used as a reactant, effective elimination of ROS is realized by platinum nanoparticle catalysis, and generated oxygen can further generate NO with arginine in situ under the catalysis of iNOS. The process can reduce oxidative stress injury, inhibit apoptosis and inflammatory factor expression, and finally realize effective intervention on HIRI. The cascade nanoenzyme activated by active oxygen not only improves the targeting property and bioavailability of NO, but also realizes the dual protection functions of ROS elimination and NO regulation, and provides a new idea and a clinical application prospect for developing safe and effective HIRI treatment medicines.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims

1. A cascade nanoenzyme, comprising a metal-organic framework support, a nitric oxide synthase bound to the metal-organic framework support, and a platinum nanoparticle bound to the metal-organic framework support.

2. The cascade nanoenzyme of claim 1, wherein the nitric oxide synthase is bound inside the metal-organic framework support, and the platinum nanoparticle is bound inside the metal-organic framework support and on the surface of the metal-organic framework support.

3. The cascade nanoenzyme of claim 1, wherein the particle size of the cascade nanoenzyme is 50-100 nm.

4. The cascade nanoenzyme of claim 1, wherein the platinum nanoparticles have a particle size of 1-10 nm.

5. The cascade nanoenzyme of claim 1, wherein the mass of the nitric oxide synthase is 1% -10% of the mass of the sum of the nitric oxide synthase and the metal-organic framework support.

6. The cascade nanoenzyme of claim 1, wherein the ratio of the mass of the metal in the metal-organic framework to the mass of the platinum nanoparticle is (1-10): 1.

7. the cascade nanoenzyme of claim 1, wherein the surface of the platinum nanoparticle comprises a first ligand selected from one or more of polyvinylpyrrolidone, polyethylene glycol, and polyoxyethylene polyoxypropylene ether block copolymer.

8. A method for preparing a cascade nanoenzyme as claimed in any one of claims 1 to 7, comprising the steps of:

providing an aqueous solution of platinum nanoparticles;

mixing a second ligand with the platinum nanoparticle aqueous solution;

9. The method for preparing cascade nanoenzyme as claimed in claim 8, wherein the second ligand is selected from one or two of 2-methylimidazole and 2-aldehydic imidazole, and/or the metal salt is selected from one or more of zinc salt, cobalt salt, zirconium salt and copper salt.

10. Use of a cascade nanoenzyme of any one of claims 1 to 7 in the manufacture of a medicament for the treatment of hepatic ischemia-reperfusion injury.