Multifunctional nano-enzyme for regulating tissue microenvironment to promote vascular transplantation and reconstruction and preparation method of multifunctional nano-enzyme

Abstract

The invention relates to the technical field of biology, in particular to a multifunctional nano-enzyme for regulating a tissue microenvironment to promote vascular transplantation and reconstruction and a preparation method of the multifunctional nano-enzyme. The multifunctional nano enzyme for adjusting the tissue microenvironment to promote vascular transplantation reconstruction comprises nano cerium dioxide and a phospholipid layer wrapping the nano cerium dioxide. According to the multifunctional nano-enzyme, a phospholipid layer and cells have good biocompatibility, nano cerium dioxide can simulate behaviors of superoxide dismutase and catalase, active oxygen generated around a tissue engineering blood vessel graft is removed, migration and survival of smooth muscle cells are facilitated, and the tissue engineering blood vessel graft has good biocompatibility. According to the present invention, the recombinant adenosine triphosphate can further regulate the microenvironment of adjacent tissues, catalyze ATP hydrolysis into ADP, further catalyze ADP hydrolysis into AMP, limit the activation and secretion of pro-inflammatory cytokines, achieve the effects of anticoagulation and antioxidation, facilitate vascular remodeling, have small platelet adhesion, and reduce the risk of thrombosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction and a preparation method thereof. Background Art

[0002] Severe vascular damage in cardiovascular disease (CVD) is a global health problem. Tissue-engineered vascular grafts (TEVG) have broad application prospects in overcoming severe vascular damage in cardiovascular disease. After the tissue-engineered vascular graft enters the body, due to the immune stress of the recipient, there is a local imbalance in the microenvironment around the tissue-engineered vascular graft, resulting in transplantation failure. This microenvironmental imbalance will make it difficult for endothelial cells (EC) and smooth muscle cells (SMC) to migrate and survive, which is not conducive to vascular reconstruction.

[0003] After research, it was found that tissue engineering vascular grafts, as exogenous materials, can cause platelet adhesion and activation after implantation, produce high concentrations of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), and the ATP released into the extracellular space can activate and secrete pro-inflammatory cytokines, trigger an inflammatory response, and have potential hazards to migrating endothelial cells and smooth muscle cells. Apyrase can effectively catalyze the hydrolysis of ATP into ADP, and catalyze the hydrolysis of ADP into adenosine monophosphate (AMP), and AMP can be converted into anti-inflammatory adenosine (ADO) under the action of 5′-nucleotidase. Adenosine can inhibit inflammatory response and reduce immune stress. Therefore, apyrase and 5′-nucleotidase can be prepared and purified, and tissue engineering vascular grafts can be modified by apyrase and 5′-nucleotidase. However, this multi-enzyme modification method has high cost and poor operational stability, which limits its clinical application.

[0004] In addition to ATP, highly reactive reactive oxygen species (ROS) are generated around tissue-engineered vascular grafts, which are potentially dangerous to migrating endothelial and smooth muscle cells and must be removed to keep their levels below a critical threshold. Cells can defend against ROS through antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase.

[0005] In order to regulate the complex microenvironment around tissue-engineered vascular grafts, researchers are exploring various methods to simulate the functions of natural endothelial cells or smooth muscle cells and accelerate the endothelialization of tissue-engineered vascular grafts. Among them, biomimetic protocells are often used to accelerate the endothelialization of tissue-engineered vascular grafts. Bionic protocells are a minimal cell-like structure that has the ability to adapt to the environment and self-awareness, and have important application prospects in tissue engineering and regenerative medicine.

[0006] In the prior art, bioactive molecules and covalently fixed cytokines are often used as biomimetic protocells to coat tissue-engineered vascular grafts. However, the vascular lumen modified with bioactive molecules not only promotes the adhesion of endothelial cells, but also adheres to other blood cells such as platelets and white blood cells, increasing the risk of thrombosis.

[0007] Therefore, the microenvironment surrounding tissue-engineered vascular grafts needs to be regulated to facilitate vascular reconstruction and reduce the risk of thrombosis. Summary of the invention

[0008] 1. Technical issues to be resolved

[0009] In order to solve the problem in the prior art that the microenvironment around tissue engineering vascular grafts has a local imbalance, which is not conducive to vascular reconstruction, and coating the tissue engineering vascular grafts with bioactive molecules and covalently fixed cytokines as bionic protocells will also increase the risk of thrombosis, the present invention provides a multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplant reconstruction and a preparation method thereof.

[0010] (II) Technical solution

[0011] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0012] A multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, comprising nano-cerium dioxide and a phospholipid layer wrapped on the outside of the nano-cerium dioxide.

[0013] The multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplant reconstruction as described above, preferably, the phospholipid layer includes uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups;

[0014] The uncharged phospholipids include 1,2-dioleoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine;

[0015] The negatively charged phospholipids include 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt);

[0016] The phospholipid containing an N3 group includes 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-PEG-azide.

[0017] As described above, in the multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplant reconstruction, preferably, the mass ratio of uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups is (5-10):(1-4):(1-4).

[0018] As described above, the multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplant reconstruction, preferably, the particle size of nano-cerium dioxide is 200-300nm, and the mass volume ratio w:v of nano-cerium dioxide and phospholipid layer is (1-10) mg:1mL.

[0019] The present invention also provides a method for preparing the multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, comprising the following steps:

[0020] S1: providing nano-cerium dioxide;

[0021] S2: preparing lipid membranes using uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups as raw materials;

[0022] S3: Add nano-cerium dioxide into the lipid membrane, so that the lipid membrane wraps the nano-cerium dioxide, and then perform particle size homogenization to obtain a multifunctional nanozyme.

[0023] In the method for preparing the multifunctional nanozyme as described above, preferably, in step S1, the method for preparing nano-cerium dioxide comprises the following steps:

[0024] S11: adding a soluble cerium salt and polyvinyl pyrrolidone into a first organic solvent at a certain temperature to obtain a cerium dioxide precursor through reaction;

[0025] S12: heating the cerium dioxide precursor in an air atmosphere to obtain a crude nano-cerium dioxide product;

[0026] S13: calcining the crude cerium dioxide product, and obtaining nano-cerium dioxide after cooling.

[0027] The method for preparing the multifunctional nanozyme as described above, preferably, in step S11, the reaction is carried out at 150-220° C. for 1-4 hours to obtain a cerium dioxide precursor; the first organic solvent is ethylene glycol, and the mass ratio of the soluble cerium salt to polyvinyl pyrrolidone is 1:1-5:1;

[0028] In step S12, the temperature of the heating treatment is 200-800°C, the duration is 1-4 hours, and the heating rate of the heating treatment is 1-10°C·min -1 ;

[0029] In step S13, the calcination temperature is 500-700°C, the duration is 2-5h, and the heating rate of the heating treatment is 1-10°C·min -1 .

[0030] The method for preparing the multifunctional nanozyme as described above, preferably, the method for preparing the lipid membrane comprises: dissolving uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups in a second organic solvent, mixing them uniformly and removing the second organic solvent to obtain a lipid membrane;

[0031] The second organic solvent is a mixture of any two of water, ethanol, ether, methanol, dichloromethane and chloroform.

[0032] In the method for preparing the multifunctional nanozyme as described above, preferably, uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups are dissolved in a second organic solvent, ultrasonically treated for 10-40 minutes and mixed evenly, and then the second organic solvent is removed by rotary evaporation at 25-60°C to obtain a lipid membrane.

[0033] The preparation method of the multifunctional nanozyme as described above preferably comprises adding nano-cerium dioxide and deionized water into the lipid membrane for hydration treatment for 20-60 minutes, and then performing particle size homogenization treatment by ultrasonic wave, shearing, homogenization or extrusion at a temperature of ≤80°C, so that the lipid membrane wraps the nano-cerium dioxide to obtain the multifunctional nanozyme.

[0034] (III) Beneficial effects

[0035] The multifunctional nanozyme of the present invention has a bionic original cell structure, including nano-cerium dioxide and a phospholipid layer wrapped on the outside of the nano-cerium dioxide, wherein the phospholipid layer has good biocompatibility with the cells, and the nano-cerium dioxide can simulate the behavior of superoxide dismutase and catalase, remove the reactive oxygen generated around the tissue engineering vascular graft, and is beneficial to the migration and survival of smooth muscle cells. It can also regulate the microenvironment of adjacent tissues, catalyze the hydrolysis of ATP into ADP, and further catalyze the hydrolysis of ADP into AMP, thereby limiting the activation and secretion of pro-inflammatory cytokines, achieving anticoagulant and antioxidant effects, promoting the functional recovery and maturation of the vascular structure, and being beneficial to vascular reconstruction.

[0036] The multifunctional nanozyme with a bionic protocell structure in the present invention can modify tissue engineering vascular grafts, regulate tissue microenvironment, accelerate vascular endothelialization, reduce platelet adhesion, and reduce the risk of thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the preparation process of the multifunctional nanozyme of the present invention;

[0038] Figure 2 a is the X-ray diffraction pattern of CeO2NPs and CeO2@M prepared in Example 1;

[0039] Figure 2 b is the Fourier transform infrared spectra of CeO2NPs and CeO2@M prepared in Example 1;

[0040] Figure 2 c is the particle size distribution histogram of CeO2NPs and CeO2@M prepared in Example 1;

[0041] Figure 2 d is the zeta potential diagram of CeO2NPs and CeO2@M prepared in Example 1;

[0042] Figure 2 e Scanning electron microscopy image of CeO2NPs prepared in Example 1;

[0043] Figure 2 f is a scanning electron microscope image of CeO2@M prepared in Example 1;

[0044] Figure 2 g is a transmission electron microscopy image of CeO2NPs prepared in Example 1;

[0045] Figure 2 h is the X-ray energy spectrum of CeO2NPs prepared in Example 1;

[0046] Figure 2 i is the X-ray photoelectron spectrum of CeO2NPs prepared in Example 1;

[0047] Figure 2 j is the distribution diagram of the valence ratio of Ce element in CeO2NPs prepared in Example 1;

[0048] Figure 3 a is the hydrolysis of ATP under the action of CeO2NPs prepared in Example 1 31 P-NMR spectrum monitoring diagram;

[0049] Figure 3 b Hydrolysis of ADP under the action of CeO2NPs prepared in Example 1 31 P-NMR spectrum monitoring diagram;

[0050] Figure 3 c is a comparison chart of the hydrolysis degree of ATP and ADP over time under the action of CeO2NPs prepared in Example 1;

[0051] Figure 3 d is the hydrolysis of AMP with ATP as substrate under the action of CeO2NPs prepared in Example 1 31 P-NMR spectrum monitoring diagram;

[0052] Figure 3 e is the hydrolysis of AMP with ADP as substrate under the action of CeO2NPs prepared in Example 1 31 P-NMR spectrum monitoring diagram;

[0053] Figure 3 f is a mass spectrometry analysis of the hydrolysis products of ATP and ADP under the action of CeO2NPs prepared in Example 1;

[0054] Figure 3 g is a schematic diagram of the principle of extracellular ADP-induced platelet adhesion and aggregation;

[0055] Figure 3 h is the schematic diagram of CeO2NPs inducing ADP dephosphorylation to form AMP, thereby hindering the adhesion and disaggregation of platelets;

[0056] Figure 4 a is a comparison chart of the changes in oxygen free radicals in the experimental group and the control group in the study of CeO2 nanoparticles and nanozymes as peroxidase-like nanozymes catalyzing TMB oxidation;

[0057] Figure 4 b is a comparison of the degree of oxygen changes in the experimental group and the control group in the study of CeO2 nanoparticles and nanozymes as peroxidase-like nanozymes catalyzing TMB oxidation;

[0058] Figure 4 c is a comparison of the degree of change of hydroxyl groups in the experimental group and the control group in the study of CeO2 nanoparticles and nanozymes catalyzing TMB oxidation as peroxidase-like nanozymes;

[0059] Figure 4 d is a schematic diagram of the absorbance in the experimental and control groups in the study of CeO2 nanoparticles as peroxidase-like nanozymes catalyzing TMB oxidation;

[0060] Figure 4 e is a comparison chart of the release amount of CeO2 nanoparticles with different concentrations as peroxidase-like nanozymes in the study of catalyzing TMB oxidation;

[0061] Figure 4 f is the DHE staining image of the experimental group and the control group in the study where the nanozyme was used as a peroxidase-like nanozyme to catalyze TMB oxidation;

[0062] Figure 4g is the DAPI staining image of the experimental group and the control group in the study where nanozymes act as peroxidase-like nanozymes to catalyze TMB oxidation;

[0063] Figure 4 h is the CD31 immunofluorescence staining image of the experimental and control groups in the study where nanozymes acted as peroxidase-like nanozymes to catalyze TMB oxidation;

[0064] Figure 4 i is Figure 4 Statistical graph of f;

[0065] Figure 4 j is Figure 4 g and Figure 4 Statistical graph of h. DETAILED DESCRIPTION

[0066] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0067] The invention provides a multifunctional nanozyme for regulating tissue microenvironment to promote blood vessel transplantation reconstruction, comprising nano cerium dioxide and a phospholipid layer wrapped outside the nano cerium dioxide.

[0068] Preferably, the phospholipid layer includes uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups. Among them, the uncharged phospholipids have the same structure as the cell membrane, the surface of the cell is negatively charged, the negatively charged phospholipids can make the nanozyme have the same electrical properties as the cell surface, and the phospholipids containing N3 groups can bond with blood vessels.

[0069] The above-mentioned uncharged phospholipids can be 1,2-dioleoylphosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), etc., preferably 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, the negatively charged phospholipids can be 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), etc., and the phospholipid containing the N3 group can be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-azide (DSPE-PEG-N3), etc.

[0070] Further preferably, the mass ratio of uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups is (5-10):(1-4):(1-4), which can be 5:1:1, 6:2:2, 7:3:3, 2:1:1, etc., and is preferably 5:1:1.

[0071] In the multifunctional nanozyme of the present invention, the particle size of nano-cerium dioxide is 200-300nm, and the mass volume ratio w:v of nano-cerium dioxide and phospholipid layer is (1-10) mg:1mL.

[0072] The multifunctional nanozyme of the present invention has a biomimetic original cell structure, including nano-cerium dioxide and a phospholipid layer wrapped on the outside of the nano-cerium dioxide, wherein the phospholipid layer has good biocompatibility with the cells and can reduce immune stress. The nano-cerium dioxide can simulate the behavior of superoxide dismutase and catalase, remove the reactive oxygen generated around the tissue engineering vascular graft, and is beneficial to the migration and survival of smooth muscle cells. It can also regulate the microenvironment of adjacent tissues, catalyze the hydrolysis of ATP into ADP, and further catalyze the hydrolysis of ADP into AMP, thereby limiting the activation and secretion of pro-inflammatory cytokines, achieving anticoagulant and antioxidant effects, promoting the functional recovery and maturation of the vascular structure, and being beneficial to vascular reconstruction.

[0073] The multifunctional nanozyme with a bionic protocell structure in the present invention can modify tissue engineering vascular grafts, regulate tissue microenvironment, accelerate vascular endothelialization, reduce platelet adhesion, and reduce the risk of thrombosis.

[0074] In addition, the multifunctional nanozyme of the present invention is also a nanoliposome. The phospholipid layer can protect the encapsulated nanocerium dioxide and improve the stability of the nanozyme. The nanoliposome can also extend the action time of nanocerium dioxide and reduce the frequency of administration.

[0075] The nanometer cerium dioxide (CeO2) prepared by the present invention has high biocompatibility and enzyme catalysis ability. In the nanometer cerium dioxide prepared by the present invention, the cerium ions are Ce 3+ and Ce 4+ The oxidized states of α and β coexist in a stable form to produce a redox couple, which has catalytic activity and can inhibit ADP-induced platelet aggregation.

[0076] like Figure 1 As shown, the present invention also provides a method for preparing the multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, comprising the following steps:

[0077] S1: Provide nano cerium dioxide.

[0078] S2: Prepare lipid membrane using uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups as raw materials.

[0079] S3: Add nano-cerium dioxide into the lipid membrane, so that the lipid membrane wraps the nano-cerium dioxide, and then perform particle size homogenization to obtain a multifunctional nanozyme.

[0080] Preferably, in the above step S1, the method for preparing nano-cerium dioxide comprises the following steps:

[0081] S11: Add soluble cerium salt and polyvinyl pyrrolidone to a first organic solvent, and then perform ultrasonic treatment to obtain a uniformly dispersed orange mixture, and then heat the orange mixture in an oil bath, the oil bath temperature is 150-220°C, and can be 180-200°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 210°C and 220°C, etc., and is preferably 190°C, heated for 1-4h, for example, 2-3h, 1h, 2h, 3h and 4h, etc., preferably heated for 2h, naturally cooled, centrifuged, washed and dried to obtain a brown cerium dioxide precursor, the washing times are 1-5 times, specifically 1-2 times, 3-4 times and 5 times, etc., preferably washed 3-4 times, and the drying time is 6-16h, for example, 10-14h, 6h, 8h, 10h, 12h, 14h and 16h, etc., and preferably dried for 12h. The first organic solvent is preferably ethylene glycol, and the ultrasonic treatment time is ≤60 min, specifically ≤10 min, 10-20 min, 20-30 min, 30-40 min, 40-50 min or 50-60 min, etc., preferably 10 min. The mass ratio of the soluble cerium salt to polyvinyl pyrrolidone is 1:1-5:1, specifically 2:1-4:1, 1.2:1, 2.2:1, 3.2:1, 4.2:1 and 5:1, etc., preferably 2.2:1. The soluble cerium salt can be ammonium cerium nitrate (Ce(NH4)2(NO3)6) and the like.

[0082] S12: In an air atmosphere, the cerium dioxide precursor is placed in a tube furnace or other device for heat treatment. The temperature of the heat treatment is 200-800°C, such as 500-600°C, 200°C, 300°C, 400°C, 500°C, 600°C, 700°C and 800°C, preferably 500°C, for 1-4h, specifically 2-3h, 1h, 2h, 3h and 4h, preferably 2h, and the heating rate of the heat treatment is 1-10°C·min -1 , for example 1-2℃·min -1 、2-4℃·min -1 、4-8℃·min -1 and 8-10℃·min -1 etc., preferably 2℃·min -1 , and a crude nano-cerium dioxide product is obtained. The particle size of the cerium dioxide precursor obtained after the oil bath heating is uneven. In this step, the cerium dioxide precursor is heated to obtain spindle-shaped nano-cerium dioxide with uniform particle size.

[0083] S13: calcining the crude cerium dioxide to remove the residual organic matter therein, and cooling to obtain nano cerium dioxide. In this step, the calcination temperature is 500-700°C, the duration is 2-5h, and the heating rate of the heating treatment is 1-10°C·min -1 .

[0084] Preferably, the method for preparing the lipid membrane in the above step S2 includes: dissolving uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups in a second organic solvent according to the above proportions, mixing them evenly by ultrasonic dispersion treatment to obtain a uniformly dispersed solution, then removing the second organic solvent by rotary evaporation treatment, and vacuum drying the residual solvent to obtain a lipid membrane.

[0085] Wherein, the second organic solvent is a mixture of any two of water, ethanol, ether, methanol, dichloromethane and chloroform, preferably a mixture of methanol and chloroform. In the second organic solvent, the volume ratio of the two solvents is (1-4): (1-4), specifically 1: 1, 1: 2, 1: 3, 1: 4, 2: 1, 2: 3, 2: 4, 3: 1, 3: 2, 3: 4, 4: 1 and 4: 3, etc., preferably 1: 1. The time of ultrasonic treatment is 10-40min, specifically ≤ 10min, 10-20min, 20-30min and 30-40min, etc. can be selected, preferably 30min. The rotary evaporation temperature is 25-60°C, specifically 35-40°C, 25°C, 30°C, 37°C, 42°C, 50°C and 60°C, etc. can be selected, preferably 37°C. The vacuum drying temperature is 25-60°C, specifically 35-40°C, 25°C, 30°C, 37°C, 42°C, 50°C and 60°C, etc., preferably 37°C.

[0086] Preferably, in the above step S3, the method for making the lipid membrane to wrap the nano-cerium dioxide can be any one of the solvent injection method, thin film hydration method, reverse evaporation method, emulsion method and microfluidic method, preferably the thin film hydration method, which is as follows: nano-cerium dioxide and deionized water are added to the lipid membrane for hydration treatment for 20-60 minutes, specifically 20-40 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes, etc., preferably 30 minutes. Then, at a temperature of ≤80°C, ultrasonic, shearing, homogenization or extrusion are used to homogenize the particle size, so that the lipid membrane wraps the nano-cerium dioxide to obtain a multifunctional nanozyme.

[0087] After adding nano-cerium dioxide to the lipid membrane, the mass volume ratio w:v of CeO2 nanoparticles to the phospholipid layer is 1-10mg:1mL, specifically 1mg / mL, 2-4mg / mL, 5mg / mL, 6-8mg / mL, 8-10mg / mL, etc., preferably 5mg / mL. The above-mentioned particle size homogenization treatment method preferably adopts extrusion, and the particle size control device can be any one of a micro-hand-push extruder, a tank-type liposome extruder and a gas-pressurized extruder, and is preferably a micro-hand-push extruder.

[0088] In order to further clarify the scheme of the present invention and its technical advancement, the following is a description in conjunction with specific embodiments and technical effects.

[0089] Example 1

[0090] This embodiment provides a method for preparing a multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, comprising the following steps:

[0091] S1: Ce(NH4)(NO3)6 and PVP were added to a round-bottom flask containing ethylene glycol. The mass ratio of Ce(NH4)(NO3)6 to PVP was 2.2:1, and the volume mass ratio of ethylene glycol to PVP was 50 mL:1 g. After ultrasonic treatment for 10 min, a uniformly dispersed orange mixture was obtained. The round-bottom flask was then placed in an oil bath at 190 °C for 2 hours. After natural cooling, it was centrifuged, washed 3 times and dried for 12 hours to obtain a brown precipitate. The brown precipitate was placed in a tube furnace under air atmosphere at 2 °C·min -1 The temperature was raised to 500 °C at a rate of 2 °C·min and maintained for 2 h. -1 The temperature was raised to 500°C at a heating rate and calcined for 2 h. After natural cooling, bright yellow spindle-shaped CeO2 nanoparticles were obtained.

[0092] S2: DPPC, DPPA, DSPE-PEG-N3 were mixed in a clean round-bottom flask at a mass ratio of 5:1:1, and then methanol and chloroform (v / v 1:1) were added and ultrasonicated for 30 min to obtain a uniform solution. Rotary evaporation and vacuum drying were performed at 37°C to obtain a lipid film layer in the flask.

[0093] S3: Add nano-cerium dioxide and deionized water to the lipid membrane for hydration for 30 minutes. The mass volume ratio of nano-cerium dioxide to the phospholipid layer is 5 mg:1 mL, and the volume ratio of deionized water to the lipid membrane is 1:1. Then, the particles are uniformly sized through an extruder to obtain liposome-encapsulated nano-CeO2.

[0094] Example 2

[0095] This embodiment provides a method for preparing a multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, comprising the following steps:

[0096] S1: Ce(NH4)(NO3)6 and PVP were added to a round-bottom flask containing ethylene glycol. The mass ratio of Ce(NH4)(NO3)6 to PVP was 1:1, and the volume mass ratio of ethylene glycol to PVP was 80 mL:1 g. After ultrasonic treatment for 30 min, a uniformly dispersed orange mixture was obtained. The round-bottom flask was then placed in an oil bath at 150 °C for 4 hours. After natural cooling, it was centrifuged, washed 4 times and dried for 10 h to obtain a brown precipitate. The brown precipitate was placed in a tube furnace under air atmosphere at 10 °C min -1 The temperature was raised to 800 °C at a rate of 8 °C min -1 The temperature was raised to 700 °C at a heating rate and calcined for 5 h. After natural cooling, bright yellow spindle-shaped CeO2 nanoparticles were obtained.

[0097] S2: DPPC, DPPA DSPE-PEG-N3 were mixed in a clean round-bottom flask at a mass ratio of 5:2:2, and then methanol and chloroform (v / v 1:1) were added and ultrasonicated for 40 min to obtain a uniform solution. Rotary evaporation and vacuum drying were performed at 37°C to obtain a lipid film layer in the flask.

[0098] S3: Add nano-cerium dioxide and deionized water to the lipid membrane for hydration for 20 minutes. The mass volume ratio of nano-cerium dioxide to the phospholipid layer is 10 mg:1 mL, and the volume ratio of deionized water to the lipid membrane is 1:1. Then, the particles are uniformly sized through an extruder to obtain liposome-encapsulated nano-CeO2.

[0099] Example 3

[0100] This embodiment provides a method for preparing a multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, comprising the following steps:

[0101] S1: Ce(NH4)(NO3)6 and PVP were added to a round-bottom flask containing ethylene glycol. The mass ratio of Ce(NH4)(NO3)6 to PVP was 5:1, and the volume mass ratio of ethylene glycol to PVP was 33 mL:1 g. After ultrasonic treatment for 60 min, a uniformly dispersed orange mixture was obtained. The round-bottom flask was then placed in an oil bath at 220 °C for 1 hour. After natural cooling, it was centrifuged, washed 3 times and dried for 16 h to obtain a brown precipitate. The brown precipitate was placed in a tube furnace under air atmosphere at 5 °C·min -1 The temperature was raised to 200 °C at a rate of 10 °C min-1 and maintained for 4 h. -1The temperature was raised to 600 °C at a heating rate and calcined for 3 h. After natural cooling, bright yellow spindle-shaped CeO2 nanoparticles were obtained.

[0102] S2: DPPC, DPPA DSPE-PEG-N3 were mixed in a clean round-bottom flask at a mass ratio of 6:3:2, and then methanol and chloroform (v / v 1:1) were added and ultrasonicated for 20 min to obtain a uniform solution. Rotary evaporation and vacuum drying were performed at 37°C to obtain a lipid film layer in the flask.

[0103] S3: Add nano-cerium dioxide and deionized water to the lipid membrane for hydration for 60 minutes. The mass volume ratio of nano-cerium dioxide to the phospholipid layer is 1 mg:1 mL, and the volume ratio of deionized water to the lipid membrane is 1:1. Then, the particles are uniformly sized through an extruder to obtain liposome-encapsulated nano-CeO2.

[0104] Test ①:

[0105] The CeO2 nanoparticles (CeO2NPs) and liposome-encapsulated nano-CeO2 (CeO2@M) prepared in Example 1 were characterized and the results were as follows: Figure 2 .

[0106] in, Figure 2 a is the X-ray diffraction (XRD) pattern of CeO2NPs and CeO2@M. Figure 2 b is the Fourier transform infrared spectra (FTIR) of CeO2NPs and CeO2@M. Figure 2 c is the particle size distribution histogram of CeO2NPs and CeO2@M. Figure 2 d is the zeta potential diagram of CeO2NPs and CeO2@M. Figure 2 As shown in d, CeO2NPs exhibited a positive potential, while CeO2@M exhibited a negative potential, indicating that Example 1 successfully prepared nano-cerium dioxide wrapped with a phospholipid layer. Figure 2 e and Figure 2 f are scanning electron microscope (SEM) images of CeO2NPs and CeO2@M, respectively. Figure 2 e and Figure 2 f shows that spindle-shaped nano-cerium dioxide was prepared in Example 1, and the element distribution in CeO2NPs and CeO2@M is shown. Figure 2 g is the transmission electron microscopy (TEM) image of CeO2NPs, showing the lattice structure of CeO2NPs. Figure 2 h is the X-ray energy spectrum (EDX) of CeO2NPs, showing the elemental composition of CeO2NPs. Figure 2i is the X-ray photoelectron spectrum (XPS) of CeO2NPs, which shows that in the nano-cerium dioxide prepared in Example 1, the cerium ions are Ce 3+ and Ce 4+ The oxidation states of the two species coexist in a stable form, thereby being able to produce redox pairs with corresponding catalytic activity. Figure 2 j is the distribution diagram of the valence state ratio of Ce in CeO2NPs. 4+ The proportion of Ce is considered as 1. 3+ The proportion is 0.38.

[0107] Test ②:

[0108] Investigating the role of CeO2 NPs as apyrase-like enzyme in catalyzing the hydrolysis of ATP and ADP:

[0109] The CeO2 NPs nanozyme prepared in Example 1 was used as apyrase to catalyze the hydrolysis of ATP and ADP. The specific test method is as follows:

[0110] HEPES (0.12 mmol, 28.6 mg) buffer was dissolved in 6 mL of D2O, and ATP (0.1 mmol, 50.7 mg) and ADP (0.1 mmol, 42.7 mg) were added to the resulting mixed solution. The pH value of the system was adjusted to 7.4 with NaOD solution. Then, 25 mg of CeO2 NPs prepared in Example 1 were mixed into the above buffer solution and stirred at 37°C. The hydrolysis reaction was monitored regularly by taking out 0.6 mL of the suspension from the reaction mixture and filtering it through a 0.22 μm filter. 31 The P-NMR spectrum records the filtrate to monitor the reaction. The substrate conversion rate of ATP and ADP can be calculated by equation (I) and equation (II). Equation (I) and equation (II) are as follows:

[0111]

[0112] In equation (I) and equation (II), I ATP ,I ADP and I AMP Respectively 31 The peak area of ​​ATP, ADP and AMP in the P-NMR spectrum is integrated. The hydrolysis of PNPP to 4-AP is a typical reaction for studying the enzyme properties of nanomaterials. 100 μL, 0.6 mmol PNPP and 100 μL, 0.6 mg mL -1CeO2 nanoparticles were added to a centrifuge tube containing 100 μL HEPES (pH=7.4) buffer and incubated at 37° C. for 2 h. The absorbance of the reaction system in the wavelength range of 200-500 nm was recorded using an ultraviolet spectrophotometer.

[0113] pass 31 P-NMR spectroscopy monitors the hydrolysis of ATP, such as Figure 3 As shown in a, the results show that the characteristic peaks associated with β-ATP gradually decreased within 24 hours and were converted into characteristic peaks of α-ADP, β-ADP, AMP and phosphate on CeO2NPs. At the same time, the characteristic peaks of converted ADP were not detected after 24 hours, indicating that ATP was converted into AMP by sequential hydrolysis of its terminal phosphate by CeO2NPs.

[0114] Likewise, through Figure 3 b It can be seen that similar catalytic phenomena were also observed when ADP was directly used as a substrate on CeO2NPs.

[0115] pass Figure 3 c As can be seen, ADP showed more efficient hydrolysis, exceeding 90% after 12 h, which may be due to the fact that ADP hydrolysis was completed in one step, while ATP hydrolysis needed to be completed in multiple steps.

[0116] Based on the above results, we continued to study the hydrolysis of AMP containing high-energy phosphate bonds catalyzed by CeO2NPs and obtained Figure 3 d-3f. The hydrolysis products of ATP and ADP were analyzed by mass spectrometry to obtain Figure 3 f. By Figure 3 f shows that at 6h, the signal peaks of ATP, ADP, AMP and HEPES molecules were recorded from the hydrolysis products; after 24h of hydrolysis reaction, the AMP signal peak appeared, the HEPES signal peak remained unchanged, and the ATP and ADP signal peaks disappeared, indicating that CeO2NPs catalyzed the hydrolysis of ATP and ADP. 6 hours after ATP and ADP dephosphorylation, the relative abundance of ADO in the product was weak, while after 24 hours, its relative abundance increased significantly, indicating that the content of ADO in the product increased with the extension of reaction time, but the relative abundance of ADO was only 0.1% of AMP. Therefore, it shows that CeO2NPs have a good ability to simulate apyrase.

[0117] pass Figure 3g-3h shows that extracellular ADP molecules activate platelets through P2 Y1, P2 Y12 and P2 X1 purinergic receptors, inducing platelet adhesion and aggregation on the surface. When CeO2NPs are modified on the glass interface, the inducer ADP is dephosphorylated to form AMP, and no large number of aggregated platelets are observed on the interface, proving that CeO2NPs can hinder platelet adhesion and disaggregation. Therefore, CeO2NPs have endogenous adenosine triphosphate diphosphatase-like activity, which can effectively reduce the physiological level of ADP and have potential application value in anti-platelet adhesion and anticoagulation of tissue engineering blood vessels.

[0118] Test 3:

[0119] Continue to study CeO2 nanoparticles and nanozymes as peroxidase-like nanozymes catalyzing TMB oxidation as follows:

[0120] TMB (3,3',5,5'-tetramethylbenzidine, a commonly used colorimetric agent, is colorless when not oxidized. When TMB is oxidized by hydrogen peroxide or other oxidants, a blue product is generated) is often used to study the enzyme activity of nanoparticles. 0.5 mL of TMB (concentration of 0.6 mM), H2O2 (10 mM) and 0.5 mL of CeO2NPs (50 μg mL -1 ) was added to a 2 mL centrifuge tube containing 0.5 mL HAc-NaAc buffer (pH = 4.0), and the mixture was incubated at 37 ° C for 30 min to promote the reaction. This group was used as experimental group 1. CeO2NPs were replaced with CeO2@M prepared in Example 1, and the same operation was performed as experimental group 2. The group without adding H2O2, CeO2NPs and CeO2@M was used as control group 1. The changes in oxygen free radicals, oxygen and hydroxyl groups in the experimental group and control group 1 were recorded, and the results were obtained. Figure 4 a- Figure 4 c. By Figure 4 a- Figure 4 c It can be seen that the multifunctional nanozyme can convert superoxide anions (O 2- ·) are converted into O2 and H2O2, exerting anti-inflammatory and antioxidant effects.

[0121] The groups with added H2O2, without added CeO2NPs and CeO2@M were used as the control groups. The results of the experimental group with added CeO2@M and the two control groups, as well as the oxygen release, were recorded by UV-visible spectrophotometer in the wavelength range of 300-800nm. Figure 4 d and Figure 4 e. By Figure 4 d and Figure 4e It can be seen that TMB will change color only when hydrogen peroxide and CeO2@M are added, and its catalytic effect will increase with the increase of nano-CeO2 concentration.

[0122] Control group 1 simulates the environment in which normal cells are located, and control group 2 can simulate the environment in which inflammatory cells are located. ROS-induced apoptosis of human umbilical vein endothelial cells and the expression of terminal metallothionein (EndMT) Objective To explore the effect of CeO2@M on ROS-induced cell apoptosis and EndMT. HUVECs were pretreated with CeO2@M and then incubated with H2O2. Specifically, cells reaching 80% confluence were separated from the cell culture flask using 0.25% trypsin and seeded on a 48-well plate. After 12 hours of culture, the cells were mixed with 50 μg.mL prepared in Example 1. -1 CeO2@M was incubated for 24 hours. After HUVECs were treated with 100 μM H2O2 for 5 hours, they were stained with DHE or LIVE / DEAD staining kit to detect intracellular ROS levels and cell apoptosis. After stimulation with 100 μM H2O2 for 24 hours, immunofluorescence staining and RT-qPCR were used to detect the expression of End MT in ROS-induced endothelial cells. Figure 4 f-4j, the results showed that compared with the Control group (control group 1) and the H2O2 group (control group 2), the experimental group containing CeO2@M could promote angiogenesis and inhibit the secretion and release of pro-inflammatory factors.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction, characterized in that: The invention comprises nano cerium dioxide and a phospholipid layer wrapped on the outside of the nano cerium dioxide.

2. The multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction according to claim 1, characterized in that: The phospholipid layer includes uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups; The uncharged phospholipids include 1,2-dioleoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine; The negatively charged phospholipids include 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt); The phospholipid containing an N3 group includes 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-PEG-azide.

3. The multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction according to claim 2, characterized in that: The mass ratio of uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups is (5-10):(1-4):(1-4).

4. The multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction according to claim 1, characterized in that: The particle size of the nano-cerium dioxide is 200-300nm, and the mass volume ratio w:v of the nano-cerium dioxide and the phospholipid layer is (1-10) mg:1mL.

5. A method for preparing a multifunctional nanozyme for regulating tissue microenvironment to promote vascular transplantation reconstruction according to any one of claims 1 to 4, characterized in that: The steps include: S1: providing nano-cerium dioxide; S2: preparing lipid membranes using uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups as raw materials; S3: Add nano-cerium dioxide into the lipid membrane, so that the lipid membrane wraps the nano-cerium dioxide, and then perform particle size homogenization to obtain a multifunctional nanozyme.

6. The method for preparing the multifunctional nanozyme according to claim 5, characterized in that: In step S1, the preparation method of nano cerium dioxide comprises the following steps: S11: adding a soluble cerium salt and polyvinyl pyrrolidone into a first organic solvent at a certain temperature to obtain a cerium dioxide precursor through reaction; S12: heating the cerium dioxide precursor in an air atmosphere to obtain a crude nano-cerium dioxide product; S13: calcining the crude cerium dioxide product, and obtaining nano-cerium dioxide after cooling.

7. The method for preparing the multifunctional nanozyme according to claim 6, characterized in that: In step S11, the reaction is carried out at 150-220° C. for 1-4 hours to obtain a cerium dioxide precursor; the first organic solvent is ethylene glycol, and the mass ratio of the soluble cerium salt to polyvinyl pyrrolidone is 1:1-5:1; In step S12, the temperature of the heating treatment is 200-800°C, the duration is 1-4 hours, and the heating rate of the heating treatment is 1-10°C·min -1 ; In step S13, the calcination temperature is 500-700°C, the duration is 2-5h, and the heating rate of the heating treatment is 1-10°C·min -1 .

8. The method for preparing the multifunctional nanozyme according to claim 5, characterized in that: In step S2, the method for preparing the lipid membrane comprises: dissolving uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups in a second organic solvent, mixing them uniformly and removing the second organic solvent to obtain the lipid membrane; The second organic solvent is a mixture of any two of water, ethanol, ether, methanol, dichloromethane and chloroform.

9. The method for preparing the multifunctional nanozyme according to claim 8, characterized in that: In step S2, uncharged phospholipids, negatively charged phospholipids and phospholipids containing N3 groups are dissolved in a second organic solvent, and mixed evenly after ultrasonic treatment for 10-40 minutes, and then the second organic solvent is removed by rotary evaporation at 25-60° C. to obtain a lipid film.

10. The method for preparing the multifunctional nanozyme according to claim 5, characterized in that: In step S3, nano-cerium dioxide and deionized water are added to the lipid membrane for hydration treatment for 20-60 minutes, and then the particle size is homogenized by ultrasound, shearing, homogenization or extrusion at a temperature of ≤80°C, so that the lipid membrane wraps the nano-cerium dioxide to obtain a multifunctional nanozyme.

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