Esterase response type nanogel modified bioprosthetic valve, preparation method and application

Abstract

The invention discloses an esterase response type nanogel modified bioprosthetic valve as well as a preparation method and application thereof, and belongs to the technical field of bioprosthetic valves. According to the invention, the problem of how to consider the infection resistance, biocompatibility and high surface modification rate of the bioprosthetic valve is solved. The preparation method comprises the following steps: synthesizing an esterase response monomer by taking methacrylate containing an ester bond and an antibiotic drug as raw materials, then synthesizing esterase response type nanogel, and then connecting the esterase response type nanogel to the surface of a bioprosthetic valve; under a pathological environment with relatively high esterase concentration, ester bond groups are decomposed, and antibiotic drugs are released in situ at fixed points, so that infection caused by bacteria is treated, inflammatory cell infiltration is reduced, and anti-infection and anti-inflammatory performance of the bioprosthetic valve is improved; in addition, the nanogel contains a large number of hydrophilic groups, a hydration layer is formed on the surface of the bioprosthetic valve, the risk of thrombus and blood coagulation can be effectively reduced, and bacterial adhesion is resisted. The prepared bioprosthetic valve has excellent anti-infection, anti-coagulation and anti-calcification capabilities and stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological valves, and particularly relates to an esterase-responsive nanogel modified biological valve, a preparation method and an application thereof. Background Art

[0002] Valvular heart disease is currently a major disease leading to death worldwide. Currently, more than 100 million people worldwide suffer from heart valve disease. Artificial heart valve replacement is one of the effective means for the clinical treatment of heart valve disease. The artificial valve prostheses used for artificial heart replacement are divided into mechanical valves and biological valves. With the development of transcatheter interventional treatment technology for heart valve disease in recent years, the demand for biological valves has been increasing continuously.

[0003] Most of the biological valves currently used clinically are made of glutaraldehyde-crosslinked bovine pericardium or porcine aortic valves, and their service life is usually only about 10 years. The failure of biological valves is caused by multiple factors, including infective endocarditis, calcification, microcoagulation and difficult endothelialization of biological valves. Among them, prosthetic valve endocarditis (PVE) is a serious and fatal complication after heart valve replacement, seriously threatening human health.

[0004] For prosthetic valve endocarditis, drug treatment is mainly adopted clinically. However, due to the large vegetations caused by PVE, patients usually need to take high-dose antibiotics for a long time, resulting in serious toxic and side effects. Constructing a biological valve with in-situ anti-infection function is expected to solve this problem. Simple drug modification of biological valves usually reduces the biocompatibility of materials, and the modification sites on the surface of biological valves are limited, and the modification efficiency is not high. How to endow artificial biological valves with anti-infection performance while improving their biocompatibility and surface modification efficiency is a problem that needs to be solved currently. Summary of the Invention

[0005] Aiming at the problem in the prior art of how to endow artificial biological valves with anti-infection performance while improving their biocompatibility and surface modification efficiency, the present invention provides an esterase-responsive nanogel modified biological valve, a preparation method and an application thereof, aiming to solve the technical problems of poor stability, short validity period, poor long-term anticoagulation, anti-inflammatory, anti-infection and anti-calcification capabilities of biological valves in the prior art.

[0006] The technical solution adopted by the present invention is as follows:

[0007] An esterase-responsive nanogel modified biological valve, wherein an esterase-responsive nanogel is connected to the nanogel modified biological valve, and the esterase-responsive nanogel contains an antibiotic drug with an ester bond group.

[0008] A preparation method of an esterase-responsive nanogel modified biological valve, comprising the following steps:

[0009] S1: Perform glutaraldehyde cross-linking treatment on the biological valve;

[0010] S2: Graft an amino cationic polymer onto the glutaraldehyde-crosslinked biological valve;

[0011] S3: Chemically bond the esterase-responsive nanogel to the surface of the grafted biological valve;

[0012] S4: Treat the biological valve with an active ester compound to obtain the nanogel-modified biological valve.

[0013] Preferably, the biological valve is acellular bovine pericardium or porcine pericardium.

[0014] Preferably, the preparation method of the esterase-responsive nanogel in S3 includes:

[0015] S2011: Dissolve a methacrylate containing an ester bond group and an antibiotic drug in a solvent, and add a condensing agent, a dehydrating agent, and a catalyst to react to obtain an esterase-responsive monomer;

[0016] S2012: Dissolve the esterase-responsive monomer, a hydrophilic monomer, an active ester monomer, a crosslinking agent, and an initiator in a solvent, and react under the protection of an inert gas to obtain an esterase-responsive nanogel.

[0017] Preferably, the dosage ratio of the methacrylate containing an ester bond, the antibiotic drug, the solvent, the condensing agent, the dehydrating agent, and the catalyst is 1 mmol: 0.2 - 5 mmol: 10 - 20 mL: 1 - 3 mmol: 1 - 3 mmol: 0.1 - 1 mmol;

[0018] The dosage ratio of the esterase-responsive monomer, the hydrophilic monomer, the active ester monomer, the crosslinking agent, the initiator, and the solvent is 1 - 20 mg: 5 - 100 mg: 1 - 10 mg: 1 - 10 mg: 1 - 10 mg: 20 - 40 mL.

[0019] Furthermore, the feed liquid ratio of the esterase-responsive monomer, the hydrophilic monomer, the active ester monomer, the crosslinking agent, the initiator, and the solvent is 1 - 10 mg: 5 - 100 mg: 1 - 10 mg: 1 - 10 mg: 1 - 10 mg: 20 - 40 mL.

[0020] Preferably, the methacrylate containing an ester bond is 2-hydroxyethyl methacrylate;

[0021] The hydrophilic monomer is 2-acrylamido-2-methylpropanesulfonic acid;

[0022] The active ester monomer is N-acryloxysuccinimide or N-hydroxysuccinimide methacrylate.

[0023] After adopting this technical solution, the hydrophilic monomer is selected as 2-acrylamido-2-methylpropanesulfonic acid, and the sulfonic acid group can inhibit platelet adhesion by simulating the anticoagulant activity similar to heparin. The active ester monomer is selected as N-acryloxysuccinimide or N-hydroxysuccinimide methacrylate, so it has good biocompatibility and strong amino reaction activity.

[0024] Preferably, in S2011, the solvent is dichloromethane; the condensing agent is one or more of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate; the dehydrating agent is one or more of N,N-dicyclohexylcarbodiimide, diisopropylcarbodiimide, and N,N'-diisopropylcarbodiimide; the catalyst is one or more of 4-dimethylaminopyridine p-toluenesulfonate, 4-dimethylaminopyridine, triethylamine, and N,N-diisopropylethylamine.

[0025] Preferably, in S2012, the crosslinking agent is N,N'-methylenebisacrylamide; the initiator is azobisisobutyronitrile; the solvent is one or more of acetonitrile, water, ethanol, dimethyl sulfoxide, and dioxane.

[0026] Preferably, the reaction conditions of S2011 are to react at room temperature for 6 to 72 h; the reaction conditions of S2012 are to react at 40 to 160 °C for 0.1 to 24 h.

[0027] Preferably, in S1, the biological valve is soaked in a glutaraldehyde solution with a concentration of 0.05 to 8 wt% for 48 to 72 h; the concentration of the esterase-responsive nanogel solution is 0.01 to 10 mg / mL;

[0028] In S2, the amino cationic polymer is branched polyethyleneimine with a molecular weight of 300 to 1,000,000, and the concentration is 5 to 15 mg / mL;

[0029] In S3, the concentration of the esterase-responsive nanogel solution is 0.01 to 10 mg / mL;

[0030] In S4, the active ester compound is branched acetic acid-N-succinimide ester or polyethylene glycol succinimide ester, and the concentration is 1 to 5 mg / mL.

[0031] Preferably, S2-3 is repeated 0-1 times. When performing S2 for the first time, the glutaraldehyde-crosslinked biological valve is soaked in 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a quantity equivalent of 0.05-0.4 times the theoretical amino group quantity and N-hydroxysuccinimide with a quantity equivalent of 0.05-0.4 times the theoretical amino group quantity.

[0032] Preferably, the reaction conditions in S2 are as follows: reacting for 1-48 h under the conditions of a temperature of 4-37 °C and a pH of 3-10;

[0033] The reaction conditions in S3 are as follows: reacting for 0.5-48 h under the conditions of a temperature of 4-37 °C and a pH of 3-10.

[0034] Further, when soaking the glutaraldehyde-crosslinked biological valve with the amino cationic polymer solution for the first time, the pH is 5.5, the concentration of the amino cationic polymer is 10 mg / mL, and the soaking time is 12 h. When soaking and treating with the amino cationic polymer solution again, the pH is 7, the concentration of the amino cationic polymer solution is 10 mg / mL, and the soaking time is 4 h.

[0035] Further, when soaking and treating the biological valve grafted with the amino cationic polymer with the esterase-responsive nanogel solution for the first time, the pH is 7, the concentration of the esterase-responsive nanogel solution is 1 mg / mL, and the soaking time is 2 h.

[0036] Further, when soaking and treating with the esterase-responsive nanogel solution again, the pH is 7, the concentration of the esterase-responsive nanogel solution is 1 mg / mL, and the soaking time is 12 h.

[0037] Further, when soaking the biological valve bonded with the esterase-responsive nanogel with the active ester compound solution, the pH is 7, the concentration of the active ester compound solution is 5 mg / mL, and the soaking time is 2 h.

[0038] Preferably, the antibiotic drug is a drug for preventing infective endocarditis, and the drug for preventing infective endocarditis is one of ofloxacin, levofloxacin, ciprofloxacin, amoxicillin, ceftriaxone, penicillin, and moxifloxacin.

[0039] An application of a nanogel-modified biological valve for preventing infective endocarditis in the preparation of an artificial valve prosthesis.

[0040] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0041] 1. The nanogels prepared in the present invention have uniform particle sizes. The nanogels contain monomers of drugs for preventing infective endocarditis with ester bonds, and the ester bonds are esterase-responsive groups. The drugs for preventing infective endocarditis are antibiotics with broad-spectrum anti-infective activities. In pathological environments with high esterase concentrations such as microbial infections, the ester bonds in the monomers decompose, and the drugs for preventing infective endocarditis are released at specific sites, thereby exerting an anti-infective effect and providing preventive measures for potential risks of infective endocarditis.

[0042] 2. The method for modifying biological valves provided by the present invention solves the problem of few modifiable sites on the surface of biological valves. By reacting biological valves with amino cationic polymers, the residual aldehyde groups on the glutaraldehyde-crosslinked biological valves are blocked, effectively reducing the problems of biological valve calcification and biotoxicity. At the same time, a large number of amino groups are introduced, providing reaction sites for the efficient modification of subsequent nanogels and solving the dilemma of few modifiable sites on biological valves.

[0043] 3. The present invention introduces a large number of hydrophilic groups on the surface of biological valves, which can form a stable hydration layer on the surface of biological valves, effectively reducing platelet adhesion, reducing the risks of thrombosis and coagulation, and resisting bacterial adhesion.

[0044] 4. The present invention modifies esterase-responsive nanogels on the surface of biological valve materials by means of chemical bonding. Compared with biological valves modified by electrostatic adsorption or physical filling, the biological valves prepared by this chemical bonding method have good stability and long-term effectiveness, can improve the long-term anti-inflammatory, anticoagulant and anti-calcification abilities of biological valves, realize the organic integration of multiple functions on the surface of biological valves, and thus extend the service life of biological valves. Description of the Drawings

[0045] Figure 1 It is a diagram of the particle size measurement results of the esterase-responsive nanogels synthesized in Example 1;

[0046] Figure 2 It is a diagram of the stability test results of the esterase-responsive nanogels synthesized in Example 1;

[0047] Figure 3 It is an SEM image of the esterase-responsive nanogels synthesized in Example 1;

[0048] Figure 4 It is an SEM image of the biological valve modified with the nanogels prepared in Example 1;

[0049] Figure 5 It is an SEM image of platelets adhering to the surface of biological valves with and without esterase-responsive nanogel modification;

[0050] Figure 6SEM images of bioprosthetic valves modified with and without esterase-responsive nanogels after ex vivo blood contact tests;

[0051] Figure 7 Immunohistochemical staining results of bioprosthetic valves modified with and without esterase-responsive nanogels at 7 and 14 days after subcutaneous implantation;

[0052] Figure 8 Statistical graphs of the number of IL-1β- and TNF-α-positive cells after immunohistochemical staining of bioprosthetic valves modified with and without esterase-responsive nanogels at 7 and 14 days after subcutaneous implantation;

[0053] Figure 9 Microscopic images of alizarin red-stained sections of bioprosthetic valves modified with and without esterase-responsive nanogels at 60 days after subcutaneous implantation;

[0054] Figure 10 Drug release experimental results of bioprosthetic valves modified with nanogels for preventing infective endocarditis;

[0055] Figure 11 Statistical graphs of the number of bacteria adhering to the surface of bioprosthetic valves modified with and without esterase-responsive nanogels after contact with bacteria;

[0056] Figure 12 SEM images of bacteria adhering to the surface of bioprosthetic valves modified with and without esterase-responsive nanogels after contact with bacteria;

[0057] Figure 13 Colony-forming unit graphs and statistical graphs of bacteria after co-culture of bioprosthetic valves modified with and without esterase-responsive nanogels with bacteria. Detailed implementation manners

[0058] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Apparently, the described embodiments are only some of the embodiments of this application, rather than all of them. Usually, the components of the embodiments of this application described and illustrated herein can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely represents selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative efforts fall within the scope of protection of this application.

[0059] Example 1

[0060] A method for preparing an esterase-responsive nanogel-modified bioprosthetic valve includes the following steps:

[0061] ①Synthetic esterase-responsive nanogel, including the following steps:

[0062] S2011: Add 222 mg of 2-hydroxyethyl methacrylate, 617 mg of ofloxacin, 458 mg of N,N'-dicyclohexylcarbodiimide, 843 mg of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, 201 mg of 4-dimethylaminopyridine p-toluenesulfonate and 20 mL of dichloromethane into a 100 mL round-bottom flask. After stirring and dissolving, react at room temperature for 48 h. After the reaction is completed, perform suction filtration, collect the filtrate, concentrate the filtrate with a rotary evaporator, redissolve it in dichloromethane, then add 20 mL of sodium chloride solution for extraction. Extract 3 times, collect the lower-layer dichloromethane solution, dehydrate and dry it with anhydrous sodium sulfate, perform suction filtration, collect the filtrate, concentrate the filtrate with a rotary evaporator until there is no dichloromethane left, and then dissolve it in 2 mL of dichloromethane; Dissolve an appropriate amount of silica gel powder in petroleum ether, load a 15 cm chromatography column, after loading the reactant dissolved in dichloromethane, pass the column successively with eluents of petroleum ether:ethyl acetate = 8:1 and 4:1, collect the product, dry it with a rotary evaporator, and dry it with an oil pump to obtain an esterase-responsive ofloxacin monomer; The structure of the esterase-responsive ofloxacin monomer is as shown in Formula I,

[0063]

[0064] S2012: Take 10 mg of the esterase-responsive ofloxacin monomer, 170 mg of 2-acrylamido-2-methylpropanesulfonic acid, 15 mg of N-acryloxysuccinimide, 20 mg of N,N'-methylenebisacrylamide, 4 mg of azobisisobutyronitrile and 40 mL of chromatographic grade acetonitrile in a thick-walled pressure-resistant bottle. Promote the dissolution of the raw materials by ultrasonic treatment, then displace nitrogen, and react at 100 °C under nitrogen protection for 1 h. After the reaction is completed, collect the suspension, centrifuge at 12000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and dry it in ventilation for 20 min to obtain an esterase-responsive nanogel, which is a pale yellow solid powder.

[0065] ②Preparation of a nanogel-modified bioprosthesis for preventing infective endocarditis, including the following steps:

[0066] S1: Take fresh porcine pericardium, remove connective tissue and perform cleaning treatment, then wash it clean with physiological saline, then soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake for 48 h, and then soak it in a 1 wt% glutaraldehyde solution and shake for 48 h for cross-linking treatment. After taking it out, wash it clean with physiological saline to obtain a glutaraldehyde-crosslinked bioprosthesis;

[0067] S2. Immerse the glutaraldehyde-crosslinked biological valve in a branched polyethyleneimine solution with a concentration of 10 mg / mL. The molecular weight of the branched polyethyleneimine is 10,000. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with an equivalent amount of 0.1 times the theoretical number of amino groups and N-hydroxysuccinimide with an equivalent amount of 0.1 times the theoretical number of amino groups. Then, immerse it at 32 °C and pH 6 for 12 h to obtain a biological valve grafted with an amino cationic polymer.

[0068] S3. Immerse the biological valve grafted with an amino cationic polymer in an esterase-responsive nanogel solution with a concentration of 1 mg / mL. Shake and react and immerse it at 32 °C and pH 6 for 12 h to obtain a biological valve bonded with an esterase-responsive nanogel.

[0069] S4. Immerse the biological valve bonded with an esterase-responsive nanogel in a branched acetic acid-N-succinimide ester solution with a concentration of 5 mg / mL twice. Each time, shake and immerse it at 32 °C and pH 6 for 2 h. After taking it out, wash it clean with physiological saline to obtain the product.

[0070] Example 2

[0071] A method for preparing an esterase-responsive nanogel-modified biological valve includes the following steps:

[0072] ① Synthesize an esterase-responsive nanogel, including the following steps:

[0073] S2011: Add 222 mg of 2-hydroxyethyl methacrylate, 566 mg of ciprofloxacin, 458 mg of N,N'-dicyclohexylcarbodiimide, 843 mg of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, 201 mg of 4-dimethylaminopyridine p-toluenesulfonate, and 20 mL of dichloromethane to a 100 mL round-bottom flask. After stirring and dissolving, react at room temperature for 48 h. After the reaction is completed, perform suction filtration, collect the filtrate, concentrate the filtrate with a rotary evaporator, redissolve it in dichloromethane, then add 20 mL of sodium chloride solution for extraction. Extract 3 times, collect the lower-layer dichloromethane solution, dehydrate and dry it with anhydrous sodium sulfate, perform suction filtration, collect the filtrate, concentrate the filtrate with a rotary evaporator until there is no dichloromethane left, and then dissolve it in 2 mL of dichloromethane; Dissolve an appropriate amount of silica gel powder in petroleum ether, load a 15 cm chromatography column, load the reactant dissolved in dichloromethane, and then pass through the column with eluents of petroleum ether:ethyl acetate = 10:1 and 5:1 in sequence. Collect the product, dry it with a rotary evaporator, and then dry it with an oil pump to obtain an esterase-responsive ciprofloxacin monomer. The structure of the esterase-responsive ciprofloxacin monomer is shown in Formula II.

[0074]

[0075] S2012: Take 20 mg of the esterase-responsive ciprofloxacin monomer, 170 mg of 2-acrylamido-2-methylpropanesulfonic acid, 15 mg of N-acryloxysuccinimide, 20 mg of N,N'-methylenebisacrylamide, 4 mg of azobisisobutyronitrile, and 40 mL of chromatographic grade acetonitrile in a thick-walled pressure-resistant bottle. Sonicate to promote the dissolution of the raw materials, then displace the nitrogen, and react under nitrogen protection at 160 °C for 0.5 h. After the reaction, collect the suspension, centrifuge at 12,000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and dry it in a ventilated place for 20 min to obtain the esterase-responsive nanogel, which is a slightly yellow solid powder.

[0076] ② Preparation of a nanogel-modified bioprosthesis for preventing infective endocarditis, including the following steps:

[0077] S1: Take fresh porcine pericardium, remove connective tissue and perform a cleaning treatment, then wash it clean with physiological saline, then soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake for 48 h, and then soak it in a 5 wt% glutaraldehyde solution and shake for 48 h for cross-linking treatment. After taking it out, wash it clean with physiological saline to obtain a glutaraldehyde-crosslinked bioprosthesis;

[0078] S2: Immerse the glutaraldehyde-crosslinked bioprosthesis in a 10 mg / mL branched polyethyleneimine solution with a molecular weight of 1,000,000, and add 0.15 theoretical amino group equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15 theoretical amino group equivalent of N-hydroxysuccinimide, and then soak it at 37 °C and pH 6 for 1 h to obtain a bioprosthesis grafted with an amino cationic polymer;

[0079] S3: Immerse the bioprosthesis grafted with an amino cationic polymer in a 0.1 mg / mL esterase-responsive nanogel solution, and shake and soak it at 37 °C and pH 10 for 0.5 h to obtain a bioprosthesis bonded with an esterase-responsive nanogel;

[0080] S4: Immerse the bioprosthesis bonded with an esterase-responsive nanogel in a 5 mg / mL branched acetic acid-N-succinimide ester solution twice, each time shaking and soaking it at 37 °C and pH 6 for 2 h. After taking it out, wash it clean with physiological saline to obtain the product.

[0081] Example 3

[0082] An esterase-responsive nanogel-modified bioprosthesis, and its preparation method includes the following steps:

[0083] ② Synthesis of an esterase-responsive nanogel, including the following steps:

[0084] S2011: Add 222 mg of 2-hydroxyethyl methacrylate, 623 mg of amoxicillin, 458 mg of N,N-dicyclohexylcarbodiimide, 843 mg of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, 201 mg of 4-dimethylaminopyridine p-toluenesulfonate and 20 mL of dichloromethane into a 100 mL round-bottom flask. After stirring and dissolving, react at room temperature for 48 h. After the reaction, perform suction filtration, collect the filtrate, concentrate the filtrate with a rotary evaporator, redissolve it in dichloromethane, then add 20 mL of sodium chloride solution for extraction. Extract three times, collect the lower layer of dichloromethane solution, dehydrate and dry it with anhydrous sodium sulfate, perform suction filtration, collect the filtrate, concentrate the filtrate with a rotary evaporator until there is no dichloromethane left, and then dissolve it in 2 mL of dichloromethane; Dissolve an appropriate amount of silica gel powder in petroleum ether, load a 15 cm chromatography column, load the reactant dissolved in dichloromethane, and then pass the column successively with eluents of petroleum ether:ethyl acetate = 6:1 and 4:1. Collect the product, dry it with a rotary evaporator, and then dry it with an oil pump to obtain the esterase-responsive amoxicillin monomer; The structure of the esterase-responsive amoxicillin monomer is shown in Formula III,

[0085]

[0086] S2012: Take 10 mg of the esterase-responsive amoxicillin monomer, 170 mg of 2-acrylamido-2-methylpropanesulfonic acid, 15 mg of N-acryloxysuccinimide, 20 mg of N,N'-methylenebisacrylamide, 4 mg of azobisisobutyronitrile and 40 mL of chromatographic grade acetonitrile in a thick-walled pressure-resistant bottle. Promote the dissolution of the raw materials by ultrasonic treatment, then displace the nitrogen, and react under nitrogen protection and at 40 °C for 6 h. After the reaction, collect the suspension, centrifuge at 12000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and dry it in a ventilated place for 20 min to obtain the esterase-responsive nanogel, which is a white solid powder.

[0087] ② Preparation of a nanogel-modified bioprosthesis for preventing infective endocarditis, including the following steps:

[0088] S1: Take fresh porcine pericardium, remove the connective tissue and perform cleaning treatment, then wash it clean with physiological saline, then soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake it for 48 h, and then soak it in a 0.05 wt% glutaraldehyde solution and shake it for 48 h for cross-linking treatment. After taking it out, wash it clean with physiological saline to obtain the glutaraldehyde-crosslinked bioprosthesis;

[0089] S2. Immerse the glutaraldehyde-crosslinked biological valve in a branched polyethyleneimine solution with a concentration of 5 mg / mL. The molecular weight of the branched polyethyleneimine is 300, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with 0.15 theoretical amino group equivalent and N-hydroxysuccinimide with 0.15 theoretical amino group equivalent. Then immerse it at 4 °C and pH 5 for 24 h to obtain a biological valve grafted with an amino cationic polymer.

[0090] S3. Immerse the biological valve grafted with an amino cationic polymer in an esterase-responsive nanogel solution with a concentration of 5 mg / mL, and shake and immerse it at 4 °C and pH 3 for 24 h to obtain a biological valve bonded with an esterase-responsive nanogel.

[0091] S4. Immerse the biological valve bonded with an esterase-responsive nanogel twice in a branched acetic acid-N-succinimide ester solution with a concentration of 3 mg / mL. Each time, shake and immerse it at 4 °C and pH 5 for 6 h, and then take it out and wash it clean with physiological saline to obtain the product.

[0092] Experimental Example

[0093] The properties of the nanogel-modified biological valve for preventing infective endocarditis prepared in the examples of the present invention are similar. Taking Example 1 as an example, the performance of related products is described as follows:

[0094] I. Structure Test

[0095] ① Use a Malvern nanoparticle size analyzer to measure the particle size and Zeta potential of the esterase-responsive nanogel prepared in Example 1. The results are as shown in Figure 1 and Figure 2 It can be seen from the figure that the particle size of the nanogel is about 320 nm, the polydispersity index is 0.0327, and the particle size is relatively uniform; the change in the particle size of the esterase-responsive nanogel within 180 days is very small, and the stability is good.

[0096] ② Conduct an electron microscopy scan on the esterase-responsive nanogel sample synthesized in Example 1 and the biological valve for preventing infective endocarditis prepared with the nanogel modification. The SEM images are as shown in Figure 3 and Figure 4 As shown in Figure 3 the esterase-responsive nanogel has a uniform particle size distribution; Figure 4 As shown in the surface seen in

[0097]

[0098] II. Performance TestPlatelet adhesion experiments were carried out on glutaraldehyde-crosslinked biological valves with and without nanogel modification. Platelet adhesion is the initiating step of thrombosis. Platelets adhering to the material surface will initiate the processes of platelet aggregation, activation, and blood coagulation by releasing cytokines and procoagulant substances. Therefore, the fewer platelets adhering to the material surface, the better its anticoagulant performance may be Figure 5 is the SEM image of platelet adhesion, as Figure 5 shown, the number of platelets (yellow globules) adhering to the surface of the glutaraldehyde-crosslinked biological valve modified with nanogel is significantly reduced, showing better anticoagulant performance

[0099] To better evaluate the blood compatibility of biological valves in a simulated physiological environment, semi-in vivo blood contact tests were carried out on biological valves with and without nanogel modification respectively. The SEM images of the biological valves after contact are as Figure 6 shown. There is a large fibrin network formed by the activation of a large number of red blood cells and platelets on the surface of the biological valve without nanogel modification. The surface of the biological valve may trigger the activation and aggregation of blood cells and promote thrombus formation; there are only a small number of adherent red blood cells and platelets on the surface of the biological valve modified with nanogel; this is because a large number of hydrophilic groups are introduced onto the surface of the biological valve by chemically bonding nanogel, forming a stable hydration layer, reducing the direct contact and interaction between blood cells and the surface of the biological valve, effectively reducing platelet adhesion, and reducing the risk of thrombus and blood coagulation

[0100] The biological valves without nanogel modification and those with nanogel modification were respectively implanted into rats, and the vital signs of the rats were restored. At 7 days and 14 days after implantation, the implanted biological valves were taken out

[0101] The taken-out biological valves were rinsed clean with physiological saline to remove the blood and impurities on the surface, and then paraffin-embedded, sectioned, and stained. Since immune inflammatory reactions will occur due to foreign body implantation, IL-1β and TNF-α positive cells will accumulate on the surface of the biological valve. Therefore, specific antibodies against IL-1β and TNF-α were used for immunohistochemical staining of the sections to compare the positive expression levels of IL-1β and TNF-α in the biological valves at 7 days and 14 days, so as to evaluate their blood compatibility and immune inflammatory reactions. The staining result images are as Figure 7 shown. The coincidence of the cell nucleus and dark yellow is the positive cell; the statistical results of the number of IL-1β and TNF-α positive cells are as Figure 8 shown; from Figure 7 and Figure 8It can be seen that whether it is 7 days or 14 days, the positive expression of IL-1β and TNF-α in the bioprosthesis without nanogel modification is higher than that in the bioprosthesis modified with esterase-responsive nanogel, which shows that the inflammatory response after implantation of the bioprosthesis modified with esterase-responsive nanogel is milder. Similarly, after 60 days of implantation, the implanted bioprosthesis was removed and stained with alizarin red to show the distribution of calcium salts in the bioprosthesis. The staining results are shown in the figure Figure 9 As shown, Alizarin Red will appear red or orange-red after combining with calcium. Figure 9 The red part without nanogel modification is very deep and covers a large area, indicating severe calcification. The test results show that after 60 days of subcutaneous implantation, the glutaraldehyde-cross-linked biological valve without nanogel modification underwent severe calcification during the subcutaneous implantation process, while the glutaraldehyde-cross-linked biological valve modified with nanogel had no obvious calcification. Therefore, the glutaraldehyde-cross-linked biological valve modified with nanogel has better anti-calcification performance.

[0102] The present application also conducted a drug release experiment on the esterase-responsive nanogel, using PBS diluent containing 1mM esterase to simulate the human physiological environment. First, 2mg of the esterase-responsive nanogel prepared in Example 1 was dissolved in 2mL of esterase diluent and placed in a dialysis bag (Mw=3500), and then the dialysis bag was placed in a 50mL centrifuge tube, and three parallel sample groups were set up. 20mL of esterase diluent of corresponding concentration was added to each centrifuge tube, and the tube was sealed and placed on a 37°C oven shaker; 2mL of diluent was taken out at the sampling time points (0, 5min, 10min, 15min, 30min, 45min, 1h, 1.5h, 2h, 4h, 6h, 12h, 24h, 48h, 72h, 120h, 168h), and the same volume of release liquid was added. Then, the absorbance of ofloxacin in the release solution at different time points was measured by ultraviolet spectrophotometer, and the release amount was calculated with reference to the standard curve, and the cumulative release rate (%) of ofloxacin was calculated. The average results of the three parallel groups are shown in Figure 10 As shown, from Figure 10 It can be seen that under the physiological environment of inflammation, ofloxacin in the nanogel can be released.

[0103] The present invention also conducts anti-infection experiments on the nanogel-modified bioprosthesis for preventing infective endocarditis, and selects typical Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli) to analyze the anti-infection performance of the bioprosthesis.

[0104] The test procedure for anti-adhesion ability is as follows: First, the biological valves with and without nano-gel modification are sterilized by ultraviolet irradiation. The Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) strains are incubated in Luria-Bertani medium at 37 °C with continuous shaking for 12 hours to obtain a bacterial suspension with a concentration of 107 CFU / mL. Take 1 mL of the above-mentioned bacterial suspension and add it to the sample, and incubate at 37 °C for 2 hours. After the incubation, rinse the sample three times with sterile saline to remove the non-adherent bacteria, and then immerse the sample in 5 mL of sterile saline and ultrasonically treat for 5 minutes to obtain a suspension. Spread 20 μL of the above suspension evenly on a nutrient agar plate and culture at 37 °C for 24 hours to obtain an image and count the colony-forming units (CFU / mL). The average results of three parallel groups are as Figure 11 shown. Whether it is for E. coli or S. aureus, the colony-forming units of bacteria on the surface of the biological valve with nano-gel modification are significantly less than those of the biological valve without nano-gel modification. The remaining samples are fixed with 2.5% glutaraldehyde solution, freeze-dried after ethanol gradient dehydration process, and used for scanning electron microscopy (SEM) observation. In Figure 12 , E. coli and S. aureus are marked by pseudo-color. The results show that whether it is for E. coli or S. aureus, the amount of bacteria adhering to the surface of the biological valve with nano-gel modification is significantly less than that of the biological valve without nano-gel modification.

[0105] The test procedure for bactericidal ability is as follows: 500 mg of the biological valves with and without nano-gel modification are cut into small pieces after being sterilized by ultraviolet irradiation and placed in a conical flask. The frozen E. coli and S. aureus strains are incubated in Luria-Bertani medium at 37 °C with continuous shaking for 12 hours to obtain a bacterial suspension with a concentration of 5×105 CFU / mL. Add 10 mL of the above bacterial suspension to the sample and incubate in a shaker incubator at 37 °C at a speed of 120 revolutions per minute for 4 hours. Take 10 μL of the bacterial suspension and spread it evenly on a nutrient agar plate, and culture for 24 hours to obtain an image and count the colony-forming units. The bacterial suspension without adding the sample is used as a control group. The anti-infection rate is determined by the following formula: Inhibitory rate (%) = (A–B) / A×100%, where A and B represent the number of colony-forming units in the control group and the experimental group, respectively. The results are as Figure 13 shown. Whether it is for E. coli or S. aureus, the colony-forming units corresponding to the biological valve with nano-gel modification are significantly less than those of the biological valve without nano-gel modification, and the inhibitory rate of the biological valve with nano-gel modification is as high as 90%.

[0106] The above-described embodiments merely represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several variations and improvements can still be made, and these all fall within the protection scope of the present application.

Claims

1. An esterase-responsive nanogel-modified biological valve, characterized in that: An esterase-responsive nanogel is connected to the nanogel-modified biological valve, and the esterase-responsive nanogel contains an antibiotic drug with an ester bond group.

2. A preparation method of an esterase-responsive nanogel modified biological valve, characterized in that: It includes the following steps: S1: Perform glutaraldehyde cross-linking treatment on the biological valve; S2: Graft an amino cationic polymer onto the glutaraldehyde-cross-linked biological valve; S3: Chemically bond the esterase-responsive nanogel to the surface of the grafted biological valve; S4: Treat the biological valve with an active ester compound to obtain the esterase-responsive nanogel-modified biological valve described in Claim 1.

3. The preparation method of an esterase-responsive nanogel-modified biological valve according to claim 2, wherein: The preparation method of the esterase-responsive nanogel in S3 includes: S2011: Dissolve a methacrylate with an ester bond group and an antibiotic drug in a solvent, and add a condensing agent, a dehydrating agent, and a catalyst to react to obtain an esterase-responsive monomer; S2012: Dissolve the esterase-responsive monomer, a hydrophilic monomer, an active ester monomer, a cross-linking agent, and an initiator in a solvent, and react under the protection of an inert gas to obtain an esterase-responsive nanogel.

4. The preparation method of an esterase-responsive nanogel-modified biological valve according to Claim 3, wherein: The dosage ratio of the methacrylate with an ester bond group, the antibiotic drug, the solvent, the condensing agent, the dehydrating agent, and the catalyst is 1 mmol: 0.2 - 5 mmol: 10 - 20 mL: 1 - 3 mmol: 1 - 3 mmol: 0.1 - 1 mmol; The dosage ratio of the esterase-responsive monomer, the hydrophilic monomer, the active ester monomer, the cross-linking agent, the initiator, and the solvent is 1 - 20 mg: 5 - 100 mg: 1 - 10 mg: 1 - 10 mg: 1 - 10 mg: 20 - 40 mL.

5. The preparation method of an esterase-responsive nanogel modified biological valve according to claim 3, characterized in that: The methacrylate with an ester bond group is 2-hydroxyethyl methacrylate or 2-hydroxyethyl acrylate; The hydrophilic monomer is 2-acrylamido-2-methylpropanesulfonic acid; The active ester monomer is N-acryloxysuccinimide or N-hydroxysuccinimide methacrylate.

6. The preparation method of an esterase-responsive nanogel-modified biological valve according to any one of Claims 2 - 5, wherein: In S1, the biological valve is soaked in a glutaraldehyde solution with a concentration of 0.05 - 8 wt% for 48 - 72 h; the concentration of the esterase-responsive nanogel solution is 0.01 - 10 mg / mL; In S2, the amino cationic polymer is branched polyethyleneimine with a molecular weight of 300 - 1000000, and the concentration is 5 - 15 mg / mL; In S3, the concentration of the esterase-responsive nanogel solution is 0.01 - 10 mg / mL; In S4, the active ester compound is branched acetic acid-N-succinimide ester or polyethylene glycol succinimide ester, and the concentration is 1 - 5 mg / mL.

7. The preparation method of an esterase-responsive nanogel-modified biological valve according to claim 6, wherein: S2 - 3 is repeated 0 - 1 times, and when S2 is performed for the first time, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with 0.05 - 0.4 times the theoretical amino quantity equivalent and N-hydroxysuccinimide with 0.05 - 0.4 times the theoretical amino quantity equivalent are added to soak the glutaraldehyde-cross-linked biological valve.

8. A method for preparing an esterase-responsive nanogel-modified biological valve according to any one of claims 2-5, characterized in that: The reaction conditions in S2 are: reacting for 1-48 h under the conditions of a temperature of 4-37 °C and a pH of 3-10; The reaction conditions in S3 are: reacting for 0.5-48 h under the conditions of a temperature of 4-37 °C and a pH of 3-10.

9. The preparation method of an esterase-responsive nanogel-modified biological valve according to any one of claims 3-5, characterized in that: The antibiotic drug is a drug for preventing infective endocarditis, and the drug for preventing infective endocarditis is one of ofloxacin, levofloxacin, ciprofloxacin, amoxicillin, ceftriaxone, penicillin, and moxifloxacin.

10. Use of an esterase-responsive nanogel-modified biological valve according to claim 1, characterized in that: For preparing an artificial valve prosthesis.

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