A drug-loaded nanogel-modified biological valve and its preparation method and application

Abstract

The present invention discloses a biological valve modified by a drug-loaded nanogel and a preparation method and application thereof, and belongs to the technical field of medical materials and medical device preparation. The present invention first uses thioketal-containing methacrylate and methylprednisolone hemisuccinate as raw materials to synthesize an active oxygen responsive monomer, then synthesizes an active oxygen responsive drug-loaded nanogel, and then introduces the active oxygen responsive drug-loaded nanogel to the surface of the biological valve by chemical bonding; under a pathological environment with a high concentration of active oxygen, the thioketal group in the monomer decomposes, and methylprednisolone hemisuccinate is released at a fixed point, thereby reducing inflammatory cell infiltration and improving the anti-inflammatory performance of the biological valve; in addition, the nanogel contains a large number of hydrophilic groups, forming a hydration layer on the surface of the biological valve, which can effectively reduce the risk of thrombosis and coagulation; the prepared biological valve has excellent anti-inflammatory, anti-coagulation, anti-calcification ability and stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of medical materials and medical devices, and in particular relates to a biological valve modified by a drug-loaded nanogel, and a preparation method and application thereof. Background Art

[0002] As the problem of population aging intensifies, the number of patients with heart valve disease in China is increasing year by year. Artificial heart valve replacement is one of the effective means of treating heart valve disease in clinical practice. Artificial valve prostheses used for artificial heart replacement are divided into mechanical valves and biological valves. Among them, mechanical valves have a long service life and are the main choice for surgical operations. Biological valves have superior hemodynamic properties, and patients do not need to take anticoagulant drugs for a long time. In recent years, with the development of transcatheter interventional treatment technology for heart valve disease, the demand for the use of biological valves has continued to increase.

[0003] Most of the bioprosthetic valves currently used in clinical practice are bovine pericardial or porcine aortic valves cross-linked with glutaraldehyde, and their service life is usually only about 10 years. The failure of bioprosthetic valves is caused by many factors, including inflammation, thrombosis, and calcification. How to enhance the performance of bioprosthetic valves and extend their service life is the focus of current research on bioprosthetic valves.

[0004] Surface functionalization is a simple and effective method that combines physical and chemical modification technologies to improve the overall effect of materials. Therefore, based on the glutaraldehyde-crosslinked biological valve, it is expected to give the material better anti-inflammatory, anticoagulant and anti-calcification capabilities by modifying it, thereby extending the service life of the valve material, which has important clinical significance. Previous studies have shown that biological valves modified by electrostatic adsorption or physical filling have poor stability and short shelf life, and it is difficult to meet the long-term anticoagulation, anti-inflammatory and anti-calcification requirements of biological valves. In addition, there are fewer modification sites on the surface of glutaraldehyde-crosslinked biological valves, which poses a challenge to improving the performance of biological valves in many aspects. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention discloses a drug-loaded nanogel-modified biological valve and its preparation method and application, so as to solve the technical problems of poor stability, short effective period, poor long-term anticoagulation, anti-inflammatory and anti-calcification capabilities of biological valves in the prior art.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present invention is: to provide a method for preparing a biological valve modified by a drug-loaded nanogel, which comprises the following steps: grafting an amino cationic polymer on a biological valve cross-linked by glutaraldehyde, chemically bonding an active oxygen responsive drug-loaded nanogel to the surface of the grafted biological valve, and finally treating the biological valve with an active ester compound to obtain; the synthesis of the active oxygen responsive drug-loaded nanogel comprises the following steps:

[0007] S1: dissolving methacrylate containing thioketal and methylprednisolone hemisuccinate in a solvent, adding a condensing agent, a dehydrating agent and a catalyst, and reacting at room temperature for 6 to 72 hours to obtain an active oxygen responsive monomer;

[0008] S2: The active oxygen responsive monomer, hydrophilic monomer, active ester monomer, crosslinking agent and initiator are dissolved in a solvent, and reacted for 0.1 to 24 hours under the protection of an inert gas to obtain an active oxygen responsive drug-loaded nanogel; the reaction temperature is 40 to 160°C; the hydrophilic monomer is 2-acrylamido-2-methylpropanesulfonic acid; the active ester monomer is N-acryloyloxysuccinimide or N-hydroxysuccinimide methacrylate.

[0009] Based on the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, in step S1, the methacrylate containing thioketal is 3-((2-((3-hydroxypropyl)thio)prop-2-yl)thio)propyl methacrylate; the solvent is dichloromethane; the condensing agent is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; the dehydrating agent is N,N-dicyclohexylcarbonyldiimide or diisopropylcarbodiimide; and the catalyst is 4-dimethylaminopyridine p-toluenesulfonate.

[0011] Furthermore, the solid-liquid ratio of thioketal-containing methacrylate, methylprednisolone hemisuccinate, solvent, shrinking agent, dehydrating agent and catalyst is 1 mmol: 0.2~5 mmol: 10~20 mL: 1~3 mmol: 1~3 mmol: 0.1~1 mmol.

[0012] Furthermore, in step S2, the crosslinking agent is N,N'-methylenebisacrylamide; the initiator is azobisisobutyronitrile; and the solvent is one or more of acetonitrile, water, ethanol, dimethyl sulfoxide and dioxane.

[0013] Furthermore, the material-liquid ratio of the active oxygen responsive monomer, the hydrophilic monomer, the active ester monomer, the crosslinking agent, the initiator and the solvent is 1~10mg:5~100mg:1~10mg:1~10mg:1~10mg:20~40mL.

[0014] Furthermore, the preparation method of the drug-loaded nanogel-modified biological valve specifically comprises the following steps:

[0015] (1) soaking the biological valve cross-linked with glutaraldehyde in an amino cationic polymer solution at 4-37° C. and pH 5-8 for 1-48 hours to obtain a biological valve grafted with amino cationic polymer;

[0016] (2) soaking the bioprosthesis grafted with amino cationic polymer in an active oxygen responsive drug-loaded nanogel solution at 4-37° C. and pH 3-10 for 0.5-48 h to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel;

[0017] (3) Repeat steps (1) to (2) 0 to 1 times, then immerse the bioprosthesis bonded with active oxygen responsive drug-loaded nanogel in the active ester compound solution for 3 times, each time at 4 to 37° C. and pH 5 to 7 for 2 to 6 hours; wash and obtain.

[0018] Furthermore, the glutaraldehyde cross-linking treatment method of the biological valve is: soak the biological valve in a glutaraldehyde solution for 48 to 72 hours, the concentration of the glutaraldehyde solution is 0.05 to 8 wt%; the biological valve is decellularized bovine pericardium or porcine pericardium; the amino cationic polymer is a branched polyethyleneimine with a molecular weight of 300 to 1,000,000; the concentration of the amino cationic polymer solution is 5 to 15 mg / mL; the concentration of the active oxygen responsive drug-loaded nanogel solution is 0.01 to 10 mg / mL; the active ester compound is a branched acetic acid-N-succinimidyl ester or polyethylene glycol succinimidyl ester, and the concentration of the active ester compound solution is 1 to 5 mg / mL.

[0019] Furthermore, when the biological valve cross-linked with glutaraldehyde is soaked in the amino cationic polymer solution for the first time, 0.1-0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-0.15 theoretical amino equivalents of N-hydroxysuccinimide are added.

[0020] Furthermore, when the biological valve treated with glutaraldehyde cross-linking was immersed in the amino cationic polymer solution for the first time, the pH was 5.5, the concentration of the amino cationic polymer was 10 mg / mL, and the immersion time was 12 h.

[0021] Further, when the amino cationic polymer was used for soaking treatment again, the pH was 7, the concentration of the amino cationic polymer solution was 10 mg / mL, and the soaking time was 4 h.

[0022] Furthermore, when the bioprosthesis grafted with amino cationic polymer was treated by immersion in the active oxygen responsive drug-loaded nanogel solution for the first time, the pH was 7, the concentration of the active oxygen responsive drug-loaded nanogel solution was 1 mg / mL, and the immersion time was 2 h.

[0023] Furthermore, when the active oxygen responsive drug-loaded nanogel solution was used for immersion treatment again, the pH was 7, the concentration of the active oxygen responsive drug-loaded nanogel solution was 1 mg / mL, and the immersion time was 12 h.

[0024] Furthermore, when the bioprosthesis bonded with the active oxygen responsive drug-loaded nanogel was immersed in the active ester compound solution, the pH was 7, the concentration of the active ester compound solution was 5 mg / mL, and the immersion time was 2 h.

[0025] The invention also discloses the application of the biological valve modified by the drug-loaded nanogel in the preparation of an artificial valve prosthesis.

[0026] The beneficial effects of the present invention are:

[0027] 1. The nanogel prepared in the present invention has a uniform particle size. The nanogel contains a methylprednisolone hemisuccinate monomer with a thioketal group. The thioketal group is a ROS-responsive group. Methylprednisolone hemisuccinate is a glucocorticoid drug with anti-inflammatory effects. Under pathological conditions such as inflammation where the concentration of reactive oxygen species is high, the thioketal group in the monomer is decomposed and the methylprednisolone hemisuccinate is released at a specific point, thereby reducing the infiltration of inflammatory cells and improving the anti-inflammatory properties of biological valves.

[0028] 2. The present invention modifies the surface of the biological valve material with active oxygen-responsive drug-loaded nanogel by chemical bonding. Compared with the biological valve modified by electrostatic adsorption or physical filling, the biological valve prepared by chemical bonding has good stability and long-term effectiveness, can improve the long-term anti-inflammatory, anticoagulant and anti-calcification capabilities of the biological valve, and extend the service life of the biological valve.

[0029] 3. The bioprosthetic valve modification method provided by the present invention can effectively seal the residual aldehyde groups on the glutaraldehyde-crosslinked bioprosthetic valve, thereby effectively reducing the bioprosthetic valve calcification and biological toxicity problems.

[0030] 4. The present invention introduces a large number of hydrophilic groups on the surface of the biological valve, which can form a stable hydration layer on the surface of the biological valve, effectively reduce platelet adhesion, and reduce the risk of thrombosis and coagulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the particle size measurement results of the active oxygen responsive drug-loaded nanogel synthesized in Example 1;

[0032] Figure 2 This is a graph showing the Zeta potential measurement results of the active oxygen responsive drug-loaded nanogel synthesized in Example 1;

[0033] Figure 3 This is a SEM image of the active oxygen responsive drug-loaded nanogel synthesized in Example 1;

[0034] Figure 4 This is a SEM image of the bioprosthesis modified with drug-loaded nanogel prepared in Example 1;

[0035] Figure 5 The SEM images of platelets adhering to the surfaces of the biological valves prepared in Example 1 and Comparative Examples 1-2;

[0036] Figure 6 This is a SEM image of the bioprosthesis prepared in Example 1 after the ex vivo blood contact test;

[0037] Figure 7 This is a SEM image of the bioprosthesis prepared in Comparative Example 1 after the ex vivo blood contact test;

[0038] Figure 8 The immunohistochemical staining results of the bioprosthetic valves prepared in Example 1 and Comparative Examples 1-2 after subcutaneous implantation for 7 days and 14 days;

[0039] Fig. 9 These are Alizarin red-stained slice microscopic images of the bioprosthetic valves prepared in Example 1 and Comparative Examples 1-2 after subcutaneous implantation for 60 days;

[0040] Fig.10 This is the result of drug release experiment. DETAILED DESCRIPTION

[0041] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0042] Example 1

[0043] A bioprosthesis modified with drug-loaded nanogel, the preparation method of which comprises the following steps:

[0044] ① Synthesizing active oxygen responsive drug-loaded nanogels, comprising the following steps:

[0045] S1: Add 500 mg of 3-((2-((3-hydroxypropyl)thio)propan-2-yl)thio)propyl methacrylate, 810 mg of methylprednisolone hemisuccinate, 460 mg of N,N-dicyclohexylcarbodiimide, 842 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 200 mg of 4-dimethylaminopyridine p-toluenesulfonate and 30 mL of dichloromethane were stirred and dissolved, and then reacted at room temperature for 48 hours. After the reaction was completed, suction filtration was performed, the filtrate was collected, and 200 mL of ethyl acetate and 20 mL of sodium chloride solution were added to the filtrate for extraction. The extraction was performed 3 times, and the upper ethyl acetate solution was collected and dehydrated with anhydrous sodium sulfate. After suction filtration, the filtrate was collected, and the filtrate was concentrated to no ethyl acetate by a rotary evaporator, and then dissolved with 2 mL of dichloromethane; an appropriate amount of silica gel powder was dissolved in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 4:1, and a 15 cm chromatography column was loaded. After the reactant dissolved in dichloromethane was sampled, the column was passed with an eluent of petroleum ether: ethyl acetate = 2:1 and 1:1 in sequence, and the product was collected, and the rotary evaporator was dried, and the oil pump was dried to obtain an active oxygen response monomer; the structure of the active oxygen response monomer is shown in Formula I,

[0046] ;

[0047] S2: 20 mg of active oxygen responsive monomer, 170 mg of 2-acrylamide-2-methylpropanesulfonic acid, 15 mg of N-acryloyloxysuccinimide, 20 mg of N,N'-methylenebisacrylamide, 4 mg of azobisisobutyronitrile and 40 mL of chromatographic grade acetonitrile were placed in a thick-walled pressure bottle, and the raw materials were dissolved by ultrasound. Then, nitrogen was replaced and the reaction was carried out under nitrogen protection and 100°C for 1 hour. After the reaction, the suspension was collected and centrifuged at 12000 rpm for 3 minutes. The supernatant was removed and the solid precipitate was retained. The solid precipitate was ventilated and dried for 20 minutes to obtain active oxygen responsive drug-loaded nanogel as a white solid powder.

[0048] ②Preparing a bioprosthesis modified with drug-loaded nanogel, comprising the following steps:

[0049] (1) Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48 hours, then soak it in a 1wt% glutaraldehyde solution and shake it for 48 hours for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde-crosslinked biological valve; then soak the glutaraldehyde-crosslinked biological valve in a 10mg / mL branched polyethyleneimine solution, the molecular weight of the branched polyethyleneimine is 10000, and add 0.1 theoretical amino equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1 theoretical amino equivalent of N-hydroxysuccinimide, then soak it at 32°C and pH 8 for 12 hours to obtain a grafted amino cationic polymer biological valve;

[0050] (2) Soaking the grafted amino cationic polymer bioprosthesis in a 0.1 mg / mL active oxygen responsive drug-loaded nanogel solution, shaking and soaking for 12 hours at 32° C. and pH 6 to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel;

[0051] (3) The bioprosthesis with active oxygen-responsive drug-loaded nanogel was repeatedly immersed in a 5 mg / mL branched-chain acetic acid-N-succinimidyl ester solution for 3 times, each time at 32°C and pH 6 for 2 h, and then taken out and cleaned with saline.

[0052] Example 2

[0053] A bioprosthesis modified with drug-loaded nanogel, the preparation method of which comprises the following steps:

[0054] ① Synthesizing active oxygen responsive drug-loaded nanogels, comprising the following steps:

[0055] S1: Add 292.4 mg of 3-((2-((3-hydroxypropyl)thio)propan-2-yl)thio)propyl methacrylate, 94.7 mg of methylprednisolone hemisuccinate, 126.2 mg of diisopropylcarbodiimide, 379.3 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 29.4 mg of 4-dimethylaminopyridine p-toluenesulfonate and 10 mL of dichloromethane were stirred and dissolved, and then reacted at room temperature for 6 hours. After the reaction was completed, suction filtration was performed, the filtrate was collected, and 200 mL of ethyl acetate and 20 mL of sodium chloride solution were added to the filtrate for extraction. The extraction was performed 3 times, and the upper ethyl acetate solution was collected and dehydrated with anhydrous sodium sulfate. After suction filtration, the filtrate was collected, and the filtrate was concentrated to no ethyl acetate by a rotary evaporator, and then dissolved with 2 mL of dichloromethane; an appropriate amount of silica gel powder was dissolved in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 4:1, and a 15 cm chromatography column was loaded. After the reactant dissolved in dichloromethane was sampled, the column was passed with an eluent of petroleum ether: ethyl acetate = 2:1 and 1:1 in sequence, and the product was collected, and the rotary evaporator was dried, and the oil pump was dried to obtain an active oxygen response monomer; the structure of the active oxygen response monomer is shown in Formula I,

[0056] ;

[0057] S2: Take 10 mg of active oxygen responsive monomer, 50 mg of 2-acrylamide-2-methylpropanesulfonic acid, 10 mg of N-acryloyloxysuccinimide, 10 mg of N,N'-methylenebisacrylamide, 10 mg of azobisisobutyronitrile and 200 mL of chromatographic grade ethanol in a thick-walled pressure bottle, use ultrasound to promote the dissolution of the raw materials, then replace the nitrogen, and react for 0.1 h under nitrogen protection and 40°C. After the reaction, collect the suspension, centrifuge at 12000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and dry it under ventilation for 20 min to obtain active oxygen responsive drug-loaded nanogel as a white solid powder.

[0058] ②Preparing a bioprosthesis modified with drug-loaded nanogel, comprising the following steps:

[0059] (1) Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48 hours, then soak it in a 5wt% glutaraldehyde solution and shake it for 48 hours for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde-crosslinked biological valve; then soak the glutaraldehyde-crosslinked biological valve in a 10mg / mL branched polyethyleneimine solution, the molecular weight of the branched polyethyleneimine is 1000000, and add 0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15 theoretical amino equivalents of N-hydroxysuccinimide, then soak it at 37°C and pH 6 for 1 hour to obtain a grafted amino cationic polymer biological valve;

[0060] (2) Soaking the grafted amino cationic polymer bioprosthesis in a 0.01 mg / mL active oxygen responsive drug-loaded nanogel solution, shaking and soaking for 0.5 h at 37° C. and pH 10, to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel;

[0061] (3) The bioprosthesis with active oxygen-responsive drug-loaded nanogel was repeatedly immersed in a 5 mg / mL branched-chain acetic acid-N-succinimidyl ester solution for 3 times, each time at 37°C and pH 6 for 2 h, and then taken out and cleaned with physiological saline.

[0062] Example 3

[0063] A bioprosthesis modified with drug-loaded nanogel, the preparation method of which comprises the following steps:

[0064] ① Synthesizing active oxygen responsive drug-loaded nanogels, comprising the following steps:

[0065] S1: Add 292.4 mg of 3-((2-((3-hydroxypropyl)thio)propan-2-yl)thio)propyl methacrylate, 2.36 g of methylprednisolone hemisuccinate, 619 mg of N,N-dicyclohexylcarbodiimide, 1.14 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 294.36 mg of 4-dimethylaminopyridine p-toluenesulfonate and 20 mL of dichloromethane were stirred and dissolved, and then reacted at room temperature for 72 hours. After the reaction was completed, suction filtration was performed, the filtrate was collected, and 200 mL of ethyl acetate and 20 mL of sodium chloride solution were added to the filtrate for extraction. The extraction was performed 3 times, and the upper ethyl acetate solution was collected and dehydrated with anhydrous sodium sulfate. After suction filtration, the filtrate was collected, and the filtrate was concentrated to no ethyl acetate by a rotary evaporator, and then dissolved with 2 mL of dichloromethane; an appropriate amount of silica gel powder was dissolved in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 4:1, and a 15 cm chromatography column was loaded. After the reactant dissolved in dichloromethane was sampled, the column was passed with an eluent of petroleum ether: ethyl acetate = 2:1 and 1:1 in sequence, and the product was collected, and the rotary evaporator was dried, and the oil pump was dried to obtain an active oxygen response monomer; the structure of the active oxygen response monomer is shown in Formula I,

[0066] ;

[0067] S2: 10 mg of active oxygen responsive monomer, 50 mg of 2-acrylamide-2-methylpropanesulfonic acid, 10 mg of methacrylate N-hydroxysuccinimide ester, 10 mg of N,N'-methylenebisacrylamide, 5 mg of azobisisobutyronitrile and 40 mL of dimethyl sulfoxide were placed in a thick-walled pressure bottle, and the raw materials were dissolved by ultrasound. Then, nitrogen was replaced and the reaction was carried out under nitrogen protection and 160°C for 12 h. After the reaction, the suspension was collected and centrifuged at 12000 rpm for 3 min. The supernatant was removed and the solid precipitate was retained. The solid precipitate was dried under ventilation for 20 min to obtain active oxygen responsive drug-loaded nanogel as a white solid powder.

[0068] ②Preparing a bioprosthesis modified with drug-loaded nanogel, comprising the following steps:

[0069] (1) Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48h, then soak it in a 0.05wt% glutaraldehyde solution and shake it for 48h for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde-crosslinked biological valve; then soak the glutaraldehyde-crosslinked biological valve in a 5mg / mL branched polyethyleneimine solution, the molecular weight of the branched polyethyleneimine is 300, and add 0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15 theoretical amino equivalents of N-hydroxysuccinimide, and then soak it at 4°C and pH 5 for 24h to obtain a grafted amino cationic polymer biological valve;

[0070] (2) Soaking the grafted amino cationic polymer bioprosthesis in a 5 mg / mL active oxygen responsive drug-loaded nanogel solution, shaking and soaking for 24 h at 4°C and pH 3, to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel;

[0071] (3) The bioprosthesis with active oxygen-responsive drug-loaded nanogel was repeatedly immersed in a 3 mg / mL branched-chain acetic acid-N-succinimidyl ester solution for 3 times, each time at 4°C and pH 5 for 6 h, and then taken out and cleaned with physiological saline.

[0072] Example 4

[0073] A bioprosthesis modified with drug-loaded nanogel, the preparation method of which comprises the following steps:

[0074] ① Synthesizing active oxygen responsive drug-loaded nanogels, comprising the following steps:

[0075] S1: Add 292.4 mg of 3-((2-((3-hydroxypropyl)thio)propan-2-yl)thio)propyl methacrylate, 947.3 mg of methylprednisolone hemisuccinate, 252 mg of diisopropylcarbodiimide, 758.6 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 147 mg of 4-dimethylaminopyridine p-toluenesulfonate and 15 mL of dichloromethane were stirred and dissolved, and then reacted at room temperature for 24 hours. After the reaction was completed, suction filtration was performed, the filtrate was collected, and 200 mL of ethyl acetate and 20 mL of sodium chloride solution were added to the filtrate for extraction. The extraction was performed 3 times, and the upper ethyl acetate solution was collected and dehydrated with anhydrous sodium sulfate. After suction filtration, the filtrate was collected, and the filtrate was concentrated to no ethyl acetate by a rotary evaporator, and then dissolved with 2 mL of dichloromethane; an appropriate amount of silica gel powder was dissolved in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 4:1, and a 15 cm chromatography column was loaded. After the reactant dissolved in dichloromethane was sampled, the column was passed with an eluent of petroleum ether: ethyl acetate = 2:1 and 1:1 in sequence, and the product was collected, and the rotary evaporator was dried, and the oil pump was dried to obtain an active oxygen response monomer; the structure of the active oxygen response monomer is shown in Formula I,

[0076] ;

[0077] S2: Take 10 mg of active oxygen responsive monomer, 100 mg of 2-acrylamide-2-methylpropanesulfonic acid, 5 mg of N-acryloyloxysuccinimide, 5 mg of N,N'-methylenebisacrylamide, 6 mg of azobisisobutyronitrile and 30 mL of chromatographic grade acetonitrile in a thick-walled pressure bottle, ultrasonically promote the dissolution of the raw materials, then replace the nitrogen, and react under nitrogen protection at 80°C for 24 hours. After the reaction, the suspension is collected and centrifuged at 12000 rpm for 3 minutes, the supernatant is removed, the solid precipitate is retained, and ventilation and drying for 20 minutes to obtain active oxygen responsive drug-loaded nanogel as a white solid powder.

[0078] ②Preparing a bioprosthesis modified with drug-loaded nanogel, comprising the following steps:

[0079] (1) Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48h, then soak it in a 8wt% glutaraldehyde solution and shake it for 48h for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde-crosslinked biological valve; then soak the glutaraldehyde-crosslinked biological valve in a 15mg / mL branched polyethyleneimine solution, the molecular weight of the branched polyethyleneimine is 10000, and add 0.1 theoretical amino equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1 theoretical amino equivalent of N-hydroxysuccinimide, then soak it at 20°C and pH 6 for 48h to obtain a grafted amino cationic polymer biological valve;

[0080] (2) Soaking the bioprosthesis grafted with amino cationic polymer in a 10 mg / mL active oxygen responsive drug-loaded nanogel solution, shaking and soaking for 48 hours at 20° C. and pH 6, to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel;

[0081] (3) The bioprosthesis with active oxygen-responsive drug-loaded nanogel was repeatedly immersed in a 1 mg / mL polyethylene glycol succinimide ester solution for 3 times, each time at 20°C and pH 7 for 2 h, and then taken out and cleaned with physiological saline.

[0082] Example 5

[0083] A bioprosthesis modified with drug-loaded nanogel, the preparation method of which comprises the following steps:

[0084] ① Synthesizing active oxygen responsive drug-loaded nanogels, comprising the following steps:

[0085] S1: Add 500 mg of 3-((2-((3-hydroxypropyl)thio)propan-2-yl)thio)propyl methacrylate, 810 mg of methylprednisolone hemisuccinate, 460 mg of N,N-dicyclohexylcarbodiimide, 650 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 200 mg of 4-dimethylaminopyridine p-toluenesulfonate and 30 mL of dichloromethane were stirred and dissolved, and then reacted at room temperature for 48 hours. After the reaction was completed, suction filtration was performed, the filtrate was collected, and 200 mL of ethyl acetate and 20 mL of sodium chloride solution were added to the filtrate for extraction. The extraction was performed 3 times, and the upper ethyl acetate solution was collected and dehydrated with anhydrous sodium sulfate. After suction filtration, the filtrate was collected, and the filtrate was concentrated to no ethyl acetate by a rotary evaporator, and then dissolved with 2 mL of dichloromethane; an appropriate amount of silica gel powder was dissolved in a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 4:1, and a 15 cm chromatography column was loaded. After the reactant dissolved in dichloromethane was sampled, the column was passed with an eluent of petroleum ether: ethyl acetate = 2:1 and 1:1 in sequence, and the product was collected, and the rotary evaporator was dried, and the oil pump was dried to obtain an active oxygen response monomer; the structure of the active oxygen response monomer is shown in Formula I,

[0086] ;

[0087] S2: 20 mg of active oxygen responsive monomer, 170 mg of 2-acrylamide-2-methylpropanesulfonic acid, 15 mg of N-acryloyloxysuccinimide, 20 mg of N,N'-methylenebisacrylamide, 4 mg of azobisisobutyronitrile and 40 mL of chromatographic grade acetonitrile were placed in a thick-walled pressure bottle, and the raw materials were dissolved by ultrasound. Then, nitrogen was replaced and the reaction was carried out under nitrogen protection and 100°C for 1 hour. After the reaction, the suspension was collected and centrifuged at 12000 rpm for 3 minutes. The supernatant was removed and the solid precipitate was retained. The solid precipitate was ventilated and dried for 20 minutes to obtain active oxygen responsive drug-loaded nanogel as a white solid powder.

[0088] ②Preparing a bioprosthesis modified with drug-loaded nanogel, comprising the following steps:

[0089] (1) Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48h, then soak it in a 0.1wt% glutaraldehyde solution and shake it for 72h for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde-crosslinked biological valve; then soak the glutaraldehyde-crosslinked biological valve in a 10mg / mL branched polyethyleneimine solution, the molecular weight of the branched polyethyleneimine is 10000, and add 0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15 theoretical amino equivalents of N-hydroxysuccinimide, and then react at 30°C and pH 5.5 for 12h to obtain a grafted amino cationic polymer biological valve;

[0090] (2) Soaking the grafted amino cationic polymer bioprosthesis in a 1 mg / mL active oxygen responsive drug-loaded nanogel solution, shaking and soaking for 2 h at 32° C. and pH 7, to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel;

[0091] (3) Repeat steps (1) and (2) once; repeat step (1) with a pH of 7 and an immersion time of 4 h, and repeat step (1) without adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; repeat step (2) with an immersion time of 12 h;

[0092] (4) The bioprosthesis with the active oxygen-responsive drug-loaded nanogel was repeatedly immersed in a 5 mg / mL branched-chain acetic acid-N-succinimidyl ester solution for 3 times, each time at 32°C and pH 7 for 2 h. After the reaction, the solution was taken out and washed with physiological saline.

[0093] Comparative Example 1

[0094] A glutaraldehyde cross-linked biological valve, the preparation method of which comprises the following steps:

[0095] Take fresh porcine pericardium, remove the connective tissue and clean it, then wash it with normal saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48 hours, then soak it in a 1wt% glutaraldehyde solution and shake it for 48 hours for cross-linking treatment, take it out and wash it with normal saline.

[0096] Comparative Example 2

[0097] A blank nanogel modified biological valve, the preparation method of which comprises the following steps:

[0098] ① Synthesizing blank nanogels, including the following steps:

[0099] Take 100 mg of 2-acrylamide-2-methylpropanesulfonic acid, 5 mg of N-acryloyloxysuccinimide, 5 mg of N,N'-methylenebisacrylamide, 6 mg of azobisisobutyronitrile and 30 mL of chromatographic grade acetonitrile in a thick-walled pressure bottle, ultrasonically promote the dissolution of the raw materials, then replace the nitrogen, and react for 24 hours under nitrogen protection and 80°C. After the reaction, the suspension is collected and centrifuged at 12000 rpm for 3 minutes, the supernatant is removed, the solid precipitate is retained, and ventilation and drying for 20 minutes to obtain blank nanogel as a white solid powder.

[0100] ②Preparing a blank nanogel-modified bioprosthesis, comprising the following steps:

[0101] (1) Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5wt% sodium dodecyl sulfate (SDS) solution and shake it for 48 hours, then soak it in a 1wt% glutaraldehyde solution and shake it for 48 hours for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde-crosslinked biological valve; then soak the glutaraldehyde-crosslinked biological valve in a 10mg / mL branched polyethyleneimine solution, the molecular weight of the branched polyethyleneimine is 10000, and add 0.1 theoretical amino equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1 theoretical amino equivalent of N-hydroxysuccinimide, then soak it at 32°C and pH 8 for 12 hours to obtain a grafted amino cationic polymer biological valve;

[0102] (2) Soak the bioprosthesis grafted with amino cationic polymer in a blank nanogel solution at a concentration of 0.1 mg / mL, shake and soak for 12 hours at 32°C and pH 6 to obtain a bioprosthesis bonded with blank nanogel;

[0103] (3) The bioprosthesis bonded with the blank nanogel was repeatedly immersed in a 5 mg / mL branched-chain acetic acid-N-succinimidyl ester solution for 3 times, each time at 32°C and pH 6 for 2 h. After being taken out, it was washed with physiological saline.

[0104] Experimental example

[0105] The performance of the biological valve modified by the drug-loaded nanogel prepared in the embodiments of the present invention is similar. Taking Embodiment 1 as an example, the performance of the related products is described.

[0106] 1. Structural test

[0107] ① The particle size and Zeta potential of the active oxygen responsive drug-loaded nanogel prepared in Example 1 were measured using a Malvern nanoparticle sizer. The results are as follows: Figure 1 and Figure 2 As shown, it can be seen that the particle size of the nanogel is about 305 nm, and the polydispersity index is 0.0565; the potential of the active oxygen responsive drug-loaded nanogel is -56.57 mV.

[0108] ② The active oxygen responsive drug-loaded nanogel sample synthesized in Example 1 and the prepared drug-loaded nanogel modified bioprosthesis were subjected to electron microscope scanning, and the SEM images were as follows: Figure 3 and Figure 4 As shown, Figure 3 As shown, the active oxygen responsive drug-loaded nanogel has a uniform particle size distribution; Figure 4 As shown, nanogels can be efficiently modified onto the surface of biological valves.

[0109] 2. Performance Test

[0110] The bioprosthetic valves prepared in Example 1 and Comparative Examples 1-2 were subjected to platelet adhesion experiments. Platelet adhesion is the initiation step of thrombosis. Platelets adhered to the surface of the material will cause platelet aggregation, activation and the initiation of the coagulation process by releasing cytokines and procoagulants. Therefore, the fewer platelets adhered to the surface of the material, the better its anticoagulant performance may be. Figure 5 is the SEM picture of platelet adhesion, such as Figure 5 As shown, compared with Comparative Examples 1-2, the number of platelet adhesion on the surface of the biological valve modified by the drug-loaded nanogel in Example 1 was significantly reduced, showing better anti-coagulation performance.

[0111] In order to better verify the blood compatibility of the bioprosthetic valve under a simulated physiological environment, the bioprosthetic valves prepared in Example 1 and Comparative Example 1 were subjected to semi-in vivo blood contact tests. The SEM images of the bioprosthetic valves at different magnifications after contact are shown in FIG. Figure 6-7 As shown, in Comparative Example 1, the surface of the biological valve without nanogel modification has a large amount of fibrin network formed by the activation of red blood cells and platelets, and the surface of the biological valve may trigger the activation and aggregation of blood cells, and promote the formation of thrombus; the surface of the biological valve modified with drug-loaded nanogel has only a small amount of adhered red blood cells and platelets; this is because a large number of hydrophilic groups are introduced into the surface of the biological valve through chemical bonding of nanogels to form a stable hydration layer, which reduces the direct contact and interaction between blood cells and the surface of the biological valve, effectively reduces platelet adhesion, and reduces the risk of thrombosis and coagulation.

[0112] The bioprosthetic valves prepared in Example 1 and Comparative Examples 1-2 were implanted into rats, respectively, and the vital signs of the rats were restored. The implanted bioprosthetic valves were removed 7 and 14 days after implantation.

[0113] The removed bioprosthesis was rinsed with saline to remove blood and impurities on the surface, then embedded in paraffin, sliced ​​and stained. Since foreign body implantation will cause immune inflammatory response, IL-1β and TNF-α positive cells will be enriched on the surface of the bioprosthesis. Therefore, specific antibodies for IL-1β and TNF-α were used to perform immunohistochemical staining on the sections, and the positive expression of IL-1β and TNF-α of the bioprosthesis at 7 days and 14 days was compared to evaluate its blood compatibility and immune inflammatory response. The staining results are shown in the figure below. Figure 8 As shown in the figure, it can be seen that the positive expression of IL-1β and TNF-α in the bioprosthesis without nanogel modification in Comparative Example 1 is higher than that in the bioprosthesis modified with drug-loaded nanogel, whether it is 7 days or 14 days, and it is concluded that the inflammatory response after implantation of the bioprosthesis modified with drug-loaded nanogel is lighter. Similarly, after 60 days of implantation, the implanted bioprosthesis was taken out and stained with alizarin red to show the distribution of calcium salt in the bioprosthesis. The staining results are shown in the figure Fig. 9 As shown, the test results show that after 60 days of subcutaneous implantation, the biological valve without nanogel modification in Comparative Example 1 underwent severe calcification during subcutaneous implantation, while the biological valve modified with glutaraldehyde cross-linked by drug-loaded nanogel had no obvious calcification. Therefore, the biological valve modified with glutaraldehyde cross-linked by drug-loaded nanogel has better anti-calcification performance.

[0114] The present application also conducted a drug release experiment on the active oxygen responsive drug-loaded nanogel, using 1mM H2O2 PBS diluent to simulate the physiological environment of human inflammation. First, 2mg of the active oxygen responsive drug-loaded nanogel prepared in Example 1 was dissolved in 2mL of 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 diluent 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 methylprednisolone hemisuccinate in the release solution at different time points was measured by UV spectrophotometer, and the release amount was calculated with reference to the standard curve. The cumulative release rate (%) of methylprednisolone hemisuccinate was calculated. The average results of the three parallel groups are shown in Fig.10 As shown in the figure, it can be seen that under the physiological environment of inflammation, methylprednisolone hemisuccinate in the nanogel can be released normally.

[0115] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing a biological valve modified with a drug-loaded nanogel, characterized in that: The following steps are involved: Grafting amino cationic polymers on glutaraldehyde cross-linked biological valves, chemically bonding active oxygen responsive drug-loaded nanogels on the surface of the grafted biological valves, and finally treating the biological valves with active ester compounds to obtain active oxygen responsive drug-loaded nanogels, comprising the following steps: S1: dissolving methacrylate containing thioketal and methylprednisolone hemisuccinate in a solvent, adding a condensing agent, a dehydrating agent and a catalyst, and reacting at room temperature for 6 to 72 hours to obtain an active oxygen responsive monomer; S2: The active oxygen responsive monomer, hydrophilic monomer, active ester monomer, crosslinking agent and initiator are dissolved in a solvent, and reacted for 0.1 to 24 hours under the protection of an inert gas to obtain an active oxygen responsive drug-loaded nanogel; the reaction temperature is 40 to 160° C.; the hydrophilic monomer is 2-acrylamido-2-methylpropanesulfonic acid; the active ester monomer is N-acryloyloxysuccinimide or N-hydroxysuccinimide methacrylate.

2. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 1, characterized in that: In step S1, the methacrylate containing thioketal is 3-((2-((3-hydroxypropyl)thio)prop-2-yl)thio)propyl methacrylate; the solvent is dichloromethane; the condensing agent is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; the dehydrating agent is N,N-dicyclohexylcarbonyldiimide or diisopropylcarbodiimide; and the catalyst is 4-dimethylaminopyridine p-toluenesulfonate.

3. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 2, characterized in that: The solid-liquid ratio of methyl acrylate containing thioketal, methylprednisolone hemisuccinate, solvent, shrinking agent, dehydrating agent and catalyst is 1mmol: 0.2~5mmol: 10~20mL: 1~3mmol: 1~3mmol: 0.1~1mmol.

4. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 1, characterized in that: In step S2, the cross-linking agent is N,N'-methylenebisacrylamide; the initiator is azobisisobutyronitrile; and the solvent is one or more of acetonitrile, water, ethanol, dimethyl sulfoxide and dioxane.

5. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 4, characterized in that: The material-liquid ratio of the active oxygen responsive monomer, the hydrophilic monomer, the active ester monomer, the cross-linking agent, the initiator and the solvent is 1~10mg:5~100mg:1~10mg:1~10mg:1~10mg:20~40mL.

6. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 5, characterized in that: The specific steps include: (1) soaking the biological valve cross-linked with glutaraldehyde in an amino cationic polymer solution at 4-37° C. and pH 5-8 for 1-48 hours to obtain a biological valve grafted with amino cationic polymer; (2) soaking the bioprosthesis grafted with amino cationic polymer in an active oxygen responsive drug-loaded nanogel solution at 4-37° C. and pH 3-10 for 0.5-48 h to obtain a bioprosthesis bonded with active oxygen responsive drug-loaded nanogel; (3) Repeat steps (1) to (2) 0 to 1 times, and then immerse the bioprosthesis bonded with active oxygen responsive drug-loaded nanogel in the active ester compound solution for 3 times, each time at 4 to 37° C. and pH 5 to 7 for 2 to 6 hours, and then wash to obtain the bioprosthesis.

7. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 6, characterized in that: The glutaraldehyde cross-linking treatment method of the biological valve is as follows: soaking the biological valve in a glutaraldehyde solution for 48 to 72 hours, wherein the concentration of the glutaraldehyde solution is 0.05 to 8 wt %; the biological valve is decellularized bovine pericardium or porcine pericardium; the amino cationic polymer is a branched polyethyleneimine with a molecular weight of 300 to 1,000,000; The concentration of the amino cationic polymer solution is 5-15 mg / mL; the concentration of the active oxygen responsive drug-loaded nanogel solution is 0.01-10 mg / mL; the active ester compound is branched acetic acid-N-succinimide ester or polyethylene glycol succinimide ester, and the concentration of the active ester compound solution is 1-5 mg / mL.

8. The method for preparing a biological valve modified with drug-loaded nanogel according to claim 7, characterized in that: When the biological valve cross-linked with glutaraldehyde is soaked in the amino cationic polymer solution for the first time, 0.1-0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-0.15 theoretical amino equivalents of N-hydroxysuccinimide are added.

9. A bioprosthetic valve modified with drug-loaded nanogel, characterized in that: The bioprosthesis is prepared by the method for preparing a drug-loaded nanogel-modified bioprosthesis according to any one of claims 1 to 8.

10. An application of a bioprosthesis modified with a drug-loaded nanogel, characterized in that: The drug-loaded nanogel-modified biological valve according to claim 9 is used to prepare an artificial valve prosthesis.

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