Esterase-responsive nanogel modified biological valve, preparation method and application
By modifying bioprosthetic valves with esterase-responsive nanogels, the problems of infective endocarditis and biocompatibility of bioprosthetic valves have been solved. This has enabled targeted release of antibiotics, reduced the risk of thrombosis and calcification, and extended the lifespan of the valves.
Patent Information
- Application Number
- CN202510546357.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2026-08-04
- Estimated Expiration
- 2045-04-28
AI Technical Summary
Existing bioprosthetic valves have problems such as severe infective endocarditis, poor biocompatibility, and short lifespan during use. In particular, the limited number of surface modification sites leads to toxic side effects from long-term antibiotic use.
Biological valves were modified with esterase-responsive nanogels. After cross-linking with glutaraldehyde, amino cationic polymers were grafted onto the nanogels, which were then chemically bonded to the esterase-responsive nanogels. Antibiotic drugs containing ester groups in the nanogels were released at specific sites, improving biocompatibility and anti-infective properties.
Nanogel-modified bioprosthetic valves can release antibiotics at specific sites in microbial infection environments, reducing platelet adhesion and coagulation risks, improving anti-inflammatory, anticoagulant and anti-calcification capabilities, and extending service life.
Smart Images
Figure CN120361308B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological valve technology, specifically relating to an esterase-responsive nanogel-modified biological valve, its preparation method, and its application. Background Technology
[0002] Valvular heart disease is a leading cause of death worldwide, affecting over 100 million people globally. Artificial heart valve replacement surgery is currently one of the most effective clinical treatments for valvular heart disease. Artificial valve prostheses used in artificial heart replacement surgery are divided into mechanical valves and bioprosthetic valves. With the development of transcatheter interventional techniques for valvular heart disease in recent years, the demand for bioprosthetic valves is continuously increasing.
[0003] Currently, most bioprosthetic valves used clinically are made of glutaraldehyde-crosslinked bovine pericardium or porcine aortic valves, and their lifespan is typically only about 10 years. Bioprosthetic valve failure is caused by a variety of factors, including infective endocarditis, calcification, microcoagulation, and difficulty in endothelialization. Among these, infective endocarditis (PVE) of prosthetic valves is a serious and fatal complication after heart valve replacement surgery, posing a significant threat to human health.
[0004] For infective endocarditis caused by prosthetic valves, clinical treatment primarily involves medication. However, due to the large vegetations resulting from prosthetic valve disease (PVE), patients often require long-term, high-dose antibiotic treatment, leading to severe toxic side effects. Constructing bioprosthetic valves with in situ anti-infective properties holds promise for addressing this issue. Simple drug modifications to bioprosthetic valves typically reduce material biocompatibility, and the limited number of modification sites on the bioprosthetic valve surface results in low modification efficiency. Therefore, improving biocompatibility and surface modification efficiency while simultaneously endowing prosthetic valves with anti-infective properties remains a key challenge. Summary of the Invention
[0005] To address the challenges in existing technologies of improving biocompatibility and surface modification efficiency while imparting anti-infection properties to artificial bioprosthetic valves, this invention provides an esterase-responsive nanogel-modified bioprosthetic valve, its preparation method, and its application. This aims to solve the technical problems of poor stability, short shelf life, and poor long-term anticoagulation, anti-inflammatory, anti-infection, and anti-calcification capabilities of existing bioprosthetic valves.
[0006] The technical solution adopted in this invention is as follows:
[0007] An esterase-responsive nanogel-modified bioprosthetic valve, wherein the esterase-responsive nanogel is attached to the bioprosthetic valve and the esterase-responsive nanogel contains an antibiotic drug with ester bond groups.
[0008] A method for preparing an esterase-responsive nanogel-modified bioprosthetic valve includes the following steps:
[0009] S1: Glutaraldehyde cross-linking treatment of the bioprosthetic valve;
[0010] S2: Amino cationic polymer grafted onto a glutaraldehyde-crosslinked bioprosthetic valve;
[0011] S3: Esterase-responsive nanogels are chemically bonded to the surface of the grafted bioprosthetic valve;
[0012] S4: The bioprosthetic valve is treated with an active ester compound to obtain the nanogel-modified bioprosthetic valve.
[0013] Preferably, the bioprosthetic valve is a decellularized bovine pericardium or porcine pericardium.
[0014] As a preferred embodiment, the preparation method of the esterase-responsive nanogel in S3 includes:
[0015] S2011: Methacrylate containing ester groups and antibiotic drugs are dissolved in a solvent, and a condensing agent, a dehydrating agent and a catalyst are added to react and obtain an esterase-responsive monomer;
[0016] S2012: The esterase-responsive monomer, hydrophilic monomer, active ester monomer, crosslinking agent and initiator are dissolved in a solvent and reacted under inert gas protection to obtain esterase-responsive nanogel.
[0017] Preferably, the ratio of ester-containing methacrylate, antibiotic, solvent, dehydrating 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;
[0018] The ratio of esterase-responsive monomer, hydrophilic monomer, active ester monomer, crosslinking agent, initiator and solvent is 1-20 mg: 5-100 mg: 1-10 mg: 1-10 mg: 1-10 mg: 20-40 mL.
[0019] Furthermore, the feed-to-liquid ratio of the esterase-responsive monomer, hydrophilic monomer, active ester monomer, crosslinking agent, initiator, and 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 the ester bond is 2-hydroxyethyl methacrylate;
[0021] The hydrophilic monomer is 2-acrylamido-2-methylpropanesulfonic acid;
[0022] The active ester monomer is N-acryloyloxysuccinimide or N-hydroxysuccinimide methacrylate.
[0023] With this technical solution, the hydrophilic monomer is selected as 2-acrylamido-2-methylpropanesulfonic acid, and the sulfonic acid group mimics the anticoagulant activity of heparin, which can inhibit platelet adhesion. The active ester monomer is selected as N-acryloyloxysuccinimide or N-hydroxysuccinimide methacrylate, thus it has good biocompatibility and strong amino reactivity.
[0024] Preferably, in S2011, the solvent is dichloromethane; the condensing agent is one or more of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(1H-benzotriazolyl-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate, and N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl)hexafluorophosphate; the dehydrating agent is one or more of N,N-dicyclohexylcarbonyldiimide, diisopropylcarbodiimide, and N,N'-diisopropylcarbodiimide; and 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; and the solvent is one or more of acetonitrile, water, ethanol, dimethyl sulfoxide, and dioxane.
[0026] As a preferred option, the reaction conditions for S2011 are 6–72 h at room temperature; and the reaction conditions for S2012 are 0.1–24 h at 40–160 °C.
[0027] Preferably, in S1, the bioprosthetic valve is immersed 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.
[0028] The amino-cationic polymer in S2 is a branched polyethyleneimine with a molecular weight of 300 to 1,000,000 and a concentration of 5 to 15 mg / mL;
[0029] The concentration of the esterase-responsive nanogel solution in S3 is 0.01–10 mg / mL;
[0030] The active ester compound in S4 is branched-chain acetic acid-N-succinimide ester or polyethylene glycol succinimide ester, with a concentration of 1–5 mg / mL.
[0031] Preferably, S2-3 is repeated 0-1 times, and during the first S2, 0.05-0.4 times the theoretical amino equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.05-0.4 times the theoretical amino equivalent of N-hydroxysuccinimide are added to soak the glutaraldehyde-crosslinked bioprosthetic valve.
[0032] As a preferred option, the reaction conditions in S2 are: a reaction at a temperature of 4–37°C and a pH of 3–10 for 1–48 hours;
[0033] The reaction conditions in S3 are: a temperature of 4–37℃ and a pH of 3–10 for 0.5–48 h.
[0034] Furthermore, when the glutaraldehyde-crosslinked bioprosthetic valve was first soaked in an amino-cationic polymer solution at pH 5.5, the amino-cationic polymer concentration was 10 mg / mL, and the soaking time was 12 h. When the amino-cationic polymer was used for a second soaking treatment, the pH was 7, the amino-cationic polymer solution concentration was 10 mg / mL, and the soaking time was 4 h.
[0035] Furthermore, when the bioprosthetic valve grafted with amino-cationic polymer was first treated with esterase-responsive nanogel solution, the pH was 7, the concentration of esterase-responsive nanogel solution was 1 mg / mL, and the soaking time was 2 h.
[0036] Furthermore, when the nanogel solution was soaked again, the pH was 7, the concentration of the nanogel solution was 1 mg / mL, and the soaking time was 12 h.
[0037] Furthermore, when the bioprosthetic valve of the bonded esterase-responsive nanogel was soaked in an active ester compound solution at pH 7, the concentration of the active ester compound solution was 5 mg / mL, and the soaking time was 2 h.
[0038] Preferably, the antibiotic 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] Application of a nanogel-modified bioprosthetic valve for the prevention of infective endocarditis, used in the preparation of artificial valve prostheses.
[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 this invention have uniform particle size. The nanogels contain monomers of drugs for preventing infective endocarditis with ester bonds. The ester bonds are esterase-responsive groups. The drugs for preventing infective endocarditis are antibiotics with broad-spectrum anti-infective activity. Under pathological conditions such as microbial infection with high esterase concentration, 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 bioprosthetic valve modification method provided by this invention solves the problem of few modifiable sites on the surface of bioprosthetic valves. By reacting the bioprosthetic valve with an amino cationic polymer, the residual aldehyde groups on the glutaraldehyde-crosslinked bioprosthetic valve are blocked, effectively reducing the problems of bioprosthetic valve calcification and biotoxicity. At the same time, a large number of amino groups are introduced, providing reaction sites for the subsequent efficient modification of nanogels, thus solving the dilemma of few modifiable sites on bioprosthetic valves.
[0043] 3. This invention introduces a large number of hydrophilic groups on the surface of the bioprosthetic valve, which can form a stable hydration layer on the surface of the bioprosthetic valve, effectively reducing platelet adhesion, reducing the risk of thrombosis and coagulation, and resisting bacterial adhesion.
[0044] 4. This invention modifies the surface of a bioprosthetic valve material by chemically bonding esterase-responsive nanogels. Compared with bioprosthetic valves modified by electrostatic adsorption or physical filling, the bioprosthetic valve prepared by chemical bonding has good stability and long-term effectiveness. It can improve the long-term anti-inflammatory, anticoagulant and anti-calcification capabilities of the bioprosthetic valve, and realize the organic integration of multiple functions on the surface of the bioprosthetic valve, thereby extending the service life of the bioprosthetic valve. Attached Figure Description
[0045] Figure 1 The image shows the particle size measurement results of the esterase-responsive nanogel synthesized in Example 1.
[0046] Figure 2 The figure shows the stability test results of the esterase-responsive nanogel synthesized in Example 1;
[0047] Figure 3 SEM image of the esterase-responsive nanogel synthesized in Example 1;
[0048] Figure 4 SEM image of the nanogel-modified bioprosthetic valve prepared in Example 1;
[0049] Figure 5 SEM images of platelets adhering to the surfaces of bioprosthetic valves modified with and without esterase-responsive nanogels;
[0050] Figure 6SEM images of bioprosthetic valves modified with and without esterase-responsive nanogels after in vivo blood contact tests;
[0051] Figure 7 Immunohistochemical staining results of bioprosthetic valves modified with and without esterase-responsive nanogels 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 7 and 14 days after subcutaneous implantation of bioprosthetic valves modified with and without esterase-responsive nanogels.
[0053] Figure 9 Microscopic images of alizarin red stained sections of bioprosthetic valves modified with and without esterase-responsive nanogels 60 days after subcutaneous implantation;
[0054] Figure 10 Figure showing the experimental results of drug release from a nanogel-modified bioprosthetic valve for the prevention of infective endocarditis;
[0055] Figure 11 A statistical chart showing 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 The images show the colony formation units and number statistics of bacteria after co-culturing bioprosthetic valves modified with esterase-responsive nanogels with and without esterase-responsive nanogels. Detailed Implementation
[0058] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally 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 inventive effort are within the scope of protection of this application.
[0059] Example 1
[0060] An esterase-responsive nanogel-modified biovalve membrane is prepared by the following steps:
[0061] ① Synthesize esterase-responsive nanogels, including the following steps:
[0062] S2011: Add 222 mg 2-hydroxyethyl methacrylate, 617 mg ofloxacin, 458 mg N,N-dicyclohexylcarbodiimide, 843 mg benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 201 mg 4-dimethylaminopyridine p-toluenesulfonate, and 20 mL dichloromethane to a 100 mL round-bottom flask. After stirring and dissolving, react at room temperature for 48 h. After the reaction is complete, filter under vacuum, collect the filtrate, concentrate the filtrate using a rotary evaporator, redissolve it in dichloromethane, and then extract it with 20 mL sodium chloride solution. Extract three times. The lower layer of dichloromethane solution was collected, dehydrated and dried with anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated until dichloromethane was removed using a rotary evaporator, and then dissolved in 2 mL of dichloromethane. An appropriate amount of silica gel powder was dissolved in petroleum ether and loaded onto a 15 cm chromatography column. The reactant dissolved in dichloromethane was loaded onto the column and eluented sequentially with petroleum ether:ethyl acetate = 8:1 and 4:1. The product was collected, evaporated to dryness using a rotary evaporator, and then dried again using an oil pump to obtain the esterase-responsive ofloxacin monomer. The structure of the esterase-responsive ofloxacin monomer is shown in Formula I.
[0063]
[0064] S2012: Take 10 mg of esterase-responsive ofloxacin 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 in a thick-walled pressure-resistant bottle, promote the dissolution of the raw materials by sonication, then replace with nitrogen gas, and react for 1 h under nitrogen protection and at 100 °C. After the reaction is completed, collect the suspension, centrifuge at 12000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and air dry for 20 min to obtain esterase-responsive nanogel, which is a light yellow solid powder.
[0065] ②Preparation of a nanogel-modified bioprosthetic valve for the prevention of infective endocarditis, including the following steps:
[0066] S1. Take fresh pig pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake for 48 hours, then soak it in a 1 wt% glutaraldehyde solution and shake for 48 hours for cross-linking treatment, take it out and wash it with physiological saline to obtain glutaraldehyde cross-linked bio-valve.
[0067] S2. The glutaraldehyde-crosslinked bioprosthetic valve was immersed in a 10 mg / mL solution of branched polyethyleneimine with a molecular weight of 10,000. 0.1 times the theoretical amino equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1 times the theoretical amino equivalent of N-hydroxysuccinimide were added. The mixture was then immersed at 32 °C and pH 6 for 12 h to obtain a bioprosthetic valve grafted with amino cationic polymer.
[0068] S3. The bioprosthetic valve grafted with amino cationic polymer was immersed in an esterase-responsive nanogel solution with a concentration of 1 mg / mL and the reaction was carried out at 32℃ and pH 6 for 12 h with shaking to obtain a bioprosthetic valve with bonded esterase-responsive nanogel.
[0069] S4. The bioprosthetic valve of the bonded esterase-responsive nanogel was repeatedly immersed twice in a 5 mg / mL branched-chain acetic acid-N-succinimide ester solution. Each time, it was shaken and immersed for 2 hours at 32℃ and pH 6. After immersion, it was taken out and cleaned with physiological saline to obtain the product.
[0070] Example 2
[0071] An esterase-responsive nanogel-modified biovalve membrane is prepared by the following steps:
[0072] ① Synthesize esterase-responsive nanogels, including the following steps:
[0073] S2011: Add 222 mg 2-hydroxyethyl methacrylate, 566 mg ciprofloxacin, 458 mg N,N-dicyclohexylcarbodiimide, 843 mg benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 201 mg 4-dimethylaminopyridine p-toluenesulfonate, and 20 mL dichloromethane to a 100 mL round-bottom flask. After stirring to dissolve, react at room temperature for 48 h. After the reaction is complete, filter under vacuum, collect the filtrate, concentrate the filtrate using a rotary evaporator, redissolve it in dichloromethane, and then extract it with 20 mL sodium chloride solution. Extract three times. The lower layer of dichloromethane solution was collected, dehydrated and dried with anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated until dichloromethane was removed using a rotary evaporator, and then dissolved in 2 mL of dichloromethane. An appropriate amount of silica gel powder was dissolved in petroleum ether and loaded onto a 15 cm chromatography column. The reactant dissolved in dichloromethane was loaded onto the column and eluented sequentially with petroleum ether:ethyl acetate = 10:1 and 5:1. The product was collected, evaporated to dryness using a rotary evaporator, and then dried again using an oil pump to obtain the esterase-responsive ciprofloxacin monomer. The structure of the esterase-responsive ciprofloxacin monomer is shown in Formula II.
[0074]
[0075] S2012: Take 20 mg of esterase-responsive ciprofloxacin 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 in a thick-walled pressure-resistant bottle. Promote the dissolution of the raw materials by sonication, then replace with nitrogen gas, and react for 0.5 h under nitrogen protection and at 160 °C. After the reaction is completed, collect the suspension, centrifuge at 12000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and air dry for 20 min to obtain esterase-responsive nanogel, which is a light yellow solid powder.
[0076] ②Preparation of a nanogel-modified bioprosthetic valve for the prevention of infective endocarditis, including the following steps:
[0077] S1. Take fresh pig pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake for 48 hours, then soak it in a 5 wt% glutaraldehyde solution and shake for 48 hours for cross-linking treatment, take it out and wash it with physiological saline to obtain glutaraldehyde cross-linked bio-valve.
[0078] S2. The glutaraldehyde-crosslinked bioprosthetic valve was immersed in a 10 mg / mL solution of branched polyethyleneimine with a molecular weight of 1,000,000. 0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15 theoretical amino equivalents of N-hydroxysuccinimide were added. The mixture was then immersed at 37 °C and pH 6 for 1 h to obtain a bioprosthetic valve grafted with amino cationic polymer.
[0079] S3. The bioprosthetic valve grafted with amino cationic polymer was immersed in a 0.1 mg / mL esterase-responsive nanogel solution and shaken for 0.5 h at 37 °C and pH 10 to obtain a bioprosthetic valve with bonded esterase-responsive nanogel.
[0080] S4. The bioprosthetic valve of the bonded esterase-responsive nanogel was repeatedly immersed twice in a 5 mg / mL branched-chain acetic acid-N-succinimide ester solution. Each time, it was shaken and immersed for 2 hours at 37°C and pH 6. After immersion, it was taken out and cleaned with physiological saline to obtain the product.
[0081] Example 3
[0082] An esterase-responsive nanogel-modified biovalve membrane is prepared by the following steps:
[0083] ② Synthesize esterase-responsive nanogels, including the following steps:
[0084] S2011: Add 222 mg 2-hydroxyethyl methacrylate, 623 mg amoxicillin, 458 mg N,N-dicyclohexylcarbodiimide, 843 mg benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 201 mg 4-dimethylaminopyridine p-toluenesulfonate, and 20 mL dichloromethane to a 100 mL round-bottom flask. After stirring to dissolve, react at room temperature for 48 h. After the reaction is complete, filter under vacuum, collect the filtrate, concentrate the filtrate using a rotary evaporator, redissolve it in dichloromethane, and then extract it with 20 mL sodium chloride solution. Extract three times. The lower layer of dichloromethane solution was collected, dehydrated and dried with anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated until dichloromethane was removed using a rotary evaporator, and then dissolved in 2 mL of dichloromethane. An appropriate amount of silica gel powder was dissolved in petroleum ether and loaded onto a 15 cm chromatography column. The reactant dissolved in dichloromethane was loaded onto the column and eluented sequentially with petroleum ether:ethyl acetate = 6:1 and 4:1. The product was collected, evaporated to dryness using a rotary evaporator, and then dried again using 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 esterase-responsive amoxicillin 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 in a thick-walled pressure-resistant bottle, promote the dissolution of the raw materials by sonication, then replace with nitrogen gas, and react for 6 h under nitrogen protection and at 40 °C. After the reaction is completed, collect the suspension, centrifuge at 12000 rpm for 3 min, remove the supernatant, retain the solid precipitate, and air dry for 20 min to obtain esterase-responsive nanogel, which is a white solid powder.
[0087] ②Preparation of a nanogel-modified bioprosthetic valve for the prevention of infective endocarditis, including the following steps:
[0088] S1. Take fresh porcine pericardium, remove connective tissue and clean it, then wash it with physiological saline, then soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake for 48 hours, then soak it in a 0.05 wt% glutaraldehyde solution and shake for 48 hours for cross-linking treatment, take it out and wash it with physiological saline to obtain a glutaraldehyde cross-linked bioprosthetic valve.
[0089] S2. The glutaraldehyde-crosslinked bioprosthetic valve was immersed in a 5 mg / mL solution of branched polyethyleneimine with a molecular weight of 300. 0.15 theoretical amino equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.15 theoretical amino equivalents of N-hydroxysuccinimide were added. The mixture was then immersed at 4 °C and pH 5 for 24 h to obtain a bioprosthetic valve grafted with amino cationic polymer.
[0090] S3. The bioprosthetic valve grafted with amino cationic polymer was immersed in a esterase-responsive nanogel solution with a concentration of 5 mg / mL and shaken for 24 h at 4 °C and pH 3 to obtain a bioprosthetic valve with bonded esterase-responsive nanogel.
[0091] S4. The bioprosthetic valve of the bonded esterase-responsive nanogel was repeatedly immersed twice in a 3 mg / mL branched-chain acetic acid-N-succinimide ester solution. Each time, it was shaken and immersed for 6 hours at 4℃ and pH 5. After immersion, it was taken out and cleaned with physiological saline to obtain the product.
[0092] Experimental Example
[0093] The nanogel-modified bioprosthetic valves for preventing infective endocarditis prepared in the embodiments of the present invention have similar performance. Taking Example 1 as an example, the performance of the relevant products will be described:
[0094] I. Structural Testing
[0095] ① The particle size and zeta potential of the esterase-responsive nanogel prepared in Example 1 were measured using a Malvern nanoparticle size analyzer. The results are as follows: Figure 1 and Figure 2 As shown in the figure, the particle size of the nanogel is around 320 nm, the polydispersity index is 0.0327, and the particle size is relatively uniform; the particle size of the esterase-responsive nanogel changes very little over 180 days, and the stability is good.
[0096] ② The esterase-responsive nanogel sample synthesized in Example 1 and the nanogel-modified bioprosthetic valve for preventing infective endocarditis were subjected to electron microscopy scanning. The SEM images are shown below. Figure 3 and Figure 4 As shown, Figure 3 As shown, the esterase-responsive nanogel has a uniform particle size distribution. Figure 4 The surface shown is entirely composed of gel layers, with a modification rate as high as 95%, demonstrating that nanogels can be efficiently modified onto the surface of biological valves.
[0097] II. Performance Testing
[0098] Platelet adhesion experiments were conducted on glutaraldehyde-crosslinked bioprosthetic valves with and without nanogel modification. Platelet adhesion is an initiating step in thrombus formation. Platelets adhering to the material surface release cytokines and procoagulant substances, initiating platelet aggregation, activation, and coagulation. Therefore, fewer platelets adhering to the material surface may indicate superior anticoagulant properties. Figure 5 SEM images of platelet adhesion, such as Figure 5 As shown, the number of platelets (yellow spherical objects) adhering to the surface of the bioprosthetic valve modified with nanogel and cross-linked with glutaraldehyde is significantly reduced, exhibiting better anticoagulant properties.
[0099] To better assess the blood compatibility of bioprosthetic valves under simulated physiological conditions, in vivo blood contact tests were conducted on bioprosthetic valves with and without nanogel modification. SEM images of the bioprosthetic valves after contact are shown below. Figure 6 As shown, the surface of the unmodified bioprosthetic valve contains a large number of fibrin networks formed by the activation of red blood cells and platelets. The surface of the bioprosthetic valve may trigger the activation and aggregation of blood cells, promoting thrombus formation. On the other hand, the surface of the bioprosthetic valve modified with nanogel has only a small number of adhered red blood cells and platelets. This is because by chemically bonding the nanogel, a large number of hydrophilic groups are introduced into the surface of the bioprosthetic valve, forming a stable hydration layer, reducing the direct contact and interaction between blood cells and the surface of the bioprosthetic valve, effectively reducing platelet adhesion, and reducing the risk of thrombosis and coagulation.
[0100] Bioprosthetic valves without nanogel modification and bioprosthetic valves with nanogel modification were implanted into rats, and the rats' vital signs were restored. The implanted bioprosthetic valves were removed 7 days and 14 days after implantation.
[0101] The removed bioprosthetic valve was rinsed thoroughly with physiological saline to remove surface blood and impurities. It was then paraffin-embedded, sectioned, and stained. Due to the immune inflammatory response triggered by foreign body implantation, IL-1β and TNF-α positive cells accumulate on the bioprosthetic valve surface. Therefore, immunohistochemical staining with specific antibodies against IL-1β and TNF-α was performed on the sections. The positive expression levels of IL-1β and TNF-α in the bioprosthetic valve were compared at 7 and 14 days to assess its hemocompatibility and immune inflammatory response. The staining results are shown in the figure below. Figure 7 As shown, cells whose nuclei overlap with the deep yellow color are positive cells; the statistical results of the number of IL-1β and TNF-α positive cells are as follows. Figure 8 As shown; from Figure 7 and Figure 8It can be seen that, regardless of whether it is 7 days or 14 days, the positive expression levels of IL-1β and TNF-α in the unmodified bioprosthetic valve are higher than those in the esterase-responsive nanogel-modified bioprosthetic valve, indicating that the esterase-responsive nanogel-modified bioprosthetic valve has a milder inflammatory response after implantation. Similarly, 60 days after implantation, the implanted bioprosthetic valve was removed, and alizarin red section staining was used to show the distribution of calcium salts in the bioprosthetic valve. The staining results are shown in the figure below. Figure 9 As shown, alizarin red turns red or orange-red when it combines with calcium. Figure 9 The red area without nanogel modification is very deep and covers a large area, indicating severe calcification. The experimental results show that after 60 days of subcutaneous implantation, the glutaraldehyde cross-linked bioprosthetic valve without nanogel modification underwent severe calcification during subcutaneous implantation, while the glutaraldehyde cross-linked bioprosthetic valve with nanogel modification showed no obvious calcification. Therefore, the glutaraldehyde cross-linked bioprosthetic valve with nanogel modification has better anti-calcification performance.
[0102] This application also conducted drug release experiments on esterase-responsive nanogels, using PBS dilution containing 1 mM esterase to simulate the human physiological environment. First, 2 mg of the esterase-responsive nanogel prepared in Example 1 was dissolved in 2 mL of esterase dilution and placed in a dialysis bag (Mw = 3500). The dialysis bag was then placed in a 50 mL centrifuge tube, with three parallel sample groups. 20 mL of the corresponding concentration of esterase dilution was added to each centrifuge tube, and the tubes were sealed and placed on a shaker in a 37°C oven. At sampling time points (0, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, 120 h, 168 h), 2 mL of dilution was taken, and the same volume of release solution was added. Then, the absorbance of ofloxacin in the release solution at different time points was measured using a UV spectrophotometer, and the release amount was calculated with reference to the standard curve. The cumulative release rate (%) of ofloxacin was calculated, and the average results of the three parallel groups are shown below. Figure 10 As shown, from Figure 10 It can be seen that ofloxacin can be released from the nanogel under inflammatory physiological conditions.
[0103] This invention also conducted anti-infective experiments on the nanogel-modified bioprosthetic valve for the prevention of infective endocarditis, selecting typical Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli to analyze the anti-infective performance of the bioprosthetic valve.
[0104] The anti-adhesion ability test procedure is as follows: First, bioprosthetic valves with and without nanogel modification were sterilized by ultraviolet irradiation. *E. coli* and *S. aureus* strains were incubated in Rhine-Bert medium at 37°C with continuous shaking for 12 hours to obtain a bacterial suspension with a concentration of 10⁷ CFU / mL. 1 mL of the bacterial suspension was added to the sample and incubated at 37°C for 2 hours. After incubation, the sample was washed three times with sterile physiological saline to remove unadhesive bacteria. The sample was then immersed in 5 mL of sterile physiological saline and sonicated for 5 minutes to obtain a suspension. 20 μL of the suspension was evenly spread on nutrient agar plates and incubated at 37°C for 24 hours to obtain images and count colony-forming units (CFU / mL). The average results of the three parallel groups are shown below. Figure 11 As shown, for both *Escherichia coli* and *Staphylococcus aureus*, the bacterial colony-forming units on the surface of the bioprosthetic valve modified with nanogel were significantly fewer than those on the unmodified bioprosthetic valve. The remaining samples were fixed with 2.5% glutaraldehyde solution, subjected to a gradient dehydration process with ethanol, and then freeze-dried for observation using scanning electron microscopy (SEM). Figure 12 In the study, *Escherichia coli* and *Staphylococcus aureus* were identified using pseudo-color labeling. The results showed that, for both *E. coli* and *Staphylococcus aureus*, the amount of bacteria adhering to the surface of the bioprosthetic valve modified with nanogel was significantly less than that of the unmodified bioprosthetic valve.
[0105] The bactericidal ability test procedure was as follows: 500 mg of bioprosthetic valves, with and without nanogel modification, were sterilized by ultraviolet irradiation, cut into small pieces, and placed in Erlenmeyer flasks. Frozen *E. coli* and *Staphylococcus aureus* strains were incubated in Rhine-Bert medium at 37°C with continuous shaking for 12 hours to obtain a bacterial suspension with a concentration of 5 × 10⁵ CFU / mL. 10 mL of the above bacterial suspension was added to the sample and incubated in a shaker at 37°C at 120 rpm for 4 hours. 10 μL of the bacterial suspension was evenly spread on a nutrient agar plate and incubated for 24 hours to obtain images and count colony-forming units. A bacterial suspension without sample was used as a control group. The anti-infection rate was determined by the following formula: Inhibition rate (%) = (A – B) / A × 100%, where A and B represent the number of colony-forming units in the control and experimental groups, respectively. Results are as follows: Figure 13 As shown, regardless of whether it is Escherichia coli or Staphylococcus aureus, the number of colony-forming units corresponding to the bioprosthetic valve modified with nanogel is significantly less than that of the bioprosthetic valve without nanogel modification, and the antibacterial rate of the bioprosthetic valve modified with nanogel is as high as 90%.
[0106] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.
Claims
1. A method for preparing an esterase-responsive nanogel-modified bioprosthetic valve, characterized by: Includes the following steps: S1: Glutaraldehyde cross-linking treatment of bioprosthetic valves; S2: Amino cationic polymer grafted onto a glutaraldehyde-crosslinked bioprosthetic valve; S3: Esterase-responsive nanogels are chemically bonded to the surface of the grafted bioprosthetic valve; S4: The bioprosthetic valve is treated with an active ester compound to obtain an esterase-responsive nanogel modified bioprosthetic valve. The esterase-responsive nanogel contains an antibiotic drug with an ester bond group. The active ester compound is branched-chain acetic acid-N-succinimide ester or polyethylene glycol succinimide ester. The preparation methods of S3 esterase-responsive nanogels include: S2011: Methacrylate containing ester groups and antibiotic drugs are dissolved in a solvent, and a condensing agent, a dehydrating agent and a catalyst are added to react and obtain an esterase-responsive monomer; S2012: Esterase-responsive monomers, hydrophilic monomers, active ester monomers, crosslinking agents and initiators are dissolved in a solvent and reacted under inert gas protection to obtain esterase-responsive nanogels. The methacrylate containing ester groups is 2-hydroxyethyl methacrylate; 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 esterase-responsive nanogel-modified bioprosthetic valves according to claim 1, characterized in that: The ratio of methacrylates containing ester groups, antibiotics, solvents, dehydrating agents, dehydrating agents, and catalysts is 1 mmol: 0.2~5 mmol: 10~20 mL: 1~3 mmol: 1~3 mmol: 0.1~1 mmol; The ratio of esterase-responsive monomer, hydrophilic monomer, active ester monomer, crosslinking agent, initiator and solvent is 1~20mg: 5~100mg: 1~10mg: 1~10mg: 1~10mg: 20~40mL.
3. The method for preparing a bioprosthetic valve modified with esterase-responsive nanogel according to any one of claims 1-2, characterized in that: In S1, the bioprosthetic valve is immersed in a glutaraldehyde solution with a concentration of 0.05~8wt% for 48~72 hours; The amino-cationic polymer in S2 is a branched polyethyleneimine with a molecular weight of 300-1,000,000 and a concentration of 5-15 mg / mL; The concentration of the esterase-responsive nanogel solution in S3 is 0.01~10 mg / mL; The concentration of the active ester compound in S4 is 1~5 mg / mL.
4. The method for preparing a bioprosthetic valve modified with esterase-responsive nanogel according to claim 3, characterized in that: S2 and S3 were repeated once. During the first S2, 0.05 to 0.4 times the theoretical amino equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.05 to 0.4 times the theoretical amino equivalent of N-hydroxysuccinimide were added to soak the glutaraldehyde-crosslinked bioprosthetic valve.
5. The method for preparing a bioprosthetic valve modified with esterase-responsive nanogel according to any one of claims 1-2, characterized in that: The reaction conditions in S2 are: a temperature of 4~37℃ and a pH of 3~10 for 1~48 hours; The reaction conditions in S3 are: a temperature of 4~37℃ and a pH of 3~10 for 0.5~48h.
6. The method of claim 1-2, wherein the esterase-responsive nanogel modified bioprosthetic valve is prepared by the following steps: (1) preparing a bioprosthetic valve; (2) preparing a nanogel; (3) modifying the bioprosthetic valve with the nanogel; and (4) modifying the nanogel with an esterase-responsive polymer. The antibiotics mentioned are drugs for the prevention of infective endocarditis, and the drugs for the prevention of infective endocarditis are one of ofloxacin, levofloxacin, ciprofloxacin, amoxicillin, ceftriaxone, penicillin, and moxifloxacin.
7. An esterase-responsive nanogel-modified bioprosthetic valve, characterized in that: It is prepared by the preparation method of any one of claims 1-6.
8. Use of the esterase-responsive nanogel of claim 7 to modify a biological valve. Used to manufacture artificial valve prostheses.