A biovalve modifier and polymer-modified biovalve and preparation method thereof

Abstract

The present invention discloses a biovalve modifier and a polymer-modified biovalve and a preparation method, and belongs to the technical field of medical material and medical device preparation. The biovalve modifier is formed by polymerization of a diclofenac monomer containing a thioketal group, a hydrophilic monomer, a crosslinking agent, an active ester monomer and an initiator. The preparation method of the polymer-modified biovalve comprises the following steps: sequentially immersing the biovalve material in a glutaraldehyde solution, an amino cationic polymer solution, and a nanogel solution, and finally immersing the biovalve twice in an active ester compound solution. The polymer-modified biovalve prepared by the present invention is a biovalve in which nanogels are modified on the valve surface by covalent bonds, has good stability and long-term effect, and has good biocompatibility and anti-inflammatory, anticoagulant and anti-calcification effects, and has good medical application prospects.

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 biovalve modifier and a polymer-modified biovalve and a preparation method thereof. Background Art

[0002] In recent years, the morbidity and mortality of cardiovascular and cerebrovascular diseases have continued to rise worldwide, and valvular heart disease has become a major problem threatening human life and health. Surgical open-chest valve replacement surgery and transcatheter minimally invasive surgery are the main methods for treating valvular heart disease. Nevertheless, no matter which surgical method is used, patients still need to receive anticoagulant drugs after surgery to reduce the risk of postoperative complications. In particular, mechanical valves used in surgical operations usually require patients to take anticoagulant drugs for life. In recent years, due to its small trauma and quick recovery, minimally invasive interventional surgery has gradually become the first choice for valve replacement, but the problem of thrombosis of biological valves has not been completely solved. Therefore, giving biological valves excellent anticoagulant properties has become the core challenge in the development of new artificial biological valves.

[0003] At present, in order to improve the anticoagulant properties of biological valves, heparin or hydrophilic molecules are often added during the preparation process, but these functional molecules are fixed by non-chemical bonds, the effect duration is limited, and the risk of thrombosis still exists. Although the stability of heparin is improved by covalent bond modification, it often significantly reduces the anticoagulant effect of heparin. In addition, modifying the surface of the cross-linked biological valve to prepare an anticoagulant coating is also a common method, but it also faces problems such as poor coating stability, short shelf life, and possible effects on the mechanical properties of the valve. It is of great clinical significance to modify the artificial biological valve with multiple functions without affecting other properties of the biological valve. Summary of the invention

[0004] In response to the above-mentioned prior art, the present invention provides a biological valve modifier and a polymer-modified biological valve and a preparation method to solve the problems that the existing biological valve materials and modified molecules are fixed by non-chemical bonds, the effect duration is limited, there is a risk of thrombosis, the modified molecules reduce the anticoagulant effect, and affect the mechanical properties of the valve.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing a bioprosthetic valve modifier, comprising the following steps: dissolving a diclofenac monomer containing a thioketal group, a hydrophilic monomer, a crosslinking agent, an active ester monomer and an initiator in a reaction solvent, reacting at 40 to 160 ° C under anaerobic conditions for 0.1 to 24 h, and polymerizing to form a bioprosthetic valve modifier;

[0006] The structural formula of the diclofenac monomer containing a thioketal group is shown in Formula III.

[0007] ;

[0008] The hydrophilic monomer is 2-methacryloyloxyethyl phosphorylcholine, the crosslinking agent is N,N'-methylenebisacrylamide, the active ester monomer is methacrylate N-hydroxysuccinimidyl ester, the initiator is azobisisobutyronitrile, and the reaction solvent is at least one of acetonitrile, water, ethanol, dimethyl sulfoxide and dioxane.

[0009] Furthermore, the diclofenac monomer containing the thioketal group is prepared by the following steps: dissolving the methacrylate containing the thioketal group, diclofenac, a dehydrating agent and a catalyst in dichloromethane, and reacting at room temperature for 6 to 72 hours to obtain;

[0010] The structural formula of the methacrylate containing thioketal group is shown in Formula I.

[0011] ;

[0012] The dehydrating agent is N,N-dicyclohexylcarbodiimide or diisopropylcarbodiimide, and the catalyst is 4-dimethylaminopyridine; the molar ratio of the methacrylate containing the thioketal group, diclofenac, the dehydrating agent and the catalyst is 1:0.2~5:1~3:0.01~0.1.

[0013] Furthermore, the reaction time of the preparation step of the diclofenac monomer containing a thioketal group is 24 h, the dehydrating agent is N,N-dicyclohexylcarbonyldiimide, and the molar ratio of the methacrylate containing a thioketal group, diclofenac, the dehydrating agent and the catalyst is 1:1:1.21:0.051.

[0014] Furthermore, the reaction temperature is 100 °C, the reaction time is 1 h; the mass ratio of 2-methacryloyloxyethyl phosphorylcholine, N,N'-methylenebisacrylamide, methacrylate N-hydroxysuccinic acid imide ester, diclofenac monomer containing thioketal group and azobisisobutyronitrile is 5~100:1~10:1~10:0~10:0.05~10.

[0015] Furthermore, the mass ratio of 2-methacryloyloxyethyl phosphorylcholine, N,N'-methylenebisacrylamide, methacrylate N-hydroxysuccinimidyl ester, diclofenac monomer containing thioketal group and azobisisobutyronitrile is 17:4:4:2.5:0.4.

[0016] Furthermore, a biovalve modifier is prepared by the above-mentioned preparation method of the biovalve modifier, and the biovalve modifier is a nanogel with an average particle size of 220 nm.

[0017] Furthermore, a method for preparing a polymer-modified bioprosthesis comprises the following steps:

[0018] A1. Soak the bioprosthetic valve material in glutaraldehyde solution and react at room temperature for 48-72 hours;

[0019] A2. Soak the bioprosthesis treated in step A1 in a solution of an amino cationic polymer for a reaction period of 0.1 to 72 hours; add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide 0.1 to 3 hours after the start of the soaking reaction;

[0020] A3. Soaking the biological valve treated in step A2 in the nanogel solution prepared by the above nanogel for 1 to 48 h;

[0021] A4. preparing a solution of the active ester compound, and then soaking the biological valve treated in step A3 with the obtained solution for 1 to 48 hours;

[0022] A5. Repeat step A4 once to obtain;

[0023] Alternatively, B1. Soak the bioprosthetic valve material in glutaraldehyde solution and react at room temperature for 48 to 72 hours;

[0024] B2. Soaking the bioprosthesis treated in step B1 in a solution of an amino cationic polymer for a reaction period of 0.1 to 72 h; adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide 0.1 to 3 h after the start of the soaking reaction;

[0025] B3. Soaking the biological valve treated in step B2 in the nanogel solution prepared by the above nanogel for 1 to 48 h;

[0026] B4. Soaking the bioprosthesis treated in step B3 in a solution prepared by an amino cationic polymer for 1 to 48 hours;

[0027] B5. Soaking the biological valve treated in step B4 in the nanogel solution prepared by the above nanogel for 1 to 48 h;

[0028] B6. preparing a solution of the active ester compound, and then soaking the biological valve treated in step B5 with the obtained solution for 1 to 48 hours;

[0029] B7. Repeat step B6 once to obtain;

[0030] The reaction temperature in steps A2-A4 and B2-B6 is 4-37°C and the reaction pH is 5-8;

[0031] The bioprosthetic valve material is decellularized bovine pericardium or porcine pericardium;

[0032] The concentration of glutaraldehyde solution is 0.05~8 wt%;

[0033] The amino cationic polymer is a branched polyethyleneimine, the molecular weight of which is 300-1,000,000 g / mol, and the concentration of the amino cationic polymer solution is 0.1-30 mg / mL;

[0034] The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide used is 0.05% to 0.3% of the amount of the amino substance in the amino cationic polymer;

[0035] The concentration of the nanogel solution was 0.1~20 mg / mL;

[0036] 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 0.1-10 mg / mL.

[0037] Furthermore, the amino cationic polymer is a branched polyethyleneimine with a molecular weight of 25000 g / mol; and the active ester compound is a branched acetic acid-N-succinimide ester.

[0038] Further, the concentration of the glutaraldehyde solution in steps A1 and B1 is 1 wt %, and the immersion time is 48 h;

[0039] The reaction temperature in steps A2-A4 and B2-B6 is 25°C;

[0040] In steps A2 and B2, the pH of the amino cationic polymer solution is 5.5, the solution concentration is 10 mg / mL, and the reaction time is 12 h; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added 0.5 h after the start of the immersion reaction, and the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 0.15% of the amount of amino substances in the amino cationic polymer;

[0041] In steps A3 and B3, the pH of the nanogel solution was 7, the solution concentration was 1 mg / mL, and the reaction time was 4 h;

[0042] In steps A4 and B6, the pH of the active ester compound solution is 7, the solution concentration is 1.5 mg / mL, and the reaction time is 3 h;

[0043] In step B4, the pH of the amino cationic polymer solution is 7, the solution concentration is 10 mg / mL, and the reaction time is 4 h;

[0044] In step B5, the pH of the nanogel solution was 7, the solution concentration was 1 mg / mL, and the reaction time was 12 h.

[0045] Furthermore, a polymer-modified biological valve is prepared by the above-mentioned method for preparing a polymer-modified biological valve.

[0046] The beneficial effects of the present invention are:

[0047] (1) The present invention uses chemical bonding to carry out amidation reaction between the amino groups on the surface of polyethyleneimine and the active ester bonds activated on the surface of the nanogel, thereby grafting the nanogel on the valve surface. This will not reduce the mechanical properties of the biological valve. Compared with biological valves modified by electrostatic action or physical filling action, the polymer-modified biological valve prepared by the present invention has a longer-lasting and more functional anti-decay ability, which is beneficial to prolonging the service life of the biological valve.

[0048] (2) The method for preparing a biological valve provided by the present invention can effectively reduce the residual aldehyde groups on the biological valve, reduce the calcification problem and biological toxicity problem of the biological valve.

[0049] (3) The present invention introduces thioketal groups and diclofenac on the surface of biological valves, which can respond to pathological environments with high reactive oxygen concentrations such as inflammation and play an anti-inflammatory role in reducing inflammatory cell infiltration and expression of inflammatory factors.

[0050] (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 the adhesion of platelets and plasma proteins, and reduce the risk of thrombosis and coagulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the graph showing the particle size change of nanogels under the stimulation of different concentrations of active oxygen;

[0052] Figure 2 This is the graph of the potential change of nanogel under the stimulation of different concentrations of active oxygen;

[0053] Figure 3 This is the conductivity change diagram of nanogel under the stimulation of different concentrations of active oxygen;

[0054] Figure 4 This is the graph showing the change of polydispersity index of nanogels under stimulation of different concentrations of reactive oxygen species;

[0055] Figure 5 The scanning electron micrographs of nanogels at different magnifications;

[0056] Figure 6 The scanning electron microscope images at different magnifications show the grafting of nanogels on the surface of biological valves;

[0057] Figure 7 This is a scanning electron microscope image of the bioprosthesis surface after nanogel grafting;

[0058] Figure 8 This is the energy spectrum of the bioprosthetic valve surface after nanogel grafting;

[0059] Fig. 9 This is a scanning electron microscope image of blood cell adhesion after the bioprosthesis was implanted into the rabbit's blood circulation;

[0060] Fig.10 The results of the toxicity test of nanogels on Hela cells were detected at 450 nm;

[0061] Fig.11 The toxicity test results of nanogels on Hela cells were detected at 490 nm;

[0062] Fig.12 The toxicity test results of nanogels on Hela cells were detected at 570 nm;

[0063] Fig.13 The toxicity test results of nanogel on Huvec cells were detected at 450 nm;

[0064] Fig.14 The toxicity test results of nanogels on Huvec cells were detected at 490 nm;

[0065] Fig.15 The toxicity test results of nanogels on Huvec cells were detected at 570 nm. DETAILED DESCRIPTION

[0066] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0067] Example 1

[0068] The synthesis of the compound of formula III, i.e., the diclofenac monomer containing the thioketal group, is as follows:

[0069]

[0070] The preparation method comprises the following steps:

[0071] Diclofenac (Formula II, 510 mg, 1.72 mmol), tert-butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (Formula I, 500 mg, 1.72 mmol), N,N-dicyclohexylcarbonyldiimide (420 mg, 2.08 mmol), 4-dimethylaminopyridine (10 mg, 0.088 mmol) were dissolved in 15 mL of dichloromethane and reacted at room temperature for 24 h. After the reaction, 1 mL of distilled water was added and stirred for 15 min, and then 200 mL of dichloromethane was added. The extraction was repeated 6 times with 20 mL of sodium chloride solution each time, and the lower dichloromethane solution was collected each time. After 6 extractions, the mixture was dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction. After being dried by rotary evaporator, it was dissolved with 2 mL of dichloromethane. An appropriate amount of silica gel powder was dissolved in pure petroleum ether solution and loaded onto a chromatography column of about 15 cm. After the sample was completely loaded, 200 mL of a mixed solution of petroleum ether: ethyl acetate in a ratio of 40:1, 20:1 and 10:1 was sequentially passed through the column to collect the target product, which was dried by a rotary evaporator and pumped dry by an oil pump to obtain 0.86 g of the compound of formula III, 3-((2-((3-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)propyl)thio)propan-2-yl)thio)propyl methacrylate, with a yield of 88.7%.

[0072] The hydrogen spectrum detection results of the compound of formula III are: 1H NMR (400 MHz, DMSO-d6) δ 7.37 (d, J =8.1 Hz, 2H), 7.09 – 7.00 (m, 2H), 6.95 – 6.86 (m, 2H), 6.69 (td, J = 7.4, 1.2Hz, 1H), 5.87 (dt, J = 1.9, 1.0 Hz, 1H), 5.60 (s, 2H), 5.50 (p, J = 1.6 Hz,1H), 3.98 (t, J = 6.3 Hz, 4H), 3.65 (s, 2H), 2.45 (dt, J = 19.4, 7.4 Hz, 4H),1.76 – 1.62 (m, 7H), 1.34 (s, 6H). The hydrogen spectrum showed that the structure of the compound was consistent with the expectation.

[0073] Example 2

[0074] The synthesis of the compound of formula III, i.e., the diclofenac monomer containing a thioketal group, has the same reaction formula as in Example 1, and the preparation method thereof comprises the following steps:

[0075] Diclofenac (Formula II, 102 mg, 0.344 mmol), tert-butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (Formula I, 500 mg, 1.72 mmol), N,N-dicyclohexylcarbonyldiimide (347 mg, 1.72 mmol), 4-dimethylaminopyridine (2 mg, 0.017 mmol) were dissolved in 15 mL of dichloromethane and reacted at room temperature for 6 h. After the reaction, 1 mL of distilled water was added and stirred for 15 min, and then 200 mL of dichloromethane was added. The extraction was repeated 6 times with 20 mL of sodium chloride solution each time, and the lower dichloromethane solution was collected each time. After 6 extractions, the mixture was dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction filtration. After being dried by rotary evaporator, it was dissolved with 2 mL of dichloromethane. An appropriate amount of silica gel powder was dissolved in pure petroleum ether solution and loaded onto a chromatography column of about 15 cm. After the sample was completely loaded, 200 mL of a mixed solution of petroleum ether: ethyl acetate in the ratio of 40:1, 20:1 and 10:1 was sequentially passed through the column to collect the target product, which was dried by a rotary evaporator and pumped dry by an oil pump to obtain 0.174 g of compound III with a yield of 90%.

[0076] The hydrogen spectrum detection results of the compound of formula III are: 1H NMR (400 MHz, DMSO-d6) δ 7.37 (d, J =8.1 Hz, 2H), 7.09 – 7.00 (m, 2H), 6.95 – 6.86 (m, 2H), 6.69 (td, J = 7.4, 1.2Hz, 1H), 5.87 (dt, J = 1.9, 1.0 Hz, 1H), 5.60 (s, 2H), 5.50 (p, J = 1.6 Hz,1H), 3.98 (t, J = 6.3 Hz, 4H), 3.65 (s, 2H), 2.45 (dt, J = 19.4, 7.4 Hz, 4H),1.76 – 1.62 (m, 7H), 1.34 (s, 6H). The hydrogen spectrum showed that the structure of the compound was consistent with the expectation.

[0077] Example 3

[0078] The synthesis of the compound of formula III, i.e., the diclofenac monomer containing a thioketal group, has the same reaction formula as in Example 1, and the preparation method thereof comprises the following steps:

[0079] Diclofenac (Formula II, 2550 mg, 8.6 mmol), tert-butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (Formula I, 500 mg, 1.72 mmol), N,N-dicyclohexylcarbonyldiimide (1042 mg, 5.16 mmol), 4-dimethylaminopyridine (20 mg, 0.172 mmol) were dissolved in 45 mL of dichloromethane and reacted at room temperature for 72 h. After the reaction, 3 mL of distilled water was added and stirred for 15 min, and then 600 mL of dichloromethane was added. The extraction was repeated 6 times with 60 mL of sodium chloride solution each time, and the lower dichloromethane solution was collected each time. After 6 extractions, the mixture was dehydrated with anhydrous sodium sulfate, and the filtrate was collected by suction filtration. After being dried by rotary evaporator, it was dissolved with 6 mL of dichloromethane. An appropriate amount of silica gel powder was dissolved in pure petroleum ether solution and loaded onto a chromatography column of about 15 cm. After complete loading, 200 mL of a mixed solution of petroleum ether: ethyl acetate in the ratio of 40:1, 20:1 and 10:1 was sequentially passed through the column to collect the target product, which was dried by a rotary evaporator and pumped dry by an oil pump to obtain 4.368 g of compound III with a yield of 91%.

[0080] The hydrogen spectrum detection results of the compound of formula III are: 1H NMR (400 MHz, DMSO-d6) δ 7.37 (d, J =8.1 Hz, 2H), 7.09 – 7.00 (m, 2H), 6.95 – 6.86 (m, 2H), 6.69 (td, J = 7.4, 1.2Hz, 1H), 5.87 (dt, J = 1.9, 1.0 Hz, 1H), 5.60 (s, 2H), 5.50 (p, J = 1.6 Hz,1H), 3.98 (t, J = 6.3 Hz, 4H), 3.65 (s, 2H), 2.45 (dt, J = 19.4, 7.4 Hz, 4H),1.76 – 1.62 (m, 7H), 1.34 (s, 6H). The hydrogen spectrum showed that the structure of the compound was consistent with the expectation.

[0081] Example 4

[0082] A biovalve modifier, namely nanogel, is prepared by the following steps:

[0083] 2-Methacryloyloxyethyl phosphorylcholine (170 mg, 0.55 mmol), N,N'-methylenebisacrylamide (40 mg, 0.25 mmol), methacrylate N-hydroxysuccinimidyl ester (40 mg, 0.20 mmol), 3-((2-((3-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)propyl)thio)propan-2-yl)thio)propyl methacrylate (Formula III, prepared in Example 1, 25 mg, 0.044 mmol) and azobisisobutyronitrile (4 mg, 0.024 mmol) were dissolved in 35 mL of acetonitrile, and ultrasonicated for 5 min to fully dissolve the reactants. Then, the mixture was added to the reaction bottle, and the mixture was reacted at 100 °C for 1 h under argon protection after 5 freeze-pump-thaw cycles. After the reaction was completed, the suspension was collected and centrifuged at 12000 rpm for 3 min. The solid precipitate was collected and ventilated for 20 min to obtain 230 mg of white powder solid.

[0084] Example 5

[0085] A biovalve modifier, namely nanogel, is prepared by the following steps:

[0086] 2-Methacryloyloxyethyl phosphorylcholine (150 mg, 0.49 mmol), N,N'-methylenebisacrylamide (30 mg, 0.19 mmol), methacrylic acid N-hydroxysuccinimidyl ester (30 mg, 0.15 mmol), 3-((2-((3-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)propyl)thio)propan-2-yl)thio)propyl methacrylate (Formula III, prepared by Example 2, 30 mg, 0.053 mmol) and azobisisobutyronitrile (1.5 mg, 0.009 mmol) were dissolved in 35 mL of dimethyl sulfoxide, and ultrasonicated for 5 min to fully dissolve the reactants. Then, the mixture was added to the reaction bottle, and the mixture was reacted at 100 °C for 1 h under argon protection after 5 freeze-pump-thaw cycles. After the reaction was completed, the suspension was collected and centrifuged at 12000 rpm for 3 min. The solid precipitate was collected and ventilated for 20 min to obtain 200 mg of white powder solid.

[0087] Example 6

[0088] A biovalve modifier, namely nanogel, is prepared by the following steps:

[0089] 2-Methacryloyloxyethyl phosphorylcholine (100 mg, 0.32 mmol), N,N'-methylenebisacrylamide (10 mg, 0.063 mmol), methacrylate N-hydroxysuccinimidyl ester (10 mg, 0.05 mmol), 3-((2-((3-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)propyl)thio)propan-2-yl)thio)propyl methacrylate (Formula III, prepared by Example 3, 10 mg, 0.018 mmol) and azobisisobutyronitrile (10 mg, 0.06 mmol) were dissolved in 35 mL of a mixed solution of ethanol and dioxane, and ultrasonicated for 5 min to fully dissolve the reactants. The mixture was then added to the reaction bottle, and the mixture was reacted at 100 °C for 1 h under argon protection after 5 freeze-pump-thaw cycles. After the reaction was completed, the suspension was collected and centrifuged at 12000 rpm for 3 min. The solid precipitate was collected and ventilated for 20 min to obtain 130 mg of white powder solid.

[0090] Example 7

[0091] A nanogel free of diclofenac anti-inflammatory monomers, the preparation method of which comprises the following steps:

[0092] 2-Methacryloyloxyethyl phosphorylcholine (170 mg, 0.55 mmol), N,N'-methylenebisacrylamide (40 mg, 0.25 mmol), methacrylic acid N-hydroxysuccinic acid imide ester (40 mg, 0.20 mmol), and azobisisobutyronitrile (4 mg, 0.024 mmol) were dissolved in 35 mL of acetonitrile, and ultrasonicated for 5 min to fully dissolve the reactants, and then added to the reaction bottle. Under argon protection, after 5 freeze-pump-thaw cycles, the reaction was carried out at 100 ° C for 1 h under argon protection. After the reaction, the suspension was collected, centrifuged at 12000 rpm for 3 min, the solid precipitate was collected, and ventilated for 20 min to obtain 200 mg of white powder solid.

[0093] Example 8

[0094] A polymer-modified bioprosthesis comprises the following steps:

[0095] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% sodium dodecyl sulfate (SDS) solution and shake it for 48 h, then soak it in a 1 wt% glutaraldehyde solution for 48 h, take it out and wash it with distilled water.

[0096] (2) At 25°C, the bovine pericardium treated in step (1) was immersed in a PBS solution containing 10 mg / mL branched polyethyleneimine and a pH of 5.5 for 12 h. The molecular weight of the branched polyethyleneimine was 25,000 g / mol. 0.5 h after the start of the reaction, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added. The amount of both was 0.05% of the amount of the amino substance in the branched polyethyleneimine. The samples were taken out and cleaned.

[0097] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared from the nanogel obtained in Example 4 at a concentration of 0.1 mg / mL and a pH of 7 at 25°C for 4 h, and then taken out and washed with distilled water.

[0098] (4) Soak the bovine pericardium treated in step (3) in a 1.5 mg / mL branched-chain acetic acid-N-succinimidyl ester solution at a pH of 7 at 25°C for 3 h.

[0099] (5) Repeat step (4) once, take out and rinse with distilled water.

[0100] Example 9

[0101] A polymer-modified bioprosthesis comprises the following steps:

[0102] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% SDS solution and shake it for 48 h, then soak it in a 0.05 wt% glutaraldehyde solution for 72 h, take it out and wash it with distilled water.

[0103] (2) Soak the bovine pericardium treated in step (1) in a PBS solution containing 30 mg / mL branched polyethyleneimine and pH 8 at 37°C for 1 h. The molecular weight of the branched polyethyleneimine is 1,000,000 g / mol. Add EDC and NHS 0.1 h after the start of the reaction. The amount of both is 0.3% of the amount of amino substances in the branched polyethyleneimine. Take out and wash clean.

[0104] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared from the nanogel obtained in Example 5 at a concentration of 20 mg / mL and a pH of 8 at 37°C for 1 h, and then taken out and washed with distilled water.

[0105] (4) Soak the bovine pericardium treated in step (3) in a 10 mg / mL polyethylene glycol succinimidyl ester solution at a pH of 8 at 37°C for 1 h.

[0106] (5) Repeat step (4) once, take out and rinse with distilled water.

[0107] Example 10

[0108] A polymer-modified bioprosthesis comprises the following steps:

[0109] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% SDS solution and shake it for 48 h, then soak it in an 8 wt% glutaraldehyde solution for 48 h, take it out and wash it with distilled water.

[0110] (2) The bovine pericardium treated in step (1) was immersed in a PBS solution containing 0.1 mg / mL branched polyethyleneimine and a pH of 7 for 72 h at 4 °C. The molecular weight of the branched polyethyleneimine was 300 g / mol. EDC and NHS were added 3 h after the reaction started. The amount of both was 0.3% of the amount of the amino substance in the branched polyethyleneimine. The membrane was taken out and cleaned.

[0111] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared from the nanogel obtained in Example 6 at a concentration of 10 mg / mL and a pH of 7 at 4°C for 48 h, and then taken out and washed with distilled water.

[0112] (4) Soak the bovine pericardium treated in step (3) in a 0.1 mg / mL branched-chain acetic acid-N-succinimidyl ester solution at a pH of 5 at 4°C for 48 h.

[0113] (5) Repeat step (4) once, take out and rinse with distilled water.

[0114] Embodiment 11

[0115] A polymer-modified bioprosthesis comprises the following steps:

[0116] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% SDS solution and shake it for 48 h, then soak it in a 4 wt% glutaraldehyde solution for 60 h, take it out and wash it with distilled water.

[0117] (2) The bovine pericardium treated in step (1) was immersed in a PBS solution containing 10 mg / mL branched polyethyleneimine and a pH of 5.5 at 25°C for 48 h. The molecular weight of the branched polyethyleneimine was 2500 g / mol. EDC and NHS were added 2 h after the reaction started. The amount of both was 0.15% of the amount of the amino substance in the branched polyethyleneimine. The membrane was taken out and cleaned.

[0118] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared from the nanogel obtained in Example 7 at a concentration of 1 mg / mL and a pH of 7 at 25°C for 24 h, and then taken out and washed with distilled water.

[0119] (4) Soak the bovine pericardium treated in step (3) in a 5 mg / mL polyethylene glycol succinimide ester solution at a pH of 7 at 25°C for 24 h.

[0120] (5) Repeat step (4) once, take out and rinse with distilled water.

[0121] Example 12

[0122] A polymer-modified bioprosthesis comprises the following steps:

[0123] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% SDS solution and shake it for 48 h, then soak it in a 1 wt% glutaraldehyde solution for 48 h, take it out and wash it with distilled water.

[0124] (2) At 25°C, the bovine pericardium treated in step (1) was immersed in a PBS solution containing 5 mg / mL branched polyethyleneimine and a pH of 5.5 for 12 h. The molecular weight of the branched polyethyleneimine was 2500 g / mol. 0.5 h after the start of the reaction, EDC and NHS were added. The amount of both was 0.05% of the amount of the amino substance in the branched polyethyleneimine. The solution was taken out and cleaned.

[0125] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared from the nanogel obtained in Example 4 at a concentration of 1 mg / mL and a pH of 7 at 25°C for 4 h, and then taken out and washed with distilled water.

[0126] (4) Soak the bovine pericardium treated in step (3) in a 1.5 mg / mL acetic acid-N-succinimidyl ester solution at a pH of 7 at 25°C for 3 h.

[0127] (5) Repeat step (4) once, take out and rinse with distilled water.

[0128] Example 13

[0129] A polymer-modified bioprosthesis comprises the following steps:

[0130] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% SDS solution and shake it for 48 h, then soak it in a 1 wt% glutaraldehyde solution for 48 h, take it out and wash it with distilled water.

[0131] (2) Soak the bovine pericardium treated in step (1) in a PBS solution containing 10 mg / mL branched polyethyleneimine and a pH of 5.5 at 25°C for 12 h. The molecular weight of branched polyethyleneimine is about 25,000 g / mol. Add EDC and NHS 0.5 h after the start of the reaction. The amount of both is 0.05% of the amount of amino substances in the branched polyethyleneimine. Take out and wash clean.

[0132] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared from the nanogel obtained in Example 4 at a concentration of 0.1 mg / mL and a pH of 7 at 25°C for 4 h, and then taken out and washed with distilled water.

[0133] (4) Soak the bovine pericardium treated in step (3) in a 10 mg / mL branched polyethyleneimine PBS solution at pH 7 for 4 h at 25°C. The molecular weight of the branched polyethyleneimine is about 25,000 g / mol. Take it out and wash it clean.

[0134] (5) The bovine pericardium treated in step (4) was immersed in a nanogel solution prepared from the nanogel obtained in Example 4 at a concentration of 0.1 mg / mL and a pH of 7 at 25°C for 12 h, and then taken out and cleaned.

[0135] (6) Soak the bovine pericardium treated in step (5) in a 1.5 mg / mL branched-chain acetic acid-N-succinimidyl ester solution at a pH of 7 at 25°C for 3 h.

[0136] (7) Repeat step (6) once, take out and rinse with distilled water.

[0137] Embodiment 14

[0138] A polymer-modified bioprosthesis comprises the following steps:

[0139] (1) Fresh bovine pericardium, remove connective tissue and clean it, then wash it with physiological saline. Soak it in a 5 wt% SDS solution and shake it for 48 h, then soak it in a 1 wt% glutaraldehyde solution for 48 h, take it out and wash it with distilled water.

[0140] (2) At 25°C, the bovine pericardium treated in step (1) was immersed in a PBS solution containing 10 mg / mL branched polyethyleneimine and pH 5.5 for 12 h. The molecular weight of branched polyethyleneimine was about 270,000 g / mol. EDC and NHS were added 0.5 h after the reaction started. The amount of both was 0.3% of the amount of amino substances in the branched polyethyleneimine. The samples were taken out and cleaned.

[0141] (3) The bovine pericardium treated in step (2) was immersed in a nanogel solution prepared by the nanogel obtained in Example 4 at a mass concentration of 1 mg / mL and a pH of 7 at 25°C for 4 h, and then taken out and washed with distilled water.

[0142] (4) Soak the bovine pericardium treated in step (3) in a PBS solution of branched polyethyleneimine with a mass concentration of 10 mg / mL and a pH of 7 for 4 h at 25°C. The molecular weight of the branched polyethyleneimine is about 270,000 g / mol. Take it out and wash it clean.

[0143] (5) The bovine pericardium treated in step (4) was immersed in a nanogel solution prepared from the nanogel obtained in Example 4 at a mass concentration of 1 mg / mL and a pH of 7 at 25°C for 12 h, and then taken out and cleaned.

[0144] (6) Soak the bovine pericardium treated in step (5) in a polyethylene glycol succinimide ester solution with a mass concentration of 1.5 mg / mL and a pH of 7 at 25°C for 3 h.

[0145] (7) Repeat step (6) once, take out and rinse with distilled water.

[0146] Comparative Example

[0147] A common glutaraldehyde cross-linked biological valve, the preparation method of which comprises the following steps:

[0148] (1) Fresh porcine pericardium was thoroughly rinsed with cold saline (4°C) to remove blood and impurities from its surface. The whole piece of pericardium was then placed directly in a mixed solution containing 0.15 wt% sodium dodecyl sulfate (SDS) and 0.05 wt% sodium deoxycholate and gently shaken in a 37°C water bath for 24 h to remove cellular components.

[0149] (2) After decellularization, the cells were rinsed three times with 0.9 wt% saline for 15 min each time to ensure that residual SDS and sodium deoxycholate were removed.

[0150] (3) Place the decellularized pericardium in a mold and clamp it to shape it, ensuring that its surface is flat. Let it stand at 4°C for 30 min.

[0151] (4) Place the formed pericardium in a 0.625% glutaraldehyde solution and gently shake it at room temperature for 12 hours to complete the cross-linking reaction and ensure uniform cross-linking. After cross-linking is completed, store the pericardium directly in a 0.625% glutaraldehyde solution at 4°C for later use.

[0152] Experimental example

[0153] The nanogels prepared in Examples 4 to 7 have similar properties, and the polymer-modified bioprosthetic valves prepared in Examples 8 to 14 have similar properties. Taking the nanogel prepared in Example 4 and the polymer-modified bioprosthetic valve prepared in Example 8 as examples, the performance of the nanogel and the polymer-modified bioprosthetic valve were tested respectively.

[0154] 1. The nanogel prepared in Example 4 was prepared into a 0.5 mg / mL aqueous solution and stimulated with active oxygen at concentrations of 10 μM, 100 μM, 500 μM, and 1000 μM, respectively. The particle size, potential, conductivity, and polydispersity index of the nanogel under different stimulation times were shown in the following table: Figures 1 to 4 As shown, the results show that the potential and particle size of the nanogels slightly increased after adding different concentrations of active oxygen species for 1 h and 6 h. Overall, there was no obvious change in particle size and potential, and the conductivity and polydispersity index remained at a low level.

[0155] 2. The nanogel prepared in Example 4 was prepared into a 0.5 mg / mL aqueous solution, and the scanning electron microscope images at magnifications of 5000, 10000, 100000, and 250000 were as follows: Figure 5 As shown, the results show that the nanogels are uniform spherical in aqueous solution and the average particle size of the nanoparticles is about 220 nm.

[0156] 3. The grafting of nanogel on the surface of the polymer-modified bioprosthesis prepared in Example 8 was detected by scanning electron microscopy with magnifications of 5000, 10000, 50000, and 100000 times, respectively. Figure 6 As shown, the nanoparticles are grafted onto the membrane surface in large quantities and evenly. Figure 7 This is a scanning electron microscope image of the bioprosthesis surface after nanogel grafting. Figure 7Spectrum Figure 3 That is Figure 8 The energy spectrum in the figure, where At% represents atomic percentage, C is 58.9%, O is 24.5%, N is 13.1%, Cl is 1.9%, P is 1.6%, and S is 0.0%; where Cl is 1.9%, indicating that diclofenac has been grafted on the surface of the bioprosthesis, and S is 0.0%, indicating that the thioketal group has been responded to by ROS; Figure 7 and Figure 8 The results showed that the amino groups on the surface of polyethyleneimine and the active ester bonds activated on the surface of the nanogels underwent amidation reaction, and the nanogels were grafted onto the valve surface by chemical bonding.

[0157] 4. The SEM image of blood cell adhesion after the polymer modified bioprosthesis prepared in Example 8 was implanted into the rabbit blood circulation is as follows: Fig. 9 As shown, the magnifications are 4000 times and 8000 times respectively. Compared with the ordinary glutaraldehyde cross-linked biological valve prepared in the comparative example, the red blood cells and immune cells adhered to the surface of the membrane treated with nanogel are significantly reduced after implantation into the rabbit blood circulation, and the nanogel grafting on the membrane surface is stable.

[0158] 5. The nanogel prepared in Example 4 was prepared into solutions with concentrations of 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM, respectively, and Hela cells and Huvec cells were treated. The cell viability was detected by MTT reagent. To prevent the material from having self-absorption affecting the MTT results, the detection was performed at wavelengths of 450 nm, 490 nm, and 570 nm, respectively. The cytotoxicity test results of the nanogel are shown in FIG. Figures 10 to 15 As shown, the results showed that there was no significant change in cell viability, indicating that the nanogel had good biocompatibility.

[0159] 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 bioprosthetic valve modifier, characterized in that: The following steps are involved: Dissolving a diclofenac monomer containing a thioketal group, a hydrophilic monomer, a crosslinking agent, an active ester monomer and an initiator in a reaction solvent, reacting at 40 to 160° C. under anaerobic conditions for 0.1 to 24 h, and polymerizing to form the bioprosthetic valve modifier; The structural formula of the diclofenac monomer containing a thioketal group is shown in Formula III: ； The hydrophilic monomer is 2-methacryloyloxyethyl phosphorylcholine, the crosslinking agent is N,N'-methylenebisacrylamide, the active ester monomer is methacrylate N-hydroxysuccinimidyl ester, the initiator is azobisisobutyronitrile, and the reaction solvent is at least one of acetonitrile, water, ethanol, dimethyl sulfoxide and dioxane.

2. The method for preparing the biovalve modifier according to claim 1, characterized in that: The diclofenac monomer containing a thioketal group is prepared by the following steps: dissolving a methacrylate containing a thioketal group, diclofenac, a dehydrating agent and a catalyst in dichloromethane, and reacting at room temperature for 6 to 72 hours to obtain the monomer; The structural formula of the methacrylate containing a thioketal group is shown in Formula I. ； The dehydrating agent is N,N-dicyclohexylcarbodiimide or diisopropylcarbodiimide, and the catalyst is 4-dimethylaminopyridine; the molar ratio of the methacrylate containing the thioketal group, diclofenac, the dehydrating agent and the catalyst is 1:0.2~5:1~3:0.01~0.

1.

3. The method for preparing the biovalve modifier according to claim 2, characterized in that: The reaction time of the preparation step of the diclofenac monomer containing a thioketal group is 24 h, the dehydrating agent is N,N-dicyclohexylcarbonyldiimide, and the molar ratio of the methacrylate containing a thioketal group, diclofenac, the dehydrating agent and the catalyst is 1:1:1.21:0.

051.

4. The method for preparing the biovalve modifier according to claim 1, characterized in that: The reaction temperature is 100° C., and the reaction time is 1 h. The mass ratio of the 2-methacryloyloxyethyl phosphorylcholine, N,N'-methylenebisacrylamide, methacrylate N-hydroxysuccinic acid imide ester, diclofenac monomer containing a thioketal group, and azobisisobutyronitrile is 5-100:1-10:1-10:0-10:0.05-10.

5. The method for preparing the bioprosthetic valve modifier according to claim 4, characterized in that: The mass ratio of the 2-methacryloyloxyethyl phosphorylcholine, N,N'-methylenebisacrylamide, methacrylate N-hydroxysuccinic acid imide ester, diclofenac monomer containing thioketal group and azobisisobutyronitrile is 17:4:4:2.5:0.

4.

6. A bioprosthetic valve modifier, characterized in that: It is prepared by the preparation method of the biological valve modifier according to any one of claims 1 to 5; the biological valve modifier is a nanogel with an average particle size of 220 nm.

7. A method for preparing a polymer-modified biological valve, characterized in that: The following steps are involved: A1. Soak the bioprosthetic valve material in glutaraldehyde solution and react at room temperature for 48-72 hours; A2. Soak the bioprosthesis treated in step A1 in a solution of an amino cationic polymer for a reaction period of 0.1 to 72 hours; add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide 0.1 to 3 hours after the start of the soaking reaction; A3. Soaking the biological valve treated in step A2 in a nanogel solution prepared by the nanogel according to claim 6 for 1 to 48 h; A4. Prepare a solution of the active ester compound, and then soak the biological valve treated in step A3 with the resulting solution for 1 to 48 hours; A5. Repeat step A4 once to obtain; Alternatively, B1. Soak the bioprosthetic valve material in glutaraldehyde solution and react at room temperature for 48 to 72 hours; B2. Soaking the bioprosthesis treated in step B1 in a solution of an amino cationic polymer for a reaction period of 0.1 to 72 h; adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide 0.1 to 3 h after the start of the soaking reaction; B3. Soaking the biological valve treated in step B2 in a nanogel solution prepared from the nanogel according to claim 6 for 1 to 48 h; B4. Soaking the bioprosthesis treated in step B3 in a solution of an amino cationic polymer for 1 to 48 hours; B5. Soaking the biological valve treated in step B4 in a nanogel solution prepared from the nanogel according to claim 6 for 1 to 48 h; B6. preparing a solution of the active ester compound, and then soaking the biological valve treated in step B5 with the resulting solution for 1 to 48 hours; B7. Repeat step B6 once to obtain; The reaction temperature in steps A2-A4 and B2-B6 is 4-37°C and the reaction pH is 5-8; The biological valve material is decellularized bovine pericardium or porcine pericardium; The concentration of the glutaraldehyde solution is 0.05-8 wt %; The amino cationic polymer is a branched polyethyleneimine, the molecular weight of which is 300-1,000,000 g / mol, and the concentration of the amino cationic polymer solution is 0.1-30 mg / mL; The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide used is 0.05% to 0.3% of the amount of amino substances in the amino cationic polymer; The concentration of the nanogel solution is 0.1-20 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 0.1-10 mg / mL.

8. The method for preparing a polymer-modified bioprosthesis according to claim 7, characterized in that: The amino cationic polymer is a branched polyethyleneimine with a molecular weight of 25000 g / mol; and the active ester compound is a branched acetic acid-N-succinimide ester.

9. The method for preparing a polymer-modified bioprosthesis according to claim 7, characterized in that: The concentration of the glutaraldehyde solution in steps A1 and B1 is 1 wt %, and the soaking time is 48 h; The reaction temperature in steps A2-A4 and B2-B6 is 25°C; In the steps A2 and B2, the pH of the amino cationic polymer solution is 5.5, the solution concentration is 10 mg / mL, and the reaction time is 12 h; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added 0.5 h after the start of the immersion reaction, and the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide used is 0.15% of the amount of amino substances in the amino cationic polymer; The pH of the nanogel solution in steps A3 and B3 is 7, the solution concentration is 1 mg / mL, and the reaction time is 4 h; The pH of the active ester compound solution in steps A4 and B6 is 7, the solution concentration is 1.5 mg / mL, and the reaction time is 3 h; In step B4, the pH of the amino cationic polymer solution is 7, the solution concentration is 10 mg / mL, and the reaction time is 4 h; The pH of the nanogel solution in step B5 is 7, the solution concentration is 1 mg / mL, and the reaction time is 12 h.

10. A polymer-modified bioprosthesis, characterized in that: Prepared by the preparation method according to any one of claims 7 to 9.

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