Preparation method of antithrombotic small-caliber artificial blood vessel with double-layer structure

Abstract

The invention relates to the technical field of artificial blood vessels, in particular to a preparation method of an anti-thrombus small-caliber artificial blood vessel with a double-layer structure, which comprises the following steps: step 1, synthesizing phosphorylcholine-containing dihydric alcohol MPCDL with an anti-biofilm adsorption effect; 2, preparing different types of polycarbonate polyurethane PCU-MPC containing phosphorylcholine groups by regulating and controlling the molar ratio of the MPCDL synthesized in the step 1 to 1, 4-butanediol, and adding the PCU-MPC into the inner layer of the small-caliber artificial blood vessel; step 3, preparing a PCU-MPC / fibroin blend synthesized in the step 2 by regulating and controlling the proportion of the water-insoluble fibroin powder; and 4, preparing the small-caliber artificial blood vessel with a double-layer structure, which takes PCU as an outer layer and takes the PCU-MPC / fibroin blend prepared in the step 3 as an inner layer, by utilizing an electrostatic spinning technology, so as to maximize the antithrombotic effect of the small-caliber artificial blood vessel. The antithrombotic small-caliber artificial blood vessel prepared by the invention has excellent biocompatibility, protein adsorption resistance, antithrombotic performance, high strength and high elasticity, and can be used for repairing or replacing lesion blood vessels.

Description

Technical Field

[0001] The invention relates to the technical field of artificial blood vessels, and in particular to a method for preparing a double-layer structured anti-thrombotic small-caliber artificial blood vessel. Background Art

[0002] Cardiovascular disease has become the leading cause of death in the world, and vascular disease is one of the important causes of cardiovascular disease. At present, the main treatment method in clinical practice besides drugs is vascular transplantation. Although autologous vascular transplantation has become the first choice due to its excellent characteristics, its application is limited due to factors such as insufficient sources, size mismatch and patient health status.

[0003] In recent years, the application of artificial blood vessels has been very extensive. The clinical research results of large and medium caliber artificial blood vessels have been relatively ideal. However, small caliber artificial blood vessels are more likely to induce thrombosis during use, lack in situ endothelialization, and lead to intimal hyperplasia, which in turn affects their patency rate in long-term use after transplantation.

[0004] In view of the above problems, it is necessary for the present application to propose a method for preparing a double-layer structured anti-thrombotic small-caliber artificial blood vessel. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for preparing a double-layer structured anti-thrombotic small-caliber artificial blood vessel. The prepared double-layer structured anti-thrombotic small-caliber artificial blood vessel has excellent biocompatibility, anti-protein adsorption performance, anti-thrombotic performance, high strength and high elasticity, can be used to repair or replace damaged blood vessels, and has good application prospects in clinical medicine.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel, wherein the double-layer anti-thrombotic small-caliber artificial blood vessel comprises an outer layer of polycarbonate polyurethane (PCU) and an inner layer of a polycarbonate polyurethane (PCU)-MPC / silk fibroin blend containing phosphorylcholine groups;

[0008] The method for preparing the double-layer anti-thrombotic small-caliber artificial blood vessel comprises the following steps:

[0009] Step 1, synthesizing phosphorylcholine diol (MPCDL) with anti-biofilm adsorption effect;

[0010] Step 2, preparing different types of polycarbonate polyurethane containing phosphorylcholine groups (PCU-MPC) by adjusting the molar ratio of MPCDL synthesized in step 1 to 1,4-butanediol, and adding them to the inner layer of a small-caliber artificial blood vessel;

[0011] Step 3, preparing the PCU-MPC / silk fibroin blend synthesized in step 2 by adjusting the proportion of water-insoluble silk fibroin powder;

[0012] Step 4: Using electrospinning technology, prepare a double-layer structure with a small-caliber artificial blood vessel, with PCU as the outer layer and the PCU-MPC / silk fibroin blend prepared in step 3 as the inner layer, to maximize its anti-thrombotic effect.

[0013] Preferably, in step 1, the mass fractions of 2-methacryloyloxyethyl phosphorylcholine and 3-mercapto-1,2-propylene glycol added during the synthesis of MPCDL are 0.01-0.03 mol and 0.01-0.03 mol, respectively.

[0014] Preferably, in step 1, the volume ratio of methanol / water eluent during synthesis is 1:5 to 2:5, the light-proof drying temperature is 100° C., the light-proof drying time is 24 to 48 hours, and the light-proof reaction time is 24 to 48 hours.

[0015] Preferably, in step 2, the molar masses of polycarbonate diol and 4,4'-diisocyanate diphenylmethane used in preparing PCU-MPC are 0.005-0.01 mol and 0.01-0.02 mol, respectively, the reaction temperature is 60-70° C., and the reaction time is 1-3 hours.

[0016] Preferably, in step 2, the molar ratio of the phosphorylcholine-containing diol to 1,4-butanediol is 0:3 to 2:3.

[0017] Preferably, in step 2, the catalyst used in the process of preparing PCU-MPC is 2-ethyltin hexanoate, and the reaction time after adding the catalyst is 5 to 7 hours; the vacuum drying time for preparing the obtained PCU-MPC viscous liquid is 48 to 72 hours.

[0018] Preferably, in step 3, the average diameter of the silk fibroin powder is 2-3 um.

[0019] Preferably, in step 3, the mass fraction of the water-insoluble silk fibroin powder is 0 to 30 wt %.

[0020] Preferably, in step 4, the concentration of the PCU-MPC / silk fibroin blend solution or PCU solution is 60-100 mg / ml, and electrospinning is carried out under the temperature and humidity conditions of 25-27°C and 45-50% humidity, the spinning voltage is set to 15-25 kV, the receiving distance is 10-25 cm, and the spinning rate is 0.8-1.5 ml / h for spinning.

[0021] Preferably, in step 4, a small-caliber artificial blood vessel with a diameter of 3 to 6 mm is prepared using a roller receiver with a diameter of 3 to 6 mm.

[0022] By adopting the above technical scheme: through double-layer structure design, using polycarbonate polyurethane containing phosphorylcholine group (PCU-MPC), and adjusting the mass fraction of non-water-soluble silk powder to prepare different types of PCU-MPC / silk blends, and finally using electrospinning technology to prepare double-layer structure small-caliber artificial blood vessels with polycarbonate polyurethane as the outer layer and PCU-MPC / silk blend as the inner layer, the anti-thrombotic effect is achieved.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention prepares different types of polycarbonate polyurethane containing phosphorylcholine groups (PCU-MPC), which are added to the inner layer of small-caliber artificial blood vessels to achieve anti-thrombotic effects.

[0025] 2. The present invention prepares different types of PCU-MPC / silk blends by adjusting the mass fraction of water-insoluble silk powder, and utilizes the coordinated effect of phosphorylcholine groups and silk to more effectively prevent thrombosis.

[0026] 3. The present invention adopts electrospinning technology to prepare a double-layer structure small-caliber artificial blood vessel with PCU as the outer layer and PCU-MPC / silk fibroin blend as the inner layer, so as to maximize its anti-thrombotic effect. The anti-thrombotic small-caliber artificial blood vessel prepared by the present invention has excellent biocompatibility, anti-protein adsorption performance, anti-thrombotic performance, high strength and high elasticity, can be used to repair or replace damaged blood vessels, and has good application prospects in clinical medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the preparation of a double-layer anti-thrombotic small-caliber artificial blood vessel of the present invention;

[0028] Figure 2 This is a schematic diagram of a double-layer anti-thrombotic small-caliber artificial blood vessel prepared by the present invention;

[0029] In the figure: 1. Outer layer of polycarbonate polyurethane (PCU); 2. Inner layer of polycarbonate polyurethane containing phosphorylcholine groups (PCU-MPC) / silk fibroin blend. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] A method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel, the specific steps are as follows:

[0033] Step 1, synthesis of phosphorylcholine diol (MPCDL): place 0.01 mol of 2-methacryloyloxyethyl phosphorylcholine in a round-bottom double-necked flask, evacuate the reaction device and replace the nitrogen three times, then add 0.01 mol of 3-mercapto-1,2-propanediol and 30 mg of diisopropylamine to the reaction device. Subsequently, add 10 mL of dimethyl sulfoxide to the reaction device, react at room temperature in the dark for 24 hours, add the reaction solution dropwise into anhydrous ether and precipitate three times, and then use methanol / water with a volume ratio of 1:5 as an eluent to obtain a colorless viscous liquid. Finally, dry the obtained viscous liquid product in a vacuum drying oven at 100°C for 24 hours;

[0034] Step 2, synthesis of polycarbonate polyurethane containing phosphorylcholine group (PCU-MPC): 0.005 mol of polycarbonate diol was added to a clean and dry three-necked flask, and placed in an 80°C oil bath to remove water by decompression and stirring. After the device was cooled to room temperature, it was evacuated and replaced with nitrogen three times. When the temperature of the reaction device rose to 60°C, 0.01 mol of 4,4'-diphenylmethane diisocyanate (MDI) was quickly added to the reaction flask, and dimethyl sulfoxide was added and stirred for 2 hours. Subsequently, the MPCDL synthesized in step 1 and 1,4-butanediol were dissolved in dimethyl sulfoxide at a molar ratio of 1:3 and added to the reaction system. After the addition was completed, 2% of 2-ethylhexanoic acid tin was added as a catalyst, and the reaction was continued at 60°C for 6 hours. Finally, the final product was precipitated in methanol, filtered to obtain a white solid product, and dried in a vacuum drying oven for 72 hours.

[0035] Step 3: Grind the silk fibroin powder to an average diameter of about 2 to 3 μm, and blend it with PCU-MPC to make the mass fraction of PCU-MPC / silk fibroin be 7:3.

[0036] Step 4, preparation of double-layer structure small-caliber artificial blood vessels: add the PCU-MPC / silk fibroin blend obtained in step 3 to dimethylformamide solvent to prepare inner spinning solution with a concentration of 80 mg / ml, dissolve polycarbonate polyurethane in dimethylformamide to prepare outer spinning solution with a solution concentration of 80 mg / ml, and perform electrospinning at an ambient temperature of 25-27°C and a humidity of 45-50%. Set the spinning voltage to 15 kV, the receiving distance to 15 cm, the inner layer spinning rate to 0.8 ml / h, and the core layer spinning rate to 1.0 ml / h for spinning; use a roller receiver with a diameter of 3-6 mm to prepare an artificial blood vessel with a diameter of 3-6 mm.

[0037] Embodiment 2:

[0038] A method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel, the specific steps are as follows:

[0039] Step 1, synthesis of phosphorylcholine diol (MPCDL): place 0.01 mol of 2-methacryloyloxyethyl phosphorylcholine in a round-bottom double-necked flask, evacuate the reaction device and replace the nitrogen three times, then add 0.01 mol of 3-mercapto-1,2-propanediol and 30 mg of diisopropylamine to the reaction device. Subsequently, add 10 mL of dimethyl sulfoxide to the reaction device, react for 48 hours at room temperature in the dark, add the reaction solution dropwise into anhydrous ether and precipitate three times, and then use methanol / water with a volume ratio of 1:5 as an eluent to obtain a colorless viscous liquid. Finally, dry the obtained viscous liquid product in a vacuum drying oven at 100°C for 24 hours;

[0040] Step 2, synthesis of polycarbonate polyurethane containing phosphorylcholine group (PCU-MPC): 0.005 mol of polycarbonate diol was added to a clean and dry three-necked flask, and placed in an 80°C oil bath to remove water by decompression and stirring. After the device was cooled to room temperature, it was evacuated and replaced with nitrogen three times. When the temperature of the reaction device rose to 60°C, 0.01 mol of 4,4'-diphenylmethane diisocyanate (MDI) was quickly added to the reaction flask, and dimethyl sulfoxide was added and stirred for 2 hours. Subsequently, the MPCDL synthesized in step 1 and 1,4-butanediol were dissolved in dimethyl sulfoxide at a molar ratio of 1:3 and added to the reaction system. After the addition was completed, 2% of 2-ethylhexanoic acid tin was added as a catalyst, and the reaction was continued at 60°C for 6 hours. Finally, the final product was precipitated in methanol, filtered to obtain a white solid product, and dried in a vacuum drying oven for 72 hours.

[0041] Step 3: Grind the silk fibroin powder to an average diameter of about 2 to 3 μm, and blend it with PCU-MPC to make the mass fraction of PCU-MPC / silk fibroin be 7:3.

[0042] Step 4, preparation of double-layer structure small-caliber artificial blood vessels: add the PCU-MPC / silk fibroin blend obtained in step 3 to dimethylformamide solvent to prepare inner spinning solution with a concentration of 80 mg / ml, dissolve polycarbonate polyurethane in dimethylformamide to prepare outer spinning solution with a solution concentration of 80 mg / ml, and perform electrospinning at an ambient temperature of 25-27°C and a humidity of 45-50%. Set the spinning voltage to 15 kV, the receiving distance to 15 cm, the inner layer spinning rate to 0.8 ml / h, and the core layer spinning rate to 1.0 ml / h for spinning; use a roller receiver with a diameter of 3-6 mm to prepare an artificial blood vessel with a diameter of 3-6 mm.

[0043] Embodiment 3:

[0044] A method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel, the specific steps are as follows:

[0045] Step 1, synthesis of phosphorylcholine diol (MPCDL): place 0.01 mol of 2-methacryloyloxyethyl phosphorylcholine in a round-bottom double-necked flask, evacuate the reaction device and replace the nitrogen three times, then add 0.01 mol of 3-mercapto-1,2-propanediol and 30 mg of diisopropylamine to the reaction device. Subsequently, add 10 mL of dimethyl sulfoxide to the reaction device, react for 48 hours at room temperature in the dark, add the reaction solution dropwise into anhydrous ether and precipitate three times, and then use methanol / water with a volume ratio of 1:5 as an eluent to obtain a colorless viscous liquid. Finally, dry the obtained viscous liquid product in a vacuum drying oven at 100°C for 24 hours;

[0046] Step 2, synthesis of polycarbonate polyurethane containing phosphorylcholine group (PCU-MPC): 0.01mol of polycarbonate diol was added to a clean and dry three-necked flask, and placed in an 80℃ oil bath to remove water by decompression and stirring. After the device was cooled to room temperature, it was evacuated and replaced with nitrogen three times. When the temperature of the reaction device rose to 60℃, 0.01mol of 4,4'-diphenylmethane diisocyanate (MDI) was quickly added to the reaction flask, and dimethyl sulfoxide was added and stirred for 2 hours. Subsequently, the MPCDL and 1,4-butanediol synthesized in step 1 were dissolved in dimethyl sulfoxide at a molar ratio of 1:3 and added to the reaction system. After the addition was completed, 2% of 2-ethylhexanoic acid tin was added as a catalyst, and the reaction was continued at 60℃ for 6 hours. Finally, the final product was precipitated in methanol, filtered to obtain a white solid product, and dried in a vacuum drying oven for 72 hours.

[0047] Step 3: Grind the silk fibroin powder to an average diameter of about 2 to 3 μm, and blend it with PCU-MPC to make the mass fraction of PCU-MPC / silk fibroin be 7:3.

[0048] Step 4, preparation of double-layer structure small-caliber artificial blood vessels: add the PCU-MPC / silk fibroin blend obtained in step 3 to dimethylformamide solvent to prepare inner spinning solution with a concentration of 100 mg / ml, dissolve polycarbonate polyurethane in dimethylformamide to prepare outer spinning solution with a solution concentration of 100 mg / ml, and perform electrospinning at an ambient temperature of 25-27°C and a humidity of 45-50%. Set the spinning voltage to 15 kV, the receiving distance to 15 cm, the inner layer spinning rate to 0.8 ml / h, and the core layer spinning rate to 1.0 ml / h for spinning; use a roller receiver with a diameter of 3-6 mm to prepare an artificial blood vessel with a diameter of 3-6 mm.

[0049] Control group:

[0050] A method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel, the specific steps are as follows:

[0051] Step 1. Preparation of a small-caliber double-layer artificial blood vessel: Add polycarbonate polyurethane (PCU) to dimethylformamide solvent to prepare inner and outer layer spinning solutions with a concentration of 80 mg / ml. Perform electrospinning at an ambient temperature of 25-27°C and a humidity of 45-50%. Set the spinning voltage to 15 kV, the receiving distance to 15 cm, the inner layer spinning rate to 0.8 ml / h, and the core layer spinning rate to 1.0 ml / h for spinning. Use a roller receiver with a diameter of 3-6 mm to prepare an artificial blood vessel with a diameter of 3-6 mm.

[0052] Performance testing:

[0053] Nuclear magnetic resonance experiments were performed on the polycarbonate polyurethane containing phosphorylcholine groups obtained in the above examples, and mechanical property tests, cytotoxicity tests, anti-protein adsorption tests, anti-platelet adsorption tests, etc. were performed on the small-caliber artificial blood vessels prepared according to the examples.

[0054] The test results show that compared with the control group, the small-caliber artificial blood vessels made of polycarbonate polyurethane / silk fibroin blend containing phosphorylcholine groups prepared by the embodiment of the present invention have significant effects in inhibiting protein adsorption, platelet adhesion and activation, indicating that by grafting phosphorylcholine groups on polycarbonate polyurethane, it can work synergistically with silk fibroin to effectively prevent thrombosis, and can be applied to fields such as small-caliber vascular stents.

[0055] In summary, the double-layer anti-thrombotic small-caliber artificial blood vessel prepared by the present invention has excellent biocompatibility, anti-protein adsorption performance, anti-thrombotic performance, high strength and high elasticity, can be used to repair or replace damaged blood vessels, and has good application prospects in clinical medicine.

[0056] The description and practice disclosed in the present invention are easy to think and understand for ordinary technicians in the technical field, and several improvements and modifications can be made without departing from the principles of the present invention. Therefore, modifications or improvements made without departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel, characterized in that: The double-layer anti-thrombotic small-caliber artificial blood vessel comprises a polycarbonate polyurethane PCU outer layer (1) and a polycarbonate polyurethane PCU-MPC / silk fibroin blend inner layer (2) containing phosphorylcholine groups; The method for preparing the double-layer anti-thrombotic small-caliber artificial blood vessel comprises the following steps: Step 1, synthesizing phosphorylcholine diol MPCDL with anti-biofilm adsorption effect; Step 2, preparing different types of polycarbonate polyurethane PCU-MPC containing phosphorylcholine groups by adjusting the molar ratio of MPCDL synthesized in step 1 and 1,4-butanediol, and adding them to the inner layer of the small-caliber artificial blood vessel; Step 3, preparing the PCU-MPC / silk fibroin blend synthesized in step 2 by adjusting the proportion of water-insoluble silk fibroin powder; Step 4: Using electrospinning technology, prepare a double-layer structure with a small-caliber artificial blood vessel, with PCU as the outer layer and the PCU-MPC / silk fibroin blend prepared in step 3 as the inner layer, to maximize its anti-thrombotic effect.

2. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 1, the mass fractions of 2-methacryloyloxyethyl phosphorylcholine and 3-mercapto-1,2-propylene glycol added during the synthesis of MPCDL are 0.01-0.03 mol and 0.01-0.03 mol, respectively.

3. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 1, during the synthesis, the volume ratio of methanol / water eluent is 1:5 to 2:5, the light-proof drying temperature is 100° C., the light-proof drying time is 24 to 48 hours, and the light-proof reaction time is 24 to 48 hours.

4. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 2, the molar masses of polycarbonate diol and 4,4'-diisocyanate diphenylmethane used in preparing PCU-MPC are 0.005-0.01 mol and 0.01-0.02 mol, respectively, the reaction temperature is 60-70° C., and the reaction time is 1-3 hours.

5. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 2, the molar ratio of the phosphorylcholine-containing diol to 1,4-butanediol is 0:3 to 2:

3.

6. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 2, the catalyst used in the process of preparing PCU-MPC is 2-ethyltin hexanoate, and the reaction time after adding the catalyst is 5 to 7 hours; the vacuum drying time of the prepared PCU-MPC viscous liquid is 48 to 72 hours.

7. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 3, the average diameter of the silk fibroin powder is 2-3 um.

8. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 3, the mass fraction of the water-insoluble silk fibroin powder is 0 to 30 wt %.

9. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 4, the concentration of PCU-MPC / silk fibroin blend solution or PCU solution is 60-100 mg / ml, and electrospinning is carried out under the temperature and humidity conditions of 25-27°C and 45-50% humidity. The spinning voltage is set to 15-25 kV, the receiving distance is 10-25 cm, and the spinning rate is 0.8-1.5 ml / h for spinning.

10. The method for preparing a double-layer anti-thrombotic small-caliber artificial blood vessel according to claim 1, characterized in that: In step 4, a small-caliber artificial blood vessel with a diameter of 3 to 6 mm is prepared using a roller receiver with a diameter of 3 to 6 mm.

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