Small-caliber artificial blood vessel with composite modified double-layer structure and preparation method of small-caliber artificial blood vessel

Abstract

The invention belongs to the technical field of artificial blood vessels, and provides a composite modified double-layer structure small-caliber artificial blood vessel which comprises an inner layer and an outer layer, the inner layer comprises a mixture of zwitterionic polyurethane and polycaprolactone, the outer layer comprises polycaprolactone, zwitterionic polyurethane contains zwitterionic sulfonic acid negative ions and ammonium positive ions, and the zwitterionic sulfonic acid negative ions and the ammonium positive ions are different. Inner and outer layers of the artificial blood vessel are modified by heparin; the invention further provides a preparation method of the artificial blood vessel, the double-layer artificial blood vessel with a zwitterionic polyurethane and polycaprolactone blend as an inner layer and polycaprolactone as an outer layer is prepared through electrostatic spinning, and heparin is used for surface modification of the double-layer artificial blood vessel. The size of the small-caliber artificial blood vessel can be effectively controlled, and the prepared small-caliber antithrombotic artificial blood vessel has excellent biocompatibility, tensile property, protein adsorption resistance and antithrombotic property and can be used for vascular tissue repair or replacement.

Description

Technical Field

[0001] The invention relates to the technical field of artificial blood vessels, and in particular to a double-layer structured small-caliber artificial blood vessel compositely modified with zwitterions and heparin and a preparation method thereof. Background Art

[0002] As human life expectancy increases, the incidence of cardiovascular disease is also gradually increasing. It has become one of the most common diseases in the world today. The development of artificial blood vessels with good anti-thrombotic properties has become one of the important ways to solve the problem of cardiovascular disease. Traditional artificial blood vessels are prone to thrombosis, leading to vascular obstruction and increasing the risk of secondary surgery for patients. The double-layer structure small-caliber artificial blood vessels have higher mechanical properties and biocompatibility. Introducing heparin modification into the double-layer structure small-caliber artificial blood vessels can not only maintain the original advantages of artificial blood vessels, but also improve their anti-thrombotic properties by reducing platelet adsorption. However, protein adsorption in the blood can also cause thrombosis and other vascular obstructions. It is still necessary to explore optimized solutions in the design of artificial blood vessels. Summary of the invention

[0003] In view of the problems in the prior art, the present invention designs a composite modified double-layer structure small-caliber artificial blood vessel, by introducing zwitterionic polyurethane into the double-layer artificial blood vessel structure and modifying the surface of the artificial blood vessel with heparin, thereby further improving the anti-thrombotic effect.

[0004] To achieve the above technical objectives, the present invention provides a composite modified double-layer structure small-caliber artificial blood vessel, the artificial blood vessel is a hollow cylindrical structure, including an inner layer and an outer layer, the inner layer includes a blend of zwitterionic polyurethane (SBPU) and polycaprolactone (PCL), the outer layer includes polycaprolactone, the inner and outer layers are both electrospun layers and are tightly combined and inseparable;

[0005] The zwitterionic polyurethane contains zwitterions which are sulfonic acid anions and ammonium cations;

[0006] The inner and outer layers of the artificial blood vessel are modified by heparin.

[0007] In some technical schemes of the present invention, the inner layer includes a zwitterionic polyurethane with a mass fraction of 1 to 20%, specifically 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, and 20%. Further, the mass fraction of the zwitterionic polyurethane is preferably 10 to 20%. The zwitterionic polyurethane can improve the anti-protein adsorption function of the artificial blood vessel, and can produce a synergistic effect with heparin to further enhance the anti-thrombotic effect of the blood vessel. However, if the amount added is too high, it will have an adverse effect on the tensile properties of the artificial blood vessel.

[0008] In some technical solutions of the present invention, the inner diameter of the artificial blood vessel is 3 to 6 mm.

[0009] In order to achieve the above technical objectives, the present invention also provides a method for preparing a composite modified double-layer structure small-caliber artificial blood vessel, which at least comprises the following steps:

[0010] Step 1: preparing zwitterionic polyurethane (SBPU);

[0011] Step 2: Prepare a double-layer artificial blood vessel with a blend of PCL and SBPU as the inner layer and PCL as the outer layer by electrospinning;

[0012] Step 3: Use heparin to modify the surface of the double-layer artificial blood vessel.

[0013] In some technical schemes of the present invention, the preparation method of the SBPU at least comprises reacting equimolar amounts of a diol containing a tertiary amine with a sultone substance; and reacting the prepared zwitterion-containing substance with a diisocyanate substance to obtain the SBPU.

[0014]

[0015] The R1 and R2 are each independently selected from at least one of -CH2- and -C2H4-;

[0016] The diisocyanate substance M2 is selected from at least one of toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), diphenylmethane diisocyanate (MDI), dicyclohexylmethane diisocyanate (HMDI), hexamethylene diisocyanate (HDI), and lysine diisocyanate (LDI).

[0017] In some technical solutions of the present invention, the method for preparing SBPU in step 1 at least comprises the following steps:

[0018] Step a1: Add dichloromethane to equal amounts of the diol containing the tertiary amine and sultone, stir at 25-27° C. for 1.8-2.2 hours, and allow the resulting precipitate to stand for at least 48 hours;

[0019] Step a2: After the product of step a1 is precipitated with dichloromethane, the filtered precipitate is washed with dichloromethane, washed with isopropanol at least 3 times, and dried in a vacuum oven at 45-55° C. for 23-25 ​​hours to obtain a zwitterionic substance (SBD);

[0020] Step a3: add equal amounts of SBD powder and diisocyanate monomer to a solvent, heat to 50-80° C. in nitrogen, dropwise add dibutyltin dilaurate with a molar fraction of SBD of 0.8-1.2%, and react for 1-30 min.

[0021] Step a4: The reaction product obtained in step 3 is treated with excess methanol as a precipitation solvent, the obtained precipitate is then washed with methanol at least 3 times, and vacuum dried at 48-52° C. for at least 24 hours to obtain the SBPU.

[0022] Furthermore, the diol M1 containing a tertiary amine structure is selected from N-methyldiethanolamine.

[0023] Furthermore, the sultone substance is selected from 1,3-propane sultone.

[0024] Furthermore, the reaction solvent in step a3 includes but is not limited to dimethyl sulfoxide (DMSO).

[0025] Furthermore, the diisocyanate M2 is preferably diphenylmethane diisocyanate (MDI).

[0026] In some technical solutions of the present invention, the electrospinning in step 2 comprises at least the following steps:

[0027] Step b1: inject SBPU / PCL spinning solution into a syringe, and adjust the ambient temperature to 25-27°C and the humidity to 45-50%;

[0028] Step b2: the spinning parameters are set to electrostatic voltage of 15-20 kV, contact distance of 15-20 cm, propulsion speed of 0.8-1.5 ml / h, collector speed of 100-1000 rpm, and inner layer structure spinning;

[0029] Step b3: PCL spinning solution is injected into the syringe, and the spinning parameters are set as electrostatic voltage 10-20 kV, receiving distance 12-20 cm, propulsion speed 0.5-1.5 ml / h, collector speed 100-1000 rpm, and the outer layer structure is spun on the surface of the inner layer structure that has been formed.

[0030] In some preferred technical solutions of the present invention, the mass fraction of SBPU in the inner layer structure prepared in step b2 is 1-20%.

[0031] In some preferred technical solutions of the present invention, the step b2 uses a roller receiver with a diameter of 3 to 6 mm to spin the inner layer structure.

[0032] In some technical solutions, in step 3, the double-layer artificial blood vessel is modified with heparin, and the specific steps are as follows:

[0033] Step c1: immersing the double-layer artificial blood vessel prepared in step 1 in a mixed solution of 1,6-hexanediamine / isopropanol and adjusting the temperature to maintain at 35-40° C. for amination;

[0034] Step c2: Dissolve heparin in a buffer solution of 4-morpholineethanesulfonic acid (MES) containing 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and place at 35-40° C. for 25-35 min to activate the carboxyl groups.

[0035] Step c3: soaking the aminated double-layer artificial blood vessel obtained in step c1 in the activated heparin solution obtained in step c2 for 24 hours at a soaking temperature of 35-40° C.;

[0036] Step c4: After the soaking is completed, the obtained product is washed with deionized water for at least 3 times, and freeze-dried for 24 hours to obtain the composite modified double-layer small-caliber artificial blood vessel.

[0037] In some technical solutions of the present invention, the concentration of the 1,6-hexanediamine / isopropanol mixed solution in step c1 is in the range of 0.4 to 0.45 g / mol.

[0038] In some technical solutions of the present invention, the concentration of the activated heparin solution prepared in step c2 is 0.8-1.2 mg / mL.

[0039] In some technical schemes of the present invention, the MES buffer solution in step c2 has a concentration of 0.05M, a pH of 5, contains an EDC concentration of 0.28 to 0.32 mol / L, and contains an NHS concentration of 0.14 to 0.16 mol / L.

[0040] The present invention also provides an application of a composite modified double-layer structured small-caliber artificial blood vessel, wherein the artificial blood vessel is applied to repair or replace vascular tissue.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The composite modified double-layer structure of the small-caliber artificial blood vessel provided by the present invention comprises an inner layer of SBPU and PCL blended to form a zwitterion modified layer on the inner layer of the blood vessel, and then the double-layer structure is modified with heparin. Through the composite effect of SBPU and heparin, the anti-protein adsorption function and anti-platelet adsorption function of the inner wall of the blood vessel are further enhanced, thereby improving the anti-thrombotic performance of the small-caliber artificial blood vessel.

[0043] 2. The composite modified double-layer structure small-caliber artificial blood vessel provided by the present invention uses PCL as the vascular material in both the outer layer and the inner layer of the blood vessel, which can further improve the tensile resistance of the small-caliber blood vessels.

[0044] 3. The present invention adopts electrospinning technology to prepare a double-layer structured small-caliber artificial blood vessel with anti-thrombotic effect. The operation is simple, safe and non-toxic. At the same time, it has excellent biocompatibility and has good application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0046] Figure 1 This is the nuclear magnetic resonance spectrum of the zwitterionic polyurethane prepared in Example 3 of the present invention;

[0047] Figure 2 The graphs are the results of the protein adsorption resistance test of the samples prepared in the embodiments of the present invention and the comparative examples;

[0048] Figure 3 This is a graph showing the results of L-LDH activity testing of samples prepared in the embodiments of the present invention and the control examples;

[0049] Figure 4 The results of the cell activity test of the samples prepared in the examples and control examples of the present invention are shown in FIG. DETAILED DESCRIPTION

[0050] The following is a clear and complete description of the technical solutions in the present invention in conjunction with the embodiments of the present invention. The described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Example 1: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0052] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 hours and then allowed to stand for 48 hours. After precipitation with dichloromethane, the mixture was washed three times with isopropanol and then dried in a vacuum oven at 50°C for 24 hours to obtain zwitterionic polyurethane (SBD). 0.01 mol SBD and 0.01 mol MDI were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80°C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 5 minutes. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol and dried in a vacuum oven at 50°C for 24 hours to obtain zwitterionic polyurethane (SBPU).

[0053] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 2wt%, and pure polycaprolactone is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0054] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0055] Example 2: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0056] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 hours, and then allowed to stand for 48 hours. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50°C for 24 hours to obtain zwitterionic polyurethane (SBD). 0.01 mol SBD and 0.01 mol MDI were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80°C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise and reacted for 10 minutes. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50°C for 24 hours to obtain zwitterionic polyurethane (SBPU).

[0057] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 20wt%, and PCL is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0058] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0059] The difference between Example 2 and Example 1 is only that the reaction time of SBD and MDI is different.

[0060] Example 3: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0061] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 20 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0062] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 20wt%, and PCL is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0063] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0064] The only difference between Example 3 and Example 1 is that the reaction time of SBD and MDI is different.

[0065] Example 4: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0066] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 20 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0067] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 15wt%, and PCL is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0068] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0069] The only difference between Example 4 and Example 3 is that the mass percentage of SBPU in the total amount of SBPU and PCL in the inner layer of the blood vessel is different.

[0070] Example 5: A method for preparing a heparin-modified double-layer small-caliber artificial blood vessel, the specific steps are as follows:

[0071] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 20 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0072] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 10wt%, and PCL is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0073] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0074] The only difference between Example 4 and Example 3 is that the mass percentage of SBPU in the total amount of SBPU and PCL in the inner layer of the blood vessel is different.

[0075] Example 6: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0076] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise and reacted for 30 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed with methanol three times, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0077] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU in the total amount of SBPU and PCL is 15wt%, and pure polycaprolactone is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45-50% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0078] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0079] The only difference between Example 6 and Example 4 is that the reaction time of SBD and MDI is different.

[0080] Example 7: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0081] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 30 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0082] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU in the total amount of SBPU and PCL is 30wt%, and pure polycaprolactone is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45-50% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0083] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0084] The difference between Example 7 and Example 6 is that the mass percentage of SBPU in the total amount of SBPU and PCL is different. Control group 1: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0085] Preparation of small-caliber artificial blood vessels: Pure polycaprolactone was added to hexafluoroisopropanol solvent to prepare the inner and outer layer spinning solutions, and electrospinning was performed under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters were as follows: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm was prepared.

[0086] Heparin modification of small-caliber artificial blood vessels: The prepared artificial blood vessels were immersed in 0.43g / mL 1,6-hexanediamine / isopropanol solution, and the temperature was maintained at 37°C. Heparin (1 mg / mL) was dissolved in MES buffer (0.05M, pH5.0) containing 0.3mol / L EDC and 0.15mol / L NHS, and placed at 37°C for 30 minutes to activate the carboxyl group. The aminated graft was immersed in the activated heparin solution at 37°C for 24 hours. After the reaction was completed, the sample was washed 3 times with deionized water and freeze-dried for 24 hours to obtain an anti-thrombotic double-layer structure small-caliber artificial blood vessel.

[0087] The difference between the control group 1 and Examples 1-6 is that there is only a PCL layer and no SBPU / PCL blended inner layer structure.

[0088] Control group 2: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0089] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 20 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0090] Preparation of small-caliber artificial blood vessels: The prepared SBPU and PCL are added to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 20wt%, and pure polycaprolactone is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25-27°C and 45-50% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; using a roller receiver with a diameter of 5mm, an artificial blood vessel with a diameter of 5mm is prepared.

[0091] The difference between control group 2 and example 3 is that the blood vessels are not modified with heparin.

[0092] Control group 3: A method for preparing a heparin-modified double-layer structure small-caliber artificial blood vessel, the specific steps are as follows:

[0093] Preparation of zwitterionic polyurethane: 0.2 mol N-methyldiethanolamine and 0.2 mol 1,3-propane sultone were added to 250 mL dichloromethane, stirred at room temperature for 2 h, and then allowed to stand for 48 h. After precipitation with dichloromethane, the mixture was washed three times with isopropanol, and then dried in a vacuum oven at 50 ° C for 24 h to obtain zwitterionic powder (SBD). 0.01 mol SBD and 0.01 mol 4,4-methylene diisocyanate were added to 30 mL dimethyl sulfoxide (DMSO) solution, heated to 80 ° C in nitrogen, and 0.0001 mol dibutyltin dilaurate was added dropwise to react for 20 min. Excess methanol was used as the precipitation solvent, and the reaction product was then washed three times with methanol, and vacuum dried at 50 ° C for 24 h to obtain zwitterionic polyurethane (SBPU).

[0094] Preparation of small-caliber artificial blood vessels: Step 1: Add the prepared SBPU and PCL to hexafluoroisopropanol solvent to prepare the inner layer spinning solution, wherein the mass percentage of SBPU to the total amount of SBPU and PCL is 30wt%, and pure polycaprolactone is dissolved in hexafluoroisopropanol to prepare the outer layer spinning solution, and electrospinning is carried out under the temperature and humidity conditions of 25°C and 45% humidity. The electrospinning parameters are: inner layer: electrostatic voltage 18kV, contact distance 20cm, propulsion speed 1ml / h, collector speed 150rpm, spinning time 30min; outer layer: electrostatic voltage 12kV, contact distance 20cm, propulsion speed 1.5ml / h, collector speed 150rpm, spinning time 30min; use a roller receiver with a diameter of 5mm to prepare an artificial blood vessel with a diameter of 5mm.

[0095] Test experiment:

[0096] 1. Anti-protein adsorption experiment: prepare bovine serum albumin (BSA) and fibrinogen (Fg) solutions. After 2 hours, aspirate PBS and drip BSA and Fg into a 24-well plate, 1 ml per well, and shake in a 37°C incubator for 1 hour; aspirate protein solution and wash three times with PBS; drip the prepared SDS into a 24-well plate, 1 ml per well, and shake in a 37°C incubator for 2 hours, aspirate and place in a 96-well plate, drip BSA solution into the wells with liquid, 0.1 ml per well, wrap with tin foil, and place in a 37°C incubator without shaking for 1-2 hours. Finally, test with an enzyme marker at 562 nm.

[0097] 2. Antiplatelet experiment: Prepare the sample and place it in a 24-well plate, sterilize it with 70% ethanol and ultraviolet light for 10 minutes. Hydrate the sample in a PBS solution at 37°C for 1 hour. Then remove the solution, add 1 ml of platelet solution to each well, and place it in a 37°C incubator for 2 hours. Remove the platelet solution, wash it three times with PBS solution, add 2% Triton-x100 solution, and place it in a 37°C incubator for 15 minutes. Then transfer the solution to a 96-well plate according to the instructions of the L-lactate dehydrogenase (L-LDH) activity detection kit, and measure the absorbance at 450nm.

[0098] 3. Cytotoxicity experiment: The cell activity of the sample was tested by CCK-8 reagent.

[0099] 4. Mechanical properties test: Use an electronic universal testing machine, adjust the distance between the two clamps to 3 cm, and adjust the tensile speed to 50 mm / min to test the tensile properties of the sample.

[0100] Test result description:

[0101] 1. Anti-protein adsorption experiment: Figure 2 As shown, the artificial blood vessels prepared by using SBPU and heparin composite modification in Examples 1-7 showed significantly better anti-protein adsorption effects than the control group in both BSA and Fg test solutions.

[0102] 2. Antiplatelet adsorption test: Figure 3 As shown, the L-LDH test value of the artificial blood vessel prepared by the composite modification of SBPU and heparin in Example 1-7 is significantly lower than that in Control Example 1-3, and it can be observed that the antiplatelet adsorption effect is better than that of the control group.

[0103] 3. The mechanical properties test results are shown in the following table:

[0104] Table 1 Tensile strength test results

[0105]

[0106] The test results show that the small-caliber artificial blood vessels prepared by the embodiment of the present invention through the introduction of SBPU substances and the surface treatment of heparin have significant anti-protein adsorption and anti-platelet adsorption effects. At the same time, the introduction of zwitterions and the surface treatment of heparin are non-toxic to small-caliber artificial blood vessels, the cell activity is more than 80%, and the tensile strength is more than 2MPa, which can be used as biomaterials for vascular tissue repair or replacement.

[0107] Finally, a few points should be explained: Although the present invention has been described in detail above with general descriptions and specific embodiments, on the basis of the present invention, the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the aforementioned embodiments, ordinary technicians in this field should understand that: it is still possible to modify the technical solutions recorded in the aforementioned embodiments, or to replace part or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.

Claims

1. A composite modified double-layer structure small-caliber artificial blood vessel, characterized in that: The artificial blood vessel is a hollow cylindrical structure, comprising an inner layer and an outer layer, wherein the inner layer comprises a blend of zwitterionic polyurethane (SBPU) and polycaprolactone (PCL), and the outer layer comprises polycaprolactone, and the inner and outer layers are both electrospun layers and are tightly combined and inseparable; The zwitterionic polyurethane contains zwitterions such as sulfonic acid anions (-SO3-) and ammonium cations (-N + ); The inner and outer layers of the artificial blood vessel are modified by heparin.

2. The artificial blood vessel according to claim 1, characterized in that: The inner diameter of the artificial blood vessel is 3 to 6 mm.

3. The artificial blood vessel according to claim 1, characterized in that: The inner layer comprises zwitterionic polyurethane in a mass fraction of 1 to 20%.

4. A method for preparing the composite modified double-layer structure small-caliber artificial blood vessel according to claim 1, characterized in that: At least the following steps are included: Step 1: preparing zwitterionic polyurethane (SBPU); Step 2: Prepare a double-layer artificial blood vessel with a blend of PCL and SBPU as the inner layer and PCL as the outer layer by electrospinning; Step 3: Use heparin to modify the surface of the double-layer artificial blood vessel.

5. The method for preparing an artificial blood vessel according to claim 4, characterized in that: The preparation method of SBPU in step 1 at least comprises reacting equimolar amounts of a diol containing a tertiary amine with a sultone substance M1 to obtain a zwitterionic substance SBD; and reacting the prepared SBD with a diisocyanate substance M2 to obtain the SBPU. Preparation of SBD material: Preparation of SBPU material: The R1 and R2 are each independently selected from at least one of -CH2- and -C2H4-; The diisocyanate substance M2 is selected from at least one of toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), diphenylmethane diisocyanate (MDI), dicyclohexylmethane diisocyanate (HMDI), hexamethylene diisocyanate (HDI), and lysine diisocyanate (LDI).

6. The method for preparing an artificial blood vessel according to claim 5, characterized in that: The reaction time of the zwitterionic substance SBD and the diisocyanate substance M2 is 1 to 30 minutes, and the reaction temperature is 50 to 80°C.

7. The method for preparing an artificial blood vessel according to claim 5, characterized in that: The diol containing tertiary amine is selected from N-methyldiethanolamine; the sultone substance is selected from 1,3-propane sultone; and the diisocyanate is selected from diphenylmethane diisocyanate (MDI).

8. The method for preparing an artificial blood vessel according to claim 4, characterized in that: The electrospinning in step 1 at least comprises the following steps: Step b1: inject SBPU / PCL spinning solution into a syringe, and adjust the ambient temperature to 25-27°C and the humidity to 45-50%; Step b2: the spinning parameters are set to electrostatic voltage of 15-20 kV, contact distance of 15-20 cm, propulsion speed of 0.8-1.5 ml / h, collector speed of 100-1000 rpm, and inner layer structure spinning; Step b3: PCL spinning solution is injected into the syringe, and the spinning parameters are set as electrostatic voltage 10-20 kV, receiving distance 12-20 cm, propulsion speed 0.5-1.5 ml / h, collector speed 100-1000 rpm, and the outer layer structure is spun on the surface of the inner layer structure that has been formed.

9. The method for preparing an artificial blood vessel according to claim 4, characterized in that: In step 3, the double-layer artificial blood vessel is modified with heparin, and the specific steps are as follows: Step c1: soaking the double-layer artificial blood vessels prepared in step 1 in a 1,6-hexanediamine / isopropanol solution respectively, and adjusting the temperature to maintain at 35-40° C.; Step c2: dissolving heparin in a buffer solution of 4-morpholineethanesulfonic acid (MES) containing 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and soaking at 35-40° C. for 24 h; Step c3: After soaking, the product was washed with deionized water and freeze-dried for 24 hours.

10. Application of a composite modified double-layer structure small-caliber artificial blood vessel, characterized in that: The artificial blood vessel is used for repairing or replacing vascular tissue.

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