Artificial blood vessel and preparation method thereof

Abstract

The invention discloses an artificial blood vessel and a preparation method thereof, and the preparation method comprises the following steps: uniformly mixing an erythrocyte ghost suspension and melanin nanoparticles to obtain a mixed solution; carrying out ultrasonic treatment and extrusion on the mixed solution to obtain a melanin nano-vesicle solution; carrying out electrostatic spinning on the polycaprolactone solution to obtain a polycaprolactone electrostatic spinning blood vessel; soaking the polycaprolactone electrostatic spinning blood vessel in an ethylenediamine solution, and then washing the polycaprolactone electrostatic spinning blood vessel with a phosphate buffer solution; then soaking in a 4-morpholine ethanesulfonic acid solution; finally, carrying out heparinization in a 4-morpholine ethanesulfonic acid solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and heparin; and incubating the heparin-modified polycaprolactone electrostatic spinning blood vessel in the melanin nano-vesicle solution to obtain the artificial blood vessel. The melanin nano-vesicles are modified, so that the anticoagulation performance of erythrocyte membranes is reserved, and meanwhile, the scavenging capacity of melanin nano-particles on reactive oxygen free radicals is reserved.

Description

Technical Field

[0001] The present invention belongs to the field of material technology, and more specifically, relates to an artificial blood vessel and a preparation method thereof. Background Art

[0002] Vascular diseases are the leading cause of death worldwide. Their pathogenesis is mainly due to the narrowing or occlusion of blood vessels, which in turn leads to a decrease in blood flow and a lack of nutrients, resulting in impaired tissue or organ function. Such diseases usually manifest as coronary heart disease, cerebrovascular disease, peripheral arterial disease, and deep vein thrombosis. According to the World Health Organization's forecast, by 2030, the number of deaths from cardiovascular-related diseases worldwide will reach 23.3 million per year. In the treatment of such diseases, vascular transplantation is one of the conventional treatments. Autologous blood vessels, as the gold standard in the field of vascular transplantation, require patients to undergo a second operation in actual applications. This not only causes additional damage to the patient's body, but also increases the cost of treatment. What's more difficult is that in some cases, the patient himself cannot provide enough autologous blood vessels for transplantation. With the continuous progress and development of fields such as biomedicine and materials science, tissue engineering artificial blood vessels have emerged, providing a new way to solve this problem.

[0003] After implantation, artificial blood vessels made of biodegradable polymer materials gradually degrade, and utilize the host's self-remodeling potential to regenerate artificial blood vessels close to natural structures in situ. This feature makes it a research hotspot in the field of vascular regeneration. However, as research continues to deepen, more and more evidence shows that small-caliber artificial blood vessels made of only a single degradable polymer material have the following problems: after implantation, on the one hand, they will induce acute coagulation reactions, and on the other hand, they will stimulate local tissues to produce significant inflammatory reactions, which is not only not conducive to the adhesion, migration and proliferation of surrounding vascular cells, but also hinders their good integration with natural vascular tissues, making it difficult to achieve truly natural vascular regeneration in a short period of time. Summary of the invention

[0004] The purpose of the present invention is to provide an artificial blood vessel and a preparation method thereof. The present invention loads melanin nanocapsules coated with red blood cell membranes onto polymer artificial blood vessels, thereby increasing the blood compatibility of polymer materials to a certain extent, resisting the formation of thrombus, and having the functions of scavenging active oxygen free radicals and regulating local inflammation.

[0005] In order to achieve the above object, one aspect of the present invention provides a method for preparing an artificial blood vessel, the method comprising:

[0006] (1) mixing the red blood cell ghost suspension and melanin nanoparticles uniformly to obtain a mixed solution; then ultrasonicating and extruding the mixed solution to obtain a melanin nanovesicle solution;

[0007] (2) electrospinning a polycaprolactone (PCL) solution to obtain a polycaprolactone electrospun blood vessel;

[0008] (3) immersing the polycaprolactone electrospun blood vessel in an ethylenediamine solution, then washing with a phosphate buffer solution; then immersing in a 4-morpholineethanesulfonic acid (MES) solution; and finally performing heparinization in a 4-morpholineethanesulfonic acid solution containing (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC), N-hydroxysuccinimide (NHS) and heparin to obtain a heparin-modified polycaprolactone electrospun blood vessel;

[0009] (4) Incubating the heparin-modified polycaprolactone electrospun blood vessel in the melanin nanovesicle solution to obtain the artificial blood vessel.

[0010] According to the present invention, preferably, the red blood cell ghost suspension is prepared by a method comprising the following steps:

[0011] a. Mix the rat blood and anticoagulant evenly, then centrifuge, discard the upper plasma, and obtain a precipitate;

[0012] b. After removing blood clots and grease from the precipitate, the precipitate was washed with phosphate buffered saline (PBS);

[0013] c. placing the washed precipitate in 0.25×PBS buffer for hypotonicity; then centrifuging and washing; finally repeating the centrifugation and washing steps for multiple times to obtain red blood cell ghosts;

[0014] d. Mix the red blood cell ghosts and 0.25×PBS buffer to obtain a red blood cell ghost suspension.

[0015] According to the present invention, preferably, in step a, the volume ratio of the rat blood to the anticoagulant is (5-10):1;

[0016] The centrifugal conditions include: centrifugal temperature of 3-5°C, rotation speed of 700-900g, and time of 4-6min;

[0017] In step c, the standing time is 0.5-1.5h; the centrifugal conditions include: centrifugal temperature is 3-5°C, rotation speed is 10000-12000rpm, and time is 4-6min.

[0018] In the present invention, mature red blood cells do not have nuclei and various organelles, so the method for purifying red blood cell membranes is relatively simple and easy. Generally, purified red blood cell membranes can be separated by hypotonic crushing and centrifugation. The amphiphilic phosphate choline and glycocalyx distributed on the membrane surface have excellent hydrophilicity, so that the red blood cell membrane can significantly inhibit the adsorption of nonspecific proteins, thereby improving the anti-fouling performance. Therefore, the red blood cell membrane can be used as a bionic coating to increase the blood compatibility of its materials. Red blood cells also show the expression of a series of immunomodulatory markers, so that the body can effectively identify it as a self-component. For example, CD47 distributed on its surface can interact with the signal-regulating α protein, reduce the activation of platelets, and thus prevent thrombosis. In addition, the functionalization of nanoparticles using red blood cell membranes can also promote the immune escape effect and significantly increase the residence time of nanoparticles in the circulatory system.

[0019] According to the present invention, preferably, the melanin nanoparticles are prepared by a method comprising the following steps:

[0020] The cuttlefish ink is first centrifuged at 1800-2200 rpm for 8-15 minutes; then centrifuged at 11000-13000 rpm for 10-20 minutes to obtain a precipitate; finally, the precipitate is washed and freeze-dried to obtain melanin nanoparticles.

[0021] The present invention utilizes the remarkable anti-oxidation property of melanin to remove active oxygen free radical components generated by implantation of artificial blood vessels.

[0022] According to the present invention, preferably, in step (1), in the mixed solution, the mass ratio of red blood cell ghosts to melanin nanoparticles calculated on a protein basis is (1-5):1.

[0023] According to the present invention, preferably, in step (1), the ultrasonic conditions include: ultrasonication at 100-110W in an ice bath, with 2s of work and 1s of rest as one cycle, each working time is 5-8 minutes, and a total of 2-3 times of working;

[0024] The extrusion is as follows: using an extruder to sequentially extrude the mixed solution after ultrasonication through a porous polycarbonate membrane with a pore size of 800 nm and a porous polycarbonate membrane with a pore size of 400 nm to obtain the melanin nanocapsule solution.

[0025] According to the present invention, preferably, in step (2), in the polycaprolactone solution, the solid-liquid ratio of polycaprolactone to solvent is (22-28): 100 g / mL; the solvent is composed of chloroform and methanol, and the volume ratio of chloroform to methanol is 5:1;

[0026] The conditions for the electrospinning include: voltage of 10-15 kV, spinning solution flow rate of 6-10 mL / h, distance between the injection needle and the receiver of 22-25 cm, and a rotation speed of the receiving device of 200-400 rpm.

[0027] According to the present invention, preferably, in step (3), the polycaprolactone electrospun blood vessel is immersed in an ethylenediamine solution, shaken at 20-30° C. for 25-35 min, and then rinsed with a phosphate buffer;

[0028] The temperature of soaking in the 4-morpholineethanesulfonic acid solution is 3-5°C, and the time is 35-45 minutes; the pH of the 4-morpholineethanesulfonic acid solution is 5-6;

[0029] In a 4-morpholineethanesulfonic acid solution containing (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC), N-hydroxysuccinimide (NHS) and heparin, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.1-0.2 mg / mL, the concentration of N-hydroxysuccinimide is 0.1-0.2 mg / mL, and the concentration of heparin is 8-12 mg / mL;

[0030] The heparinization temperature is 3-5°C and the time is 2-4h.

[0031] According to the present invention, preferably, in step (4), the incubation temperature is 3-5°C and the time is 3-5h.

[0032] Another aspect of the present invention provides an artificial blood vessel prepared by the above preparation method.

[0033] The technical solution of the present invention has the following beneficial effects:

[0034] (1) The present invention modifies the surface of blood vessels with melanin nanocapsules through electrospinning of polycaprolactone, thereby retaining the anticoagulant properties of the red blood cell membrane and the ability of melanin nanoparticles to scavenge reactive oxygen free radicals.

[0035] (2) The artificial blood vessel of the present invention has good biocompatibility, does not cause obvious hemolytic reaction when incubated with blood in vitro, and does not have obvious biological toxicity when incubated with cells.

[0036] (3) The artificial blood vessel of the present invention has a good ability to scavenge active oxygen free radicals and can reduce the damage caused by oxidative stress to a certain extent.

[0037] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention in conjunction with the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0039] Figure 1 TEM images of MN and MN@RM according to Test Example 1 of the present invention are shown; wherein the scale bar is 100 μm.

[0040] Figure 2 The UV-visible spectra of MN, RM and MN@RM according to Test Example 1 of the present invention are shown; wherein Absorbance represents absorbance, and Wavelenghth represents wavelength.

[0041] Figure 3 The cell viability diagram of HUVECs after being treated with different concentrations of MN@RM according to Test Example 2 of the present invention is shown; wherein Cell viability represents cell viability, and Ctrl represents the control group.

[0042] Figure 4 The live / dead staining image of HUVECs after co-incubation with MN@RM according to Test Example 2 of the present invention is shown; wherein the scale bar is 100 μm.

[0043] Figure 5 The staining images of superoxide anion radical activity (DHE) and total intracellular ROS (DCFH-DA) in HUVECs cells under MN@RM treatment according to Test Example 3 of the present invention are shown; wherein the scale bar is 100 μm, No treat represents the negative control group, and PBS represents the positive control group.

[0044] Figure 6 Digital photos of three blood vessels, PCL, PCL-Hep and PCL-Hep / MN@RM, prepared according to embodiments of the present invention are shown.

[0045] Figure 7 SEM images of three types of blood vessels, PCL, PCL-Hep and PCL-Hep / MN@RM, prepared according to embodiments of the present invention are shown; wherein the scale bar is 20 μm, and the scale bar of the enlarged image is 2 μm.

[0046] Figure 8The blood compatibility diagram of three vascular materials, PCL, PCL-Hep and PCL-Hep / MN@RM, according to test example 4 of the present invention is shown; wherein a is fibrinogen adsorption; b is activated partial thromboplastin time (APTT); c is thrombin time (TT); d is prothrombin time (PT), and Fibrinogen absorption represents fibrinogen adsorption.

[0047] Above Figure 1-Figure 8 In the figure, MN represents melanin nanoparticles, RM represents red blood cell membrane ghosts, MN@RM represents melanin nanovesicles, PCL represents PCL electrospun blood vessels, PCL-Hep represents heparin-modified PCL electrospun blood vessels, and PCL-Hep / MN@RM represents PCL blood vessels modified with heparin and loaded with melanin nanovesicles. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0049] The present invention is further described below by examples:

[0050] Example

[0051] 1. Extraction of melanin nanoparticles: Collect ink from fresh cuttlefish, centrifuge at 2000 rpm for 10 minutes to remove larger particles; centrifuge at 12000 rpm for 15 minutes to collect the precipitate; wash the precipitate three times with deionized water, and freeze-dry to obtain melanin nanoparticles.

[0052] 2. Extract the red blood cell membrane by hypotonic centrifugation. The specific operation is as follows: a. Take out fresh SD rat blood, immediately mix it with the anticoagulant on ice (the volume ratio of SD rat blood to anticoagulant is 5:1), centrifuge at 800g for 5 minutes at 4℃, discard the upper plasma, and obtain a precipitate; b. Remove the blood clots and off-white oil on the precipitate as soon as possible, and wash the precipitate three times with cold PBS (1×, pH=7.4); c. Add 0.25×PBS (pH=7.4) to the washed precipitate and let it stand for 1 hour for hypotonicity; then centrifuge at 10000rpm for 5 minutes at 4℃, wash once with 1×PBS; finally repeat the centrifugation and washing steps in step c 3-4 times until the supernatant is colorless and a pink red blood cell ghost is obtained. d. Mix the red blood cell ghost with 0.25×PBS buffer to obtain a red blood cell ghost suspension, and store it at -80℃. The protein content was determined by BCA protein quantification method, and the protein concentration of the prepared red blood cell ghost suspension was 5 mg / mL.

[0053] 3. Preparation of MN@RM: The red blood cell ghost suspension and melanin nanoparticles were mixed evenly to obtain a mixed solution, in which the concentration of red blood cell ghosts in terms of protein was 1 mg / mL, and the concentration of melanin nanoparticles was 1 mg / mL. The mixed solution was ultrasonicated at 14% power (about 105W) under an ice bath, with a cycle of 2s working and 1s resting, each working time was 5 minutes, and a total of two working times; the ultrasonicated mixed solution was extruded through a porous polycarbonate membrane with a pore size of 800nm ​​and a porous polycarbonate membrane with a pore size of 400nm in turn with an extruder, and finally a melanin nanovesicle solution (MN@RM solution) was obtained.

[0054] 4. Preparation of PCL-Hep / MN@RM: (1) Use a 10 mL syringe to load the PCL solution (the liquid-to-solvent ratio of PCL is 25:100 g / mL, the solvent is composed of chloroform and methanol, and the volume ratio of chloroform and methanol is 5:1); after connecting a 21-G injection needle, fix the syringe containing the PCL solution on the microinjection pump, and adjust the height of the syringe and the distance between the needle and the receiver. Both the receiver and the high-voltage electrostatic device are connected to the ground wire to ensure electrical safety; the connector of the high-voltage electrostatic device is connected to the injection needle, and the parameters are set: the voltage is 12 kV, the spinning solution flow rate is 8.0 mL / h, the receiving distance (the distance between the injection needle and the receiver) is 23 cm, and the speed of the receiving device is about 300 rpm; after turning on the receiver and the high-voltage electrostatic device in turn, the PCL solution is slowly ejected under the push of the injection pump, and fibers are generated under the action of the high-voltage electric field and collected on a stainless steel core rod with a diameter of 2 mm to obtain PCL electrospun blood vessels, and the prepared PCL electrospun blood vessels are placed in a vacuum drying oven for more than 120 hours for standby use.

[0055] (2) The above PCL electrospun blood vessels were immersed in an aqueous solution of ethylenediamine (0.1 mol / L) and gently shaken at room temperature 25°C for 30 min. Rinse three times with 1×PBS. The material was transferred to an MES aqueous solution (pH = 5.6) and immersed at 4°C for 40 min. Subsequently, it was transferred to an MES aqueous solution (pH = 5.6) containing EDC (0.144 mg / mL), NHS (0.144 mg / mL) and heparin (10 mg / mL), and heparinized on a shaker at 4°C for 3 hours. Finally, it was thoroughly washed with PBS to complete the heparin modification and obtain heparin-modified PCL electrospun blood vessels (PCL-Hep). The heparin-modified PCL electrospun blood vessels were transferred to a sterile melanin nanovesicle solution, incubated at 4°C for 4 h, and then washed three times with phosphate buffered saline (PBS) to remove excess unmodified vesicles, thereby obtaining heparin-modified and melanin nanovesicle-loaded PCL blood vessels (artificial blood vessels that can scavenge reactive oxygen free radicals and have good anticoagulant properties) (PCL-Hep / MN@RM).

[0056] The PCL electrospun blood vessels, heparin-modified PCL electrospun blood vessels, and heparin-modified and melanin nanovesicle-loaded PCL blood vessels prepared in this example are named PCL, PCL-Hep, and PCL-Hep / MN@RM, respectively. The digital photos of the above three blood vessels are shown in Figure 6 shown.

[0057] The microstructures of the three blood vessels were observed by scanning electron microscopy. Figure 7 As shown in the figure, after heparin modification, the surface of PCL fibers remained smooth without significant deformation. A large number of spherical particles were distributed on the surface of PCL-Hep / MN@RM fibers, indicating that melanin nanovesicles had been successfully modified.

[0058] Test Example 1

[0059] Transmission electron microscope (TEM) and ultraviolet-visible molecular absorption spectroscopy (UV-Vis) were used to verify whether the red blood cell membrane was successfully coated on the melanin nanoparticles in the melanin nanovesicle solution prepared in the example. The specific results are shown in Figure 1 and Figure 2 .

[0060] Depend on Figure 1 and Figure 2 It can be seen that the red blood cell membrane has been successfully coated on the surface of melanin nanoparticles.

[0061] Test Example 2

[0062] Biocompatibility testing of MN@RM

[0063] Human umbilical vein endothelial cells (HUVECs) were cultured at 1×10 4 The cells were seeded at a density of 100 μg / well in a 96-well cell culture plate and incubated at 37°C with 5% CO 2 The cells were incubated for 24 h under the conditions of 40 °C and 5% CO, and then treated with distilled water and melanin nanovesicle solutions (100 μL / well) of different concentrations (10, 20, 40, 60 μg / mL) for 24 h (the melanin nanovesicle solutions of different concentrations were prepared by diluting the melanin nanovesicle solutions prepared in step 3 of Example 3 with distilled water). After that, CCK-8 / culture medium (10 μL / 100 μL) was added to each well and incubated at 37 °C and 5% CO 2 The cells were incubated for another hour under the same conditions. The absorbance value (OD) of each well at 450 nm was measured by enzyme-linked immunosorbent assay. 450 The cell viability was calculated as follows: cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%, where As, Ac, and Ab represent the OD values ​​of the treatment group (the group treated with different concentrations of melanin nanovesicle solution), the control group (distilled water group), and the blank well (the group with only culture medium but no cells), respectively. 450 Compared with the control group, the cell viability did not change significantly with the increase of nanoparticle concentration, indicating that the melanin biomimetic nanoparticles (melanin nanovesicles) coated with red blood cell membranes have good cell compatibility (see Figure 3 ).

[0064] Human umbilical vein endothelial cells (HUVECs) were cultured at 2×10 5 The cells were seeded at a density of 1 / well in a 12-well cell culture plate and incubated at 37°C with 5% CO 2 The cells were incubated for 24 h under the conditions of 5% CO and then treated with nanoparticles (100 μL / well) at different concentrations (0, 10, 20, 40, 60 μg / mL) for 24 h. Afterwards, the cells were washed with PBS, and 200 μL of Calcein AM / PI detection working solution was added to each well. The cells were incubated at 37°C and 5% CO. 2 The cells were incubated in the dark for 30 min under the condition of 40 °C. After incubation, the cells were observed under a fluorescence microscope. As the concentration of nanoparticles increased, the cells were in good condition and no significant apoptosis occurred. This also shows that biomimetic nanoparticles (melanin nanovesicles) have good cell compatibility (see Figure 4 ).

[0065] Test Example 3

[0066] Detection of active oxygen scavenging ability

[0067] Human umbilical vein endothelial cells (HUVECs) were cultured at 2×10 5The cells were seeded at a density of 100 μg / well in a 24-well cell culture plate and incubated at 37°C with 5% CO 2 Incubate in an incubator for 24 h. Remove the plate from the incubator, discard the original culture medium, and wash the cells with preheated PBS. After absorbing the PBS as clean as possible, add 200 μL of H to the experimental wells and control wells, respectively. 2 O 2 Water, distilled water and melanin nanovesicle solutions of different concentrations (10, 20, 40, 60 μg / mL) were added to the experimental wells respectively (the melanin nanovesicle solutions of different concentrations were prepared by diluting the melanin nanovesicle solutions prepared in step 3 of Example 3 with distilled water). New complete culture medium was added to the blank wells, and then placed in a 37°C, 5% CO 2 Incubate in an incubator at 37°C and 5% CO for 24 hours. Remove the plate from the incubator, discard the stock solution in the plate, wash the cells with preheated PBS, and after absorbing the PBS as clean as possible, add 10 μmol DHE or 10 μmol DCFH-DA solution to each well. Place in a 37°C, 5% CO 2 After incubation in an incubator for 30 min, the slides were sealed with a sealing medium containing DAPI. The staining effect was observed under a fluorescence microscope (see Figure 5 ). Without H 2 O 2 The stimulated negative control group cells had almost no fluorescence in the cells; in the positive control group (distilled water group), the cells showed significant red or green fluorescence due to the large amount of reactive oxygen free radicals produced in the cells; after adding bionic nanoparticles, the fluorescence intensity gradually weakened with the increase in concentration. The results show that bionic nanoparticles can protect cells by quickly removing reactive oxygen free radicals.

[0068] Test Example 4

[0069] Blood compatibility test:

[0070] The PCL electrospinning membrane was prepared according to the method of step 4 of Example 1, with the only difference that fibers were generated separately under the action of a high voltage electric field and collected on a drum receiver with a diameter of 10 cm to obtain a PCL electrospinning membrane, and the prepared PCL electrospinning membrane was placed in a vacuum drying oven for more than 120 hours for standby use.

[0071] The PCL electrospinning membrane prepared above was used to prepare a heparin-modified PCL electrospinning membrane loaded with melanin nanovesicles according to the method of step 4 of Example.

[0072] PCL electrospun membrane or heparin-modified and melanin nanovesicle-loaded PCL electrospun membrane was spread in a 12-well plate; 200 μL of rat whole blood was added to each well; incubated at 37°C for 1 hour; centrifuged at 1000 g for 10 minutes, and the supernatant was collected; the absorbance of the supernatant at 540 nm was measured. Rat whole blood was treated with PBS solution and ultrapure water, respectively, for negative and positive controls.

[0073] The PCL electrospun membrane or the PCL electrospun membrane modified with heparin and loaded with melanin nanovesicles was flattened in a 12-well plate; a fibrinogen solution (composed of fibrinogen and PBS solution, with a solid-liquid ratio of 1 mg / mL) was added; the sample was incubated at 37°C for 2 h; after being rinsed with PBS solution three times, the sample was placed in an SDS solution (2 wt %); the sample was incubated at 37°C for 1 h; the protein concentration in the SDS solution was determined by the BCA method (C 0 , μg / mL); protein concentration of sample adsorbed (C 1 , μg / mL) is calculated as C 1 =(C 0 ×V) / A, where V represents the volume of SDS solution (mL) and A represents the area of ​​electrospinning membrane (cm 2 ).

[0074] In the coagulation time assay, PCL electrospun membranes or PCL electrospun membranes modified with heparin and loaded with melanin nanovesicles were placed in conical tubes. 500 μL of commercial human plasma was added and incubated with the graft at 37°C for 1 hour. The conical tube containing only human plasma was used as a control. After 1 hour of incubation, the graft was removed from the conical tube. Then, the prothrombin time (PT), thromboplastin time (TT) and activated partial thromboplastin time (APTT) of human plasma in contact with the graft were analyzed using an automatic SYSMEXCS-5100 coagulation analyzer. Compared with the unmodified group, the modification with heparin and vesicles significantly reduced the adsorption of fibrinogen and prolonged the duration of APTT, TT and PT, indicating that RM@MN has excellent anticoagulant properties (see Figure 8 ).

[0075] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing an artificial blood vessel, characterized in that: The preparation method comprises: (1) mixing the red blood cell ghost suspension and melanin nanoparticles uniformly to obtain a mixed solution; then ultrasonicating and extruding the mixed solution to obtain a melanin nanovesicle solution; (2) electrospinning the polycaprolactone solution to obtain a polycaprolactone electrospun blood vessel; (3) immersing the polycaprolactone electrospun blood vessel in an ethylenediamine solution, then washing with a phosphate buffer solution; then immersing in a 4-morpholineethanesulfonic acid solution; and finally performing heparinization in a 4-morpholineethanesulfonic acid solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and heparin to obtain a heparin-modified polycaprolactone electrospun blood vessel; (4) Incubating the heparin-modified polycaprolactone electrospun blood vessel in the melanin nanovesicle solution to obtain the artificial blood vessel.

2. The preparation method according to claim 1, wherein The red blood cell ghost suspension is prepared by a method comprising the following steps: a. Mix the rat blood and anticoagulant evenly, then centrifuge, discard the upper plasma, and obtain a precipitate; b. After removing blood clots and grease from the precipitate, washing with phosphate buffer; c. The washed precipitate is placed in a 0.25×PBS buffer solution for hypotonicity; then centrifuged and washed; finally, the centrifugation and washing steps are repeated multiple times to obtain red blood cell ghosts; d. Mix the red blood cell ghosts and 0.25×PBS buffer to obtain a red blood cell ghost suspension.

3. The preparation method according to claim 2, wherein In step a, the volume ratio of the rat blood to the anticoagulant is (5-10):1; The centrifugal conditions include: centrifugal temperature of 3-5°C, rotation speed of 700-900g, and time of 4-6min; In step c, the standing time is 0.5-1.5h; the centrifugal conditions include: centrifugal temperature is 3-5°C, rotation speed is 10000-12000rpm, and time is 4-6min.

4. The preparation method according to claim 1, wherein The melanin nanoparticles are prepared by a method comprising the following steps: The cuttlefish ink is first centrifuged at 1800-2200 rpm for 8-15 minutes; then centrifuged at 11000-13000 rpm for 10-20 minutes to obtain a precipitate; finally, the precipitate is washed and freeze-dried to obtain melanin nanoparticles.

5. The preparation method according to claim 1, wherein In step (1), in the mixed solution, the mass ratio of red blood cell ghosts to melanin nanoparticles calculated on a protein basis is (1-5):

1.

6. The preparation method according to claim 1, wherein In step (1), the ultrasonic conditions include: ultrasonication at 100-110W in an ice bath, with 2s of work and 1s of rest as one cycle, each working time is 5-8 minutes, and a total of 2-3 times of working; The extrusion is as follows: using an extruder to sequentially extrude the mixed solution after ultrasonication through a porous polycarbonate membrane with a pore size of 800 nm and a porous polycarbonate membrane with a pore size of 400 nm to obtain the melanin nanocapsule solution.

7. The preparation method according to claim 1, wherein In step (2), in the polycaprolactone solution, the solid-liquid ratio of polycaprolactone to solvent is (22-28): 100 g / mL; the solvent consists of chloroform and methanol, and the volume ratio of chloroform to methanol is 5:1; The conditions for the electrospinning include: voltage of 10-15 kV, spinning solution flow rate of 6-10 mL / h, distance between the injection needle and the receiver of 22-25 cm, and a rotation speed of the receiving device of 200-400 rpm.

8. The preparation method according to claim 1, wherein In step (3), the polycaprolactone electrospun blood vessel is immersed in an ethylenediamine solution, shaken at 20-30° C. for 25-35 min, and then rinsed with a phosphate buffer solution; The temperature of soaking in the 4-morpholineethanesulfonic acid solution is 3-5°C, and the time is 35-45 minutes; the pH of the 4-morpholineethanesulfonic acid solution is 5-6; In a 4-morpholineethanesulfonic acid solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and heparin, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.1-0.2 mg / mL, the concentration of N-hydroxysuccinimide is 0.1-0.2 mg / mL, and the concentration of heparin is 8-12 mg / mL; The heparinization temperature is 3-5°C and the time is 2-4h.

9. The preparation method according to claim 1, wherein In step (4), the incubation temperature is 3-5°C and the time is 3-5h.

10. An artificial blood vessel prepared by the preparation method according to any one of claims 1 to 9.

Images