Artificial blood vessel and preparation method thereof

Abstract

The invention discloses an artificial blood vessel and a preparation method thereof, and relates to the technical field of artificial materials. The artificial blood vessel comprises a polymer spinning blood vessel base; the polymer coating with positive charges is attached to the surface of the polymer spinning blood vessel substrate; and the erythrocyte membrane vesicles of the eversion phosphatidylserine are attached to the surface of the positively charged polymer coating through electrostatic adsorption. The artificial blood vessel provided by the invention can inhibit blood vessel restenosis by virtue of the immune regulation and control capability of phosphatidylserine, and finally the long-term patency rate of the artificial blood vessel is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial materials, and in particular to an artificial blood vessel and a preparation method thereof. Background Art

[0002] In the prior art, the materials used to prepare small-caliber artificial blood vessels are generally divided into two categories: one is artificial synthetic polymer materials, such as polyester, expanded polytetrafluoroethylene, polyurethane, PGA, PHA, etc.; the other is natural biomaterials, such as collagen, hyaluronic acid, silk fibroin, chitosan, bacterial cellulose, etc. However, the foreign body reaction of the material itself induces macrophages to become pro-inflammatory phenotypes for a long time, destroys the homeostasis of smooth muscle cells, and causes vascular restenosis, which is one of the important reasons for the failure of small-caliber artificial blood vessel transplantation. Pro-inflammatory macrophages release a large number of inflammatory factors, such as IL1β or TNF-α, which induce smooth muscle cell homeostasis disorders and transform from contractile to secretory types, promote their migration, excessive proliferation and secretion of a large amount of extracellular matrix, causing long-term stenosis of small-caliber artificial blood vessels. Summary of the invention

[0003] The main purpose of the present invention is to provide an artificial blood vessel and a preparation method thereof, aiming to solve the problem that small-caliber artificial blood vessels in the prior art are prone to long-term stenosis.

[0004] To achieve the above object, the present invention provides an artificial blood vessel, comprising:

[0005] polymer-spun vascular substrate;

[0006] A positively charged polymer coating is attached to the surface of the polymer-spun blood vessel substrate; and

[0007] The erythrocyte membrane vesicles with externalized phosphatidylserine are attached to the surface of the positively charged polymer coating by electrostatic adsorption.

[0008] In some embodiments, the material of the polymer-spun blood vessel substrate includes polycaprolactone, polylactic acid, polyvinyl alcohol, and polylactic acid / glycolic acid copolymer.

[0009] In some embodiments, the material of the positively charged polymer coating includes polydopamine, polyethyleneimine, and chitosan.

[0010] In some embodiments, the loading amount of the erythrocyte membrane vesicles with externalized phosphatidylserine is 2-5 mg / m 2 .

[0011] In some embodiments, the inner diameter of the artificial blood vessel is 0-6 mm.

[0012] In another aspect, the present application provides a method for preparing an artificial blood vessel, comprising the following steps:

[0013] S1, using hypotonic and ultrasonic methods to obtain erythrocyte membrane vesicles with externalized phosphatidylserine;

[0014] S2, preparation of polymer-spun vascular substrate using electrospinning technology;

[0015] S3, placing the polymer spinning blood vessel substrate in a polymer monomer solution, and polymerizing the polymer monomers in the polymer monomer solution under alkaline conditions to form a positively charged polymer coating attached to the surface of the polymer spinning blood vessel substrate;

[0016] S4, placing the polymer-spun blood vessel substrate attached with a positively charged polymer coating in a solution in which the erythrocyte membrane vesicles with the everted phosphatidylserine are dispersed, to obtain an artificial blood vessel modified with the erythrocyte membrane vesicles with the everted phosphatidylserine.

[0017] In some embodiments, step S1 comprises:

[0018] obtaining a red blood cell precipitate, and subjecting the red blood cell precipitate to a hypotonic treatment and centrifugation in sequence to obtain a precipitate;

[0019] The precipitate is subjected to ultrasonic treatment, and then centrifuged and resuspended in sequence to obtain the erythrocyte membrane vesicles with externalized phosphatidylserine;

[0020] The ultrasonic treatment time is 5-20 min, and the ultrasonic treatment frequency is 8-10 kHz.

[0021] In some embodiments, in step S2, in the electrospinning technology, the injection speed of the syringe pump is 0.3-0.5 mm / min, the applied voltage is 6.5-7 kV, the receiver includes a stainless steel rod with a diameter of 2-3 mm, and the rotation speed of the receiver is 30-35 r / min.

[0022] In some embodiments, in step S3, the polymer monomer solution includes a dopamine solution, and the dopamine solution is prepared by the following method:

[0023] Adding dopamine hydrochloride to a Tris-HCl buffer solution with a pH of 8.5-9 and a concentration of 1-1.2 mg / mL to obtain a solution with a dopamine concentration of 0.25-0.5 mg / mL; and / or,

[0024] The polymerization reaction time in step S3 is 6-12 hours, and the polymerization reaction is carried out under shaking conditions.

[0025] In some embodiments, in step S4, the concentration of the erythrocyte membrane vesicles with externalized phosphatidylserine in the solution in which the erythrocyte membrane vesicles with externalized phosphatidylserine are dispersed is 10-100 μg / mL; and / or,

[0026] The polymer spinning blood vessel substrate attached with the positively charged polymer coating is placed in a solution of red blood cell membrane vesicles dispersed with the everted phosphatidylserine, and incubated with shaking, and the shaking incubation time is 12-15 hours.

[0027] In the technical solution of the present invention, the artificial blood vessel includes erythrocyte membrane vesicles with everted phosphatidylserine, and the erythrocyte membrane vesicles with everted phosphatidylserine are attached to the surface of the positively charged polymer coating by electrostatic adsorption, thereby relying on the immune regulatory ability of phosphatidylserine to inhibit the activation of M1 macrophages and enhance the activation of M2 macrophages, thereby regulating the growth behavior of smooth muscle cells, inhibiting vascular restenosis, and ultimately improving the long-term patency rate of small-caliber artificial blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 the structures shown in these drawings without paying creative work.

[0029] Figure 1 This is a flow chart of the preparation process of the artificial blood vessel provided in Example 1 of the present invention.

[0030] Figure 2 Figure a is a graph showing the results of the determination of the number of externalized phosphatidylserine erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1 in the present invention; Figure 2 Figure b is a graph showing the results of the determination of the externalization rate of the externalized phosphatidylserine erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1 in the present invention.

[0031] Figure 3 Figure a is a bar graph showing the expression results of IL-1β and iNOS in the erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1. Figure 3 b is a bar graph showing the expression results of CD206 and YM1 of the erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1; Figure 3 Figure c is a bar graph showing the expression results of the secretory proteins TNF-α and IL10 of the erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1.

[0032] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0034] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0035] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0036] In the prior art, the materials used to prepare small-caliber artificial blood vessels are generally divided into two categories: one is artificial synthetic polymer materials, such as polyester, expanded polytetrafluoroethylene, polyurethane, PGA, PHA, etc.; the other is natural biomaterials, such as collagen, hyaluronic acid, silk fibroin, chitosan, bacterial cellulose, etc. However, the foreign body reaction of the material itself induces macrophages to become pro-inflammatory phenotypes for a long time, destroys the homeostasis of smooth muscle cells, and causes vascular restenosis, which is one of the important reasons for the failure of small-caliber artificial blood vessel transplantation. Pro-inflammatory macrophages release a large number of inflammatory factors, such as IL1β or TNF-α, which induce smooth muscle cell homeostasis disorders and transform from contractile to secretory types, promote their migration, excessive proliferation and secretion of a large amount of extracellular matrix, causing long-term stenosis of small-caliber artificial blood vessels.

[0037] At present, the research on small-caliber artificial blood vessels for regulating immunity is mainly carried out by loading bioactive factors, such as IL10, exosomes and nitric oxide, etc. However, the high cost or potential ethical issues of bioactive factors greatly limit their application.

[0038] In view of this, on the one hand, the present invention provides an artificial blood vessel, comprising:

[0039] polymer-spun vascular substrate;

[0040] A positively charged polymer coating is attached to the surface of the polymer-spun blood vessel substrate; and

[0041] The erythrocyte membrane vesicles with externalized phosphatidylserine are attached to the surface of the positively charged polymer coating by electrostatic adsorption.

[0042] In the technical solution of the present invention, the artificial blood vessel includes erythrocyte membrane vesicles with everted phosphatidylserine, and the erythrocyte membrane vesicles with everted phosphatidylserine are attached to the surface of the positively charged polymer coating by electrostatic adsorption, thereby relying on the immune regulatory ability of phosphatidylserine to inhibit the activation of M1 macrophages and enhance the activation of M2 macrophages, thereby regulating the growth behavior of smooth muscle cells, inhibiting vascular restenosis, and ultimately improving the long-term patency rate of small-caliber artificial blood vessels.

[0043] In some embodiments, the material of the polymer-spun blood vessel substrate includes polycaprolactone, polylactic acid, polyvinyl alcohol, and polylactic acid / glycolic acid copolymer.

[0044] In some embodiments, the material of the positively charged polymer coating includes polydopamine, polyethyleneimine, and chitosan. Since the erythrocyte membrane vesicles with everted phosphatidylserine are negatively charged, the use of the above-mentioned suitable positively charged polymer coating can effectively attach the erythrocyte membrane vesicles with everted phosphatidylserine to the positively charged polymer coating through electrostatic adsorption.

[0045] In some embodiments, the loading amount of the erythrocyte membrane vesicles with externalized phosphatidylserine is 2-5 mg / m 2 .

[0046] In some embodiments, the inner diameter of the artificial blood vessel is 0-6 mm.

[0047] In another aspect, the present application provides a method for preparing an artificial blood vessel, comprising the following steps:

[0048] S1, using hypotonic and ultrasonic methods to obtain erythrocyte membrane vesicles with externalized phosphatidylserine;

[0049] S2, preparation of polymer-spun vascular substrate using electrospinning technology;

[0050] S3, placing the polymer spinning blood vessel substrate in a polymer monomer solution, and polymerizing the polymer monomers in the polymer monomer solution under alkaline conditions to form a positively charged polymer coating attached to the surface of the polymer spinning blood vessel substrate;

[0051] S4, placing the polymer-spun blood vessel substrate attached with a positively charged polymer coating in a solution in which the erythrocyte membrane vesicles with the everted phosphatidylserine are dispersed, to obtain an artificial blood vessel modified with the erythrocyte membrane vesicles with the everted phosphatidylserine.

[0052] The present application uses a hypotonic + ultrasonic method to treat the erythrocyte sedimentation, and can obtain more than 70% of the erythrocyte membrane vesicles with phosphatidylserine eversion. The hypotonic + ultrasonic method of the present application can obtain everted phosphatidylserine erythrocyte membrane vesicles with immune properties.

[0053] In the preparation method proposed in the present invention, firstly, the polymer spun blood vessel substrate is prepared by electrospinning technology, then a polymer coating is generated in situ on the polymer spun blood vessel substrate, and finally an artificial blood vessel is obtained by placing it in a solution in which the erythrocyte membrane vesicles of the everted phosphatidylserine are dispersed. The steps of this preparation method are simple and the preparation efficiency is high; in addition, the artificial blood vessel modified with the erythrocyte membrane vesicles of the everted phosphatidylserine and having good long-term patency performance prepared by the preparation method proposed in the present invention has all the beneficial effects of the artificial blood vessel modified with the erythrocyte membrane vesicles of the everted phosphatidylserine and having good long-term patency performance, which will not be described one by one here.

[0054] In some embodiments, step S1 comprises:

[0055] obtaining a red blood cell precipitate, and subjecting the red blood cell precipitate to a hypotonic treatment and centrifugation in sequence to obtain a precipitate;

[0056] The precipitate is subjected to ultrasonic treatment, and then centrifuged and resuspended in sequence to obtain the erythrocyte membrane vesicles with externalized phosphatidylserine;

[0057] The ultrasonic treatment time is 5-20 min, and the ultrasonic treatment frequency is 8-10 kHz.

[0058] In some embodiments, in step S2, in the electrospinning technology, the injection speed of the syringe pump is 0.3-0.5 mm / min, the applied voltage is 6.5-7 kV, the receiver includes a stainless steel rod with a diameter of 2-3 mm, and the rotation speed of the receiver is 30-35 r / min.

[0059] In some embodiments, in step S3, the polymer monomer solution includes a dopamine solution, and the dopamine solution is prepared by the following method: adding dopamine hydrochloride to a Tris-HCl buffer with a pH of 8.5-9 and a concentration of 1-1.2 mg / mL to obtain a solution with a dopamine concentration of 0.25-0.5 mg / mL; the polymerization reaction time in step S3 is 6-12 hours, and the polymerization reaction is carried out under oscillation conditions.

[0060] In some embodiments, in step S4, the concentration of erythrocyte membrane vesicles with externalized phosphatidylserine in the solution of erythrocyte membrane vesicles with externalized phosphatidylserine is 10-100 μg / mL; the polymer-spun blood vessel substrate attached with a positively charged polymer coating is placed in the solution of erythrocyte membrane vesicles with externalized phosphatidylserine and incubated with shaking, and the time of shaking incubation is 12-15 hours.

[0061] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0062] Example 1

[0063] This embodiment provides a method for preparing an artificial blood vessel, and an artificial blood vessel prepared by the method. Figure 1 , the preparation method comprises the following steps:

[0064] (1) Preparation of erythrocyte membrane vesicles with externalized phosphatidylserine: New Zealand white rabbit blood was collected using a blood collection tube and centrifuged at 2000 r / min for 15 min. The erythrocyte precipitate was collected and resuspended in 0.25X PBS and subjected to hypotonic treatment for 1.5 h. The precipitate was collected by centrifugation at 8000 g / min for 10 min, centrifuged twice, and then ultrasonically disrupted for 5 min. The disrupted erythrocyte membrane was centrifuged at 10000 g / min for 10 min, the precipitate was collected, resuspended in PBS, and centrifuged twice to finally obtain erythrocyte membrane vesicles with externalized phosphatidylserine.

[0065] (2) Preparation of artificial blood vessels modified with erythrocyte membrane vesicles of externalized phosphatidylserine: Polycaprolactone (PCL, average molecular weight 80,000) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 15% (w / v) electrospinning solution. Subsequently, the electrospinning solution was loaded into a 5 ml syringe and injected using a syringe pump. The injection speed was 0.3 mm / min and the applied voltage was 6.5 kV. The receiver was a stainless steel rod with a diameter of 2 mm, rotating at a speed of 30 r / min. After 70 minutes, the tubular stent was removed from the rod to obtain a polymer-spun blood vessel substrate.

[0066] Dopamine hydrochloride was added to a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 1 mg / mL to obtain a solution with a concentration of 0.25 mg / mL. The polymer-spun blood vessel substrate was immersed in the dopamine reaction solution and incubated with shaking for 6-12 hours. After being taken out, it was washed three times with distilled water. Then, it was immersed in a PBS solution containing 50 μg / mL of erythrocyte membrane vesicles of everted phosphatidylserine, incubated with shaking for 12 hours, and washed three times with distilled water to finally obtain an artificial blood vessel modified with erythrocyte membrane vesicles of everted phosphatidylserine. The inner diameter of the artificial blood vessel in this embodiment is 2.5 mm, and the loading amount of the erythrocyte membrane vesicles of everted phosphatidylserine in this embodiment is 2.2 mg / m 2 .

[0067] Example 2

[0068] This embodiment provides a method for preparing an artificial blood vessel, and an artificial blood vessel prepared by the method. The preparation method comprises the following steps:

[0069] (1) Preparation of erythrocyte membrane vesicles with externalized phosphatidylserine: New Zealand white rabbit blood was collected using a blood collection tube and centrifuged at 2000 r / min for 15 min. The erythrocyte precipitate was collected and resuspended in 0.25X PBS and subjected to hypotonic treatment for 1.5 h. The precipitate was collected by centrifugation at 8000 g / min for 10 min, centrifuged twice, and then ultrasonically disrupted for 5 min. The disrupted erythrocyte membrane was centrifuged at 10000 g / min for 10 min, the precipitate was collected, resuspended in PBS, and centrifuged twice to finally obtain erythrocyte membrane vesicles with externalized phosphatidylserine.

[0070] (2) Preparation of artificial blood vessels modified with erythrocyte membrane vesicles of externalized phosphatidylserine: Polycaprolactone (PCL, average molecular weight 80,000) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 15% (w / v) electrospinning solution. Subsequently, the electrospinning solution was loaded into a 5 ml syringe and injected using a syringe pump. The injection speed was 0.3 mm / min and the applied voltage was 6.5 kV. The receiver was a stainless steel rod with a diameter of 2 mm, rotating at a speed of 30 r / min. After 70 minutes, the tubular stent was removed from the rod to obtain a polymer-spun blood vessel substrate.

[0071] Dopamine hydrochloride was added to a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 1 mg / mL to obtain a 0.25 mg / mL solution. The polymer-spun blood vessel substrate was immersed in the dopamine reaction solution and incubated with shaking for 6-12 hours. After being taken out, it was washed 3 times with distilled water. Then it was immersed in a red blood cell membrane vesicle solution containing 10 μg / mL of externalized phosphatidylserine, incubated with shaking for 12 hours, and washed 3 times with distilled water to finally obtain an artificial blood vessel modified with red blood cell membrane vesicles of externalized phosphatidylserine. The inner diameter of the artificial blood vessel in this embodiment is 2.3 mm, and the loading amount of the red blood cell membrane vesicles of externalized phosphatidylserine in this embodiment is 3.0 mg / m 2 .

[0072] Example 3

[0073] This embodiment provides a method for preparing an artificial blood vessel, and an artificial blood vessel prepared by the method. The preparation method comprises the following steps:

[0074] (1) Preparation of erythrocyte membrane vesicles with externalized phosphatidylserine: New Zealand white rabbit blood was collected using a blood collection tube and centrifuged at 2000 r / min for 15 min. The erythrocyte precipitate was collected and resuspended in 0.25X PBS and subjected to hypotonic treatment for 1.5 h. The precipitate was collected by centrifugation at 8000 g / min for 10 min, centrifuged twice, and then ultrasonically disrupted for 5 min. The disrupted erythrocyte membrane was centrifuged at 10000 g / min for 10 min, the precipitate was collected, resuspended in PBS, and centrifuged twice to finally obtain erythrocyte membrane vesicles with externalized phosphatidylserine.

[0075] (2) Preparation of artificial blood vessels modified with erythrocyte membrane vesicles of externalized phosphatidylserine: Polycaprolactone (PCL, average molecular weight 80,000) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 15% (w / v) electrospinning solution. Subsequently, the electrospinning solution was loaded into a 5 ml syringe and injected using a syringe pump. The injection speed was 0.3 mm / min and the applied voltage was 6.5 kV. The receiver was a stainless steel rod with a diameter of 2 mm, rotating at a speed of 30 r / min. After 70 minutes, the tubular stent was removed from the rod to obtain a polymer-spun blood vessel substrate.

[0076] Dopamine hydrochloride was added to a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 1 mg / mL to obtain a solution with a dopamine concentration of 0.25 mg / mL, and a dopamine reaction solution was obtained. The polymer-spun blood vessel substrate was immersed in the dopamine reaction solution and incubated with shaking for 6-12 hours, and then washed with distilled water for 3 times; then immersed in a erythrocyte membrane vesicle solution containing 100 μg / mL of externalized phosphatidylserine, incubated with shaking for 12 hours, and washed with distilled water for 3 times, and finally an artificial blood vessel modified with erythrocyte membrane vesicles of externalized phosphatidylserine was obtained. The inner diameter of the artificial blood vessel in this embodiment is 2.6 mm, and the loading amount of the erythrocyte membrane vesicles of externalized phosphatidylserine in this embodiment is 2.5 mg / m 2 .

[0077] Example 4

[0078] This embodiment provides a method for preparing an artificial blood vessel, and an artificial blood vessel prepared by the method. The preparation method comprises the following steps:

[0079] (1) Preparation of erythrocyte membrane vesicles with externalized phosphatidylserine: 20 ml of New Zealand rabbit blood was collected using a blood collection tube and centrifuged at 2000 r / min for 15 min. The erythrocyte precipitate was collected and resuspended in 0.25X PBS and subjected to hypotonic treatment for 1.5 h. The precipitate was collected by centrifugation at 8000 g / min for 10 min, and the process was repeated twice. The precipitate was then ultrasonically disrupted for 5 min. The disrupted erythrocyte membrane was centrifuged at 10000 g / min for 10 min, the precipitate was collected, and the process was repeated twice. Finally, erythrocyte membrane vesicles with externalized phosphatidylserine were obtained.

[0080] (2) Preparation of artificial blood vessels modified with erythrocyte membrane vesicles of externalized phosphatidylserine: Polycaprolactone (PCL, average molecular weight 80,000) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 15% (w / v) electrospinning solution. Subsequently, the electrospinning solution was loaded into a 5 ml syringe and injected using a syringe pump. The injection speed was 0.3 mm / min and the applied voltage was 6.5 kV. The receiver was a stainless steel rod with a diameter of 2 mm, rotating at a speed of 30 r / min. After 70 minutes, the tubular stent was removed from the rod to obtain a polymer-spun blood vessel substrate.

[0081] (3) Add dopamine hydrochloride to a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 1 mg / mL to obtain a solution with a concentration of 0.25 mg / mL, immerse the polymer-spun blood vessel substrate in the dopamine reaction solution and shake and incubate for 6-12 hours, then take it out and wash it three times with distilled water to obtain a polymer-spun blood vessel substrate with a positively charged polymer coating;

[0082] (4) dispersing the erythrocyte membrane vesicles with externalized phosphatidylserine obtained in step (1) in PBS to obtain a dispersion, immersing the polymer-spun blood vessel substrate attached with a positively charged polymer coating in the dispersion, incubating with shaking for 12 hours, and washing with distilled water for 3 times to finally obtain an artificial blood vessel modified with erythrocyte membrane vesicles with externalized phosphatidylserine.

[0083] Comparative Example 1

[0084] This embodiment provides a method for preparing an artificial blood vessel, and an artificial blood vessel prepared by the method. The preparation method comprises the following steps:

[0085] (1) 20 ml of New Zealand rabbit blood was collected using a blood collection tube, centrifuged at 2000 r / min for 15 min, the red blood cell pellet was collected, resuspended in 0.25X PBS, and subjected to hypotonic treatment for 1.5 h; the pellet was collected by centrifugation at 8000 g / min for 10 min, and the process was repeated twice. The red blood cell membrane was then filtered using a filter membrane with a pore size of 200 nm to obtain red blood cell membrane vesicles with no or a small amount of externalized phosphatidylserine.

[0086] (2) Preparation of artificial blood vessels: Polycaprolactone (PCL, average molecular weight 80,000) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 15% (w / v) electrospinning solution. Subsequently, the electrospinning solution was loaded into a 5 ml syringe and injected using a syringe pump. The injection speed was 0.3 mm / min and the applied voltage was 6.5 kV. The receiver was a stainless steel rod with a diameter of 2 mm, rotating at a speed of 30 r / min. After 70 minutes, the tubular stent was removed from the rod to obtain a polymer-spun blood vessel substrate.

[0087] (3) adding dopamine hydrochloride to a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 1 mg / mL to obtain a dopamine solution with a concentration of 0.25 mg / mL, thereby obtaining a dopamine reaction solution, immersing the polymer-spun blood vessel substrate in the dopamine reaction solution and incubating the substrate under shaking for 6-12 hours, taking the substrate out and washing it with distilled water for 3 times to obtain a polymer-spun blood vessel substrate with a positively charged polymer coating;

[0088] (4) The red blood cell membrane vesicles obtained in step (1) are dispersed in PBS to obtain a dispersion, and the polymer-spun blood vessel substrate with a positively charged polymer coating is immersed in the dispersion, incubated with shaking for 12 hours, and washed with distilled water for 3 times to finally obtain an artificial blood vessel.

[0089] Comparative Example 2

[0090] Polycaprolactone (PCL, average molecular weight 80000) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to prepare a 15% (w / v) electrospinning solution. Subsequently, the electrospinning solution was loaded into a 5ml syringe and injected with a syringe pump. The injection speed was 0.3mm / min and the applied voltage was 6.5kV. The receiver was a stainless steel rod with a diameter of 2mm, rotating at a speed of 30r / min. After 70 minutes, the tubular stent was removed from the rod to obtain an artificial blood vessel.

[0091] Performance Testing

[0092] (1) The externalization of the erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1 was measured respectively, and the measurement method was: the externalization of phosphatidylserine of the erythrocyte membrane vesicles was measured by flow cytometry.

[0093] Results Figure 2 A and Figure 2 In b, by comparison, the treatment method used in Example 4 (PS-EMVs) of the present application greatly stimulates the externalization of phosphatidylserine. The treatment method of Example 4 (PS-EMVs) of the present application causes more than 70% of the erythrocyte membrane vesicles to undergo externalization of phosphatidylserine, and flow cytometry analysis shows that the externalization rate of phosphatidylserine of FITC-Annexin V-labeled PS-EMVs is 73.68±1.37%. While the treatment method of Comparative Example 1, the externalization rate of erythrocyte membrane vesicles (EMVs) is less than 10%.

[0094] (2) The immune function of the externalized phosphatidylserine erythrocyte membrane vesicles obtained in step (1) of Example 4 and Comparative Example 1 was measured respectively. The measuring method was as follows: a macrophage verification model was constructed (macrophages were treated with 1 mg / ml for 4 hours in advance), the externalized phosphatidylserine erythrocyte membrane vesicles in Example 4 and Comparative Example 1 were incubated with inflammatory macrophages for 24 hours, and the externalized phosphatidylserine of the erythrocyte membrane vesicles was measured by flow cytometry.

[0095] Results Figure 3ac, wherein PS-EMVs is the everted phosphatidylserine erythrocyte membrane vesicle obtained in Example 4, EMVs is the everted phosphatidylserine erythrocyte membrane vesicle obtained in Comparative Example 1, PBS is the erythrocyte membrane vesicle without everted phosphatidylserine, and PBS solution is used. The results show that PS-EMVs reduce the expression of macrophage proinflammatory genes IL-1β and inos, and increase the expression of anti-inflammatory genes CD206 and YM1. PS-EMVs inhibit the expression of proinflammatory factor TNF-α and promote the expression of anti-inflammatory factor IL10. The treatment method used in Example 4 (PS-EMVs) of the present application will greatly stimulate the eversion of phosphatidylserine, so that it has immune regulation ability. The treatment method used in Comparative Example 1 does not have immune regulation ability. The immune regulation effect of the erythrocyte membrane vesicle in Comparative Example 1 tends to be immune escape, which is more suitable as a camouflage for nanocarriers and prolongs its blood circulation time. The erythrocyte membrane vesicles of Example 4 are more suitable for application in tissue repair and regeneration.

[0096] It can be seen that the everted phosphatidylserine erythrocyte membrane vesicles obtained by the treatment method adopted in Example 4 (PS-EMVs) of the present application are suitable for application in tissue repair and regeneration due to their immunomodulatory ability. Thus, by including the erythrocyte membrane vesicles of everted phosphatidylserine in the artificial blood vessels of the present application embodiment, the erythrocyte membrane vesicles of everted phosphatidylserine are attached to the surface of the positively charged polymer coating by electrostatic adsorption, thereby inhibiting the activation of M1 macrophages and enhancing the activation of M2 macrophages by virtue of the immunomodulatory ability of phosphatidylserine, thereby regulating the growth behavior of smooth muscle cells, inhibiting vascular restenosis, and ultimately improving the long-term patency rate of small-caliber artificial blood vessels.

[0097] The above description is only an exemplary embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An artificial blood vessel, It is characterized in that include: polymer-spun vascular substrate; A positively charged polymer coating is attached to the surface of the polymer-spun blood vessel substrate; as well as, The erythrocyte membrane vesicles with everted phosphatidylserine are attached to the surface of the positively charged polymer coating by electrostatic adsorption.

2. The artificial blood vessel according to claim 1, It is characterized in that The material of the polymer spinning blood vessel substrate includes polycaprolactone, polylactic acid, polyvinyl alcohol, and polylactic acid / glycolic acid copolymer.

3. The artificial blood vessel according to claim 1, It is characterized in that The material of the positively charged polymer coating includes polydopamine, polyethyleneimine and chitosan.

4. The artificial blood vessel according to claim 1, It is characterized in that The loading amount of the erythrocyte membrane vesicles with externalized phosphatidylserine is 2-5 mg / m 2 .

5. The artificial blood vessel according to claim 1, It is characterized in that The inner diameter of the artificial blood vessel is 0-6 mm.

6. A method for preparing an artificial blood vessel according to any one of claims 1 to 5, It is characterized in that The following steps are involved: S1, using hypotonic and ultrasonic methods to obtain erythrocyte membrane vesicles with externalized phosphatidylserine; S2, preparation of polymer-spun vascular substrate using electrospinning technology; S3, placing the polymer spinning blood vessel substrate in a polymer monomer solution, and polymerizing the polymer monomers in the polymer monomer solution under alkaline conditions to form a positively charged polymer coating attached to the surface of the polymer spinning blood vessel substrate; S4, placing the polymer-spun blood vessel substrate attached with a positively charged polymer coating in a solution in which the erythrocyte membrane vesicles with the everted phosphatidylserine are dispersed, to obtain an artificial blood vessel modified with the erythrocyte membrane vesicles with the everted phosphatidylserine.

7. The preparation method according to claim 6, It is characterized in that The step S1 comprises: obtaining a red blood cell precipitate, and subjecting the red blood cell precipitate to a hypotonic treatment and centrifugation in sequence to obtain a precipitate; The precipitate is subjected to ultrasonic treatment, and then centrifuged and resuspended in sequence to obtain the erythrocyte membrane vesicles with externalized phosphatidylserine; The ultrasonic treatment time is 5-20 min, and the ultrasonic treatment frequency is 8-10 kHz.

8. The preparation method according to claim 6, It is characterized in that In the step S2, in the electrospinning technology, the injection speed of the injection pump is 0.3-0.5 mm / min, the applied voltage is 6.5-7 kV, the receiver includes a stainless steel rod with a diameter of 2-3 mm, and the rotation speed of the receiver is 30-35 r / min.

9. The preparation method according to claim 6, It is characterized in that In step S3, the polymer monomer solution includes a dopamine solution, and the dopamine solution is prepared by the following method: Adding dopamine hydrochloride to a Tris-HCl buffer solution with a pH of 8.5-9 and a concentration of 1-1.2 mg / mL to obtain a solution with a dopamine concentration of 0.25-0.5 mg / mL; and / or, The polymerization reaction time in step S3 is 6-12 hours, and the polymerization reaction is carried out under shaking conditions.

10. The preparation method according to claim 6, It is characterized in that In the step S4, the concentration of the erythrocyte membrane vesicles with externalized phosphatidylserine in the solution in which the erythrocyte membrane vesicles with externalized phosphatidylserine are dispersed is 10-100 μg / mL; and / or, The polymer spinning blood vessel substrate attached with the positively charged polymer coating is placed in a solution of red blood cell membrane vesicles dispersed with the everted phosphatidylserine, and incubated with shaking, and the shaking incubation time is 12-15 hours.

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