Small-caliber tissue engineering intravascular stent and preparation method thereof

Abstract

The invention relates to the technical field of intravascular stents, in particular to a small-caliber tissue engineering intravascular stent and a preparation method thereof. The ACR is added into the spinning solution before spinning, the spinning solution is cured into the fibers by using the high-voltage electric field to prepare the PCL / SF / ACR intravascular stent, and then the PCL / SF / ACR intravascular stent is modified with Rm and Gly in a weakly alkaline or neutral environment, so that the problem of poor combination effect of an existing modification material and the PCL / SF composite intravascular stent is solved. By means of the method, thrombus, inflammation and calcification of the artificial blood vessel are not prone to forming, slow release of ACR in the body can be achieved, and the long-term effect and safety of the small-caliber tissue engineering intravascular stent in practical application are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of vascular stents, and in particular to a small-caliber tissue engineering vascular stent with anti-inflammatory, anti-coagulation and anti-calcification properties and a preparation method thereof. Background Art

[0002] At present, the main clinical treatment for cardiovascular diseases is vascular reconstruction, including stent intervention and coronary artery bypass grafting. The blood vessels used in vascular bypass grafting often come from the patient's own body, which has certain shortcomings, such as the requirement for high quality of the patient's own blood vessels, the limited number of available blood vessels, and the many complications of the use of autologous blood vessels. Therefore, the demand for artificial blood vessel substitutes is more urgent. However, artificial blood vessels are prone to calcification, infection, and low long-term patency. Therefore, it is necessary to develop small-caliber artificial blood vessel stents with an inner diameter of <6mm suitable for coronary artery bypass grafting.

[0003] Due to the limitation of materials and technology, small-caliber artificial blood vessels have always been a difficult problem in research and development. The current vascular materials are not only prone to cause coagulation and thrombosis due to blood compatibility issues, leading to vascular lumen stenosis or vascular occlusion, but also unable to promote the adhesion and growth of endothelial cells. Therefore, it is difficult for artificial blood vessels to achieve rapid endothelialization after implantation in the body, which is prone to cause vascular coagulation and embolism problems.

[0004] Polycaprolactone, referred to as PCL, is a high molecular organic polymer polymerized from ε-caprolactone monomers. It has excellent mechanical properties and is considered a potential biomedical material. However, pure PCL materials have deficiencies in biocompatibility, anti-calcification, and anti-inflammation. Silk fibroin, referred to as SF, is a natural high molecular fibrous protein extracted from silk. It has good biocompatibility, low immunogenicity, and no toxic side effects. It is a potential tissue engineering vascular scaffold.

[0005] Based on this, some researchers have compounded PCL and SF to make PCL / SF composite vascular stents. Although PCL / SF composite vascular stents have excellent biocompatibility and can induce vascular regeneration when transplanted into animals, in practical applications, they are still insufficient in anti-calcification, anti-inflammation and long-term blood flow permeability. In order to solve the above problems, researchers have tried to modify the PCL / SF composite vascular stents. However, the types of modification materials currently available are limited, such as nucleoside derivatives, α-amino acids, etc. These modification materials face the problem of unsatisfactory binding effect when combined with PCL and SF on the PCL / SF composite vascular stents. This unsatisfactory binding effect leads to poor stability of the composite vascular stent material obtained after modification, which in turn affects the long-term effect and safety of the composite vascular stent material in practical applications. Summary of the invention

[0006] In order to solve the problem that existing modified materials have poor combination effect with PCL / SF composite vascular stents, the purpose of the present invention is to provide a small-caliber tissue engineering vascular stent with anti-inflammatory, anti-coagulation and anti-calcification properties and a preparation method thereof.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] The first aspect of the present invention provides a method for preparing a small-caliber tissue engineering vascular stent, comprising the following steps:

[0009] A polycaprolactone-silk fibroin mixed solution was mixed with a 5-amino-4-imidazolecarboxamide nucleoside solution to prepare a spinning solution; the spinning solution was solidified into fibers using a high-voltage electrospinning technique to prepare a PCL / SF / ACR vascular scaffold; a red blood cell membrane dispersion with a pH value of 7.5 to 8 was mixed with the PCL / SF / ACR vascular scaffold, and the mixture was incubated at 2°C to 6°C to prepare a Rm@PCL / SF / ACR vascular scaffold; the Rm@PCL / SF / ACR vascular scaffold was placed in a glycine solution with a pH value of 7 to 10, and incubated at 30°C to 40°C to prepare a small-caliber tissue engineering vascular scaffold.

[0010] The present invention mainly uses polycaprolactone and silk fibroin as basic raw materials, adds 5-amino-4-imidazolecarboxamide nucleoside with anti-inflammatory ability, and prepares a vascular stent by electrospinning technology; then co-incubates it with red blood cell membrane to enhance its anti-thrombotic ability; modifies the vascular stent with glycine to enhance its anti-calcification ability; thereby obtaining a multifunctional small-caliber tissue engineering vascular stent with anti-inflammatory, anti-calcification and anti-coagulation properties, which is denoted as Gly-(Rm@PCL / SF / ACR). Among them, Gly represents glycine; Rm represents red blood cell membrane; PCL represents polycaprolactone; SF represents silk fibroin; ACR represents 5-amino-4-imidazolecarboxamide nucleoside.

[0011] In the method of the present invention, the reaction order of each step cannot be interchanged. First, the red blood cell membrane is loaded onto the vascular stent at a pH of 7.5 to 8, and then the pH of the glycine solution is adjusted to 7 to 10. Since the proline in the silk fibroin is an acidic amino acid, it is negatively charged in an alkaline environment and can be combined with the positively charged head of Rm to load Rm onto the vascular stent. The isoelectric point of glycine is 5.97. When the pH is greater than 5.97, glycine is negatively charged and can be combined with the positively charged head of Rm, thereby loading glycine onto the vascular stent, solving the problem that the existing modified materials have poor binding effects with the PCL / SF composite vascular stent. The method of the present invention can not only make the artificial blood vessel less prone to thrombosis, inflammation and calcification, but also can achieve the slow release of 5-amino-4-imidazole carboxamide nucleoside, red blood cell membrane and glycine in vivo, thereby improving the long-term effect and safety of the small-caliber tissue engineering vascular stent in practical applications.

[0012] Preferably, the mass ratio of polycaprolactone to silk fibroin is 8-8.5:2-1.5.

[0013] Preferably, the polycaprolactone-silk fibroin mixed solution is obtained by dissolving polycaprolactone and silk fibroin in hexafluoroisopropanol; the total mass of polycaprolactone and silk fibroin accounts for 8% to 10% of the mass percentage of the polycaprolactone-silk fibroin mixed solution.

[0014] Preferably, the 5-amino-4-imidazolecarboxamide nucleoside solution is obtained by dissolving 5-amino-4-imidazolecarboxamide nucleoside in dimethyl sulfoxide; the concentration of 5-amino-4-imidazolecarboxamide nucleoside in the 5-amino-4-imidazolecarboxamide nucleoside solution is 3 mmol / L to 5 mmol / L.

[0015] Preferably, the volume ratio of the polycaprolactone-silk fibroin mixed solution to the 5-amino-4-imidazolecarboxamide nucleoside solution is 100:1.

[0016] Preferably, in the red blood cell membrane dispersion, the concentration of red blood cell membrane is 6 mg / mL to 8 mg / mL; the mass ratio of the red blood cell membrane dispersion to the PCL / SF / ACR vascular scaffold is 1 to 1.5:2; and the mass ratio of the Rm@PCL / SF / ACR vascular scaffold to the glycine solution is 1:2 to 3.

[0017] Preferably, the electrospinning parameters of the high-voltage electrospinning technology are: voltage 16 kV to 20 kV, rotation speed 150 rpm / min to 200 rpm / min, extrusion speed 10 mm / s to 12 mm / s, and curing distance 14 cm to 16 cm.

[0018] When using an electrospinning instrument for electrostatic spinning, it was found that if the voltage was below 16kV, the electrospinning liquid could not solidify into electrospun fibers under the action of high voltage electricity, but dripped in the form of droplets. When the voltage was increased to 16-20kV, the electrospinning liquid could be successfully solidified into fibers and collected. However, when the voltage was too high, that is, greater than 20kV, flying fibers would appear, and the fibers could not be collected to make vascular stents.

[0019] Preferably, the preparation process of the red blood cell membrane dispersion is as follows: centrifuge the whole blood to remove the plasma and obtain the red blood cell precipitate; mix the red blood cell precipitate with PBS buffer, incubate at 2°C to 6°C, centrifuge, collect the red blood cell membranes, mix and disperse with PBS buffer to obtain a red blood cell membrane dispersion with a pH of 7.5 to 8.

[0020] Preferably, the mass ratio of the red blood cell membrane precipitate to the PBS buffer is 1:1-2; and the pH value of the PBS buffer is 7.5-8.

[0021] Preferably, the specific operation of centrifuging the whole blood is: centrifuging the whole blood once at a centrifugal force of 200 g, and then centrifuging it twice at a centrifugal force of 1000 g.

[0022] Preferably, the pH value of the PBS buffer is about 6.5, and the concentration of the PBS buffer is 0.01 mol / L to 0.1 mol / L.

[0023] The second aspect of the present invention provides a small-caliber tissue engineering vascular stent prepared by the preparation method described in the first aspect.

[0024] Beneficial effects of the present invention:

[0025] 1. The present invention adds ACR to the spinning solution before spinning, solidifies the spinning solution into fibers using a high voltage electric field to prepare a PCL / SF / ACR vascular stent, and then modifies Rm and Gly on the PCL / SF / ACR vascular stent under a weakly alkaline or neutral environment, thereby solving the problem of poor binding between the existing modified materials and the PCL / SF composite vascular stent. The method of the present invention not only makes it difficult for artificial blood vessels to form thrombosis, inflammation and calcification, but also realizes the slow release of ACR in the body, thereby improving the long-term effect and safety of small-caliber tissue engineering vascular stents in practical applications.

[0026] 2. The present invention modifies Rm and Gly on the PCL / SF / ACR vascular stent to induce directional growth of endothelial cells, achieve rapid vascular endothelialization, inhibit thrombosis and intimal hyperplasia, and reduce the occurrence of thrombosis, inflammation and calcification in artificial blood vessels while being beneficial to autologous vascular regeneration, thus having potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the preparation principle of a Gly-(Rm@PCL / SF / ACR) vascular stent provided in one embodiment of the present invention.

[0028] Figure 2 In the figure, A is a microscopic morphology of the PCL / SF vascular stent prepared in Example 1; B is a microscopic morphology of the PCL / SF / ACR vascular stent prepared in Example 4; C is a microscopic morphology of the Rm@PCL / SF / ACR vascular stent prepared in Example 5; D is a microscopic morphology of the Gly-

[0029] Microscopic morphology of (Rm@PCL / SF / ACR) vascular stent.

[0030] Figure 3 This is a graph showing the ACR release results of the Gly-(Rm@PCL / SF / ACR) vascular stent prepared in Example 11.

[0031] Figure 4 The graphs are the results of blood compatibility tests. A is the result of APTT test; B is the result of TT test; C is the result of HR test; and D is the result of PRT test.

[0032] Figure 5 This is a graph showing the gene expression results of the macrophage marker CCR7.

[0033] Figure 6 This is a graph showing the gene expression results of the macrophage marker CD163.

[0034] Figure 7 This is a graph showing the expression ratio of CCR7 / CD163.

[0035] Figure 8 The figure shows the expression results of IL-10 cytokine.

[0036] Fig. 9 The graph shows the expression results of IL-1α cytokine.

[0037] Fig.10 This is a graph showing the changing trend of calcium content in four groups of vascular stent samples. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0039] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0040] Polycaprolactone, or PCL for short, is a high molecular weight organic polymer made by ring-opening polymerization of ε-caprolactone monomers using metal organic compounds such as tetraphenyltin as catalysts. It has excellent mechanical properties and is considered a potential biomedical material. However, pure PCL materials have deficiencies in biocompatibility, anti-calcification, and anti-inflammation.

[0041] Silk fibroin, whose full English name is Silk Fibroin, abbreviated as SF, is a natural high-molecular fibrin extracted from silk. It has good biocompatibility, low immunogenicity and non-toxic side effects. It is a potential tissue engineering vascular scaffold material.

[0042] Based on this, some researchers have combined PCL and SF to make PCL / SF composite vascular stent materials. PCL / SF composite vascular stent materials have good biocompatibility and can induce vascular regeneration when transplanted into animals, but their anti-calcification, anti-inflammatory and long-term blood flow permeability still have certain problems.

[0043] 5-Amino-4-imidazolecarboxamideribonucleoside, also known as ACR, is an AMPK activator. The Chinese name of AMPK is adenosine monophosphate-activated protein kinase. It is reported that 5-amino-4-imidazolecarboxamideribonucleoside is a nucleoside derivative that has anti-inflammatory effects on cultured muscle cells, adipocytes and macrophages, as well as muscle and adipose tissue of rats treated with lipopolysaccharide.

[0044] Red blood cell membrane, referred to as Rm, contains proteins, glycoproteins and phospholipids that can effectively regulate the functions of biological organisms. Therefore, Rm has good blood compatibility, low immunogenicity and cell compatibility.

[0045] Glycine, also known as aminoacetic acid or aminoacetic acid, has a simplified structural formula of H2N-CH2-COOH. The full name of glycine in English is Glycine, abbreviated as Gly. It is the simplest amino acid and belongs to the α-amino acid class. It is widely involved in various metabolisms of the body and has an inhibitory effect on apoptosis. The anti-apoptotic effect of glycine is that it causes a large influx of chloride ions after binding to the glycine receptor, thereby causing hyperpolarization of the cell membrane. The hyperpolarization of the cell membrane will inevitably cause a large number of voltage-gated calcium channels to be inactivated, thereby alleviating the excessive state of intracellular calcium ion concentration, and then inhibiting its downstream pathways. Therefore, glycine can inhibit the formation of calcification by inhibiting cell apoptosis.

[0046] like Figure 1In one embodiment of the invention, PCL and SF are blended as basic raw materials, and after adding ACR with anti-inflammatory ability, a vascular stent is prepared by electrospinning technology; then it is co-incubated with red blood cell membrane to enhance its anti-thrombotic ability; the vascular stent is modified with glycine to enhance its anti-calcification ability; thereby obtaining a multifunctional small-caliber tissue engineering vascular stent with anti-inflammatory, anti-calcification and anti-coagulation properties, which is recorded as Gly-(Rm@PCL / SF / ACR). The method of the present invention can not only make artificial blood vessels less prone to thrombosis, inflammation and calcification, but also achieve the slow release of ACR in the body.

[0047] The technical solution of the present invention is described in detail below through specific examples. In the following examples, the methods described are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0048] In the following embodiments, the sodium citrate anticoagulant tube is a sodium citrate 9NC anticoagulant tube with a capacity of 2 mL, which contains approximately 0.2 mL of 3.8% sodium citrate solution. The Chinese name of PBS buffer is phosphate buffered saline; the pH value of PBS buffer is 7.5-8, and the concentration is 0.01 mol / L.

[0049] In the following examples, the sources of the raw materials used are shown in Table 1.

[0050] Table 1 Sources of raw materials

[0051]

[0052] Note: Gly is glycine; Ser is serine; Ala is alanine.

[0053] Example 1

[0054] A method for preparing a small-caliber tissue engineering vascular stent comprises the following steps:

[0055] 8g of polycaprolactone and 2g of silk fibroin were dissolved in 100g of hexafluoroisopropanol to obtain a polycaprolactone-silk fibroin mixed solution with a mass percentage of 9%. The polycaprolactone-silk fibroin mixed solution was then used as a spinning solution and fixed on a propeller. The electrospinning parameters were adjusted to: voltage 20kV, rotation speed 150rpm / min, extrusion speed 10mm / s, and solidification distance 14cm. The polymer spinning solution was solidified into fibers using a high-voltage electric field to obtain a small-caliber tissue engineering vascular scaffold, which was recorded as a PCL / SF vascular scaffold.

[0056] Example 2

[0057] A method for preparing a small-caliber tissue engineering vascular stent comprises the following steps:

[0058] 8g of polycaprolactone and 2g of silk fibroin were dissolved in 115g of hexafluoroisopropanol to obtain a polycaprolactone-silk fibroin mixed solution with a mass percentage of 8%. The polycaprolactone-silk fibroin mixed solution was then used as a spinning solution and fixed on a propeller. The electrospinning parameters were adjusted to: voltage 16kV, rotation speed 200pm / min, extrusion speed 12mm / s, and solidification distance 16cm. The polymer spinning solution was solidified into fibers using a high-voltage electric field to obtain a small-caliber tissue engineering vascular scaffold, which was recorded as a PCL / SF vascular scaffold.

[0059] Example 3

[0060] A method for preparing a small-caliber tissue engineering vascular stent comprises the following steps:

[0061] 8.5g of polycaprolactone and 1.5g of silk fibroin were dissolved in 90g of hexafluoroisopropanol to obtain a polycaprolactone-silk fibroin mixed solution with a mass percentage of 10%. The polycaprolactone-silk fibroin mixed solution was then used as a spinning solution and fixed on a propeller. The electrospinning parameters were adjusted to: voltage 18kV, rotation speed 170pm / min, extrusion speed 11mm / s, and solidification distance 15cm. The polymer spinning solution was solidified into fibers using a high-voltage electric field to obtain a small-caliber tissue engineering vascular scaffold, which was recorded as a PCL / SF vascular scaffold.

[0062] During electrospinning, it was found that the total mass of polycaprolactone and silk fibroin accounted for 8% to 10% of the mass percentage of the polycaprolactone-silk fibroin mixture. Within this concentration range, it can be used as the spinning solution for high-voltage electrospinning.

[0063] When using an electrospinning instrument for electrostatic spinning, it was found that if the voltage is below 16kV, the electrospinning liquid cannot be solidified into electrospun fibers under the action of high voltage electricity, but drips in the form of droplets. When the voltage is increased to 16-20kV, the electrospinning liquid can be smoothly solidified into fibers and collected. However, when the voltage is too large, that is, greater than 20kV, flying fibers will appear, and the fibers cannot be collected to make vascular stents. Therefore, the electrospinning parameters of high-voltage electrospinning technology are preferably: voltage 16kV~20kV, rotation speed 150rpm / min~200rpm / min, extrusion speed 10mm / s~12mm / s, and solidification distance 14cm~16cm.

[0064] Example 4

[0065] A method for preparing a small-caliber tissue engineering vascular stent comprises the following steps:

[0066] ACR was dissolved in dimethyl sulfoxide to form an ACR solution with a concentration of 4 mmol / L.

[0067] 8 g of polycaprolactone and 2 g of silk fibroin were dissolved in 100 g of hexafluoroisopropanol to obtain a polycaprolactone-silk fibroin mixed solution with a mass percentage of 9%.

[0068] The polycaprolactone-silk fibroin mixed solution and the ACR solution were mixed at a volume ratio of 100:1 to form a spinning solution. The spinning solution was then fixed on the propeller, and the electrospinning parameters were adjusted to: voltage 20kV, rotation speed 150rpm / min, extrusion speed 10mm / s, and curing distance 14cm. The solution was cured into fibers using a high-voltage electric field to obtain a small-caliber tissue engineering vascular scaffold, which was recorded as a PCL / SF / ACR vascular scaffold.

[0069] Example 5

[0070] A method for preparing a small-caliber tissue engineering vascular stent comprises the following steps

[0071] Step 1, preparation of PCL / SF / ACR vascular stent:

[0072] ACR was dissolved in dimethyl sulfoxide to form an ACR solution with a concentration of 4 mmol / L.

[0073] 8 g of polycaprolactone and 2 g of silk fibroin were dissolved in 100 g of hexafluoroisopropanol to obtain a polycaprolactone-silk fibroin mixed solution with a mass percentage of 9%.

[0074] The polycaprolactone-silk fibroin mixed solution and the ACR solution were mixed at a volume ratio of 100:1 to form a spinning solution. The spinning solution was then fixed on the propeller, and the electrospinning parameters were adjusted to: voltage 20kV, rotation speed 150rpm / min, extrusion speed 10mm / s, and curing distance 14cm. The solution was cured into fibers using a high-voltage electric field to obtain a small-caliber tissue engineering vascular scaffold, which was recorded as a PCL / SF / ACR vascular scaffold.

[0075] Step 2, preparation of Rm@PCL / SF / ACR vascular stent:

[0076] Whole blood from the ear vein of SD rats was placed in a sodium citrate anticoagulation tube. The whole blood was centrifuged at 200g for 10 minutes to remove platelet-rich plasma, and then centrifuged at 1000g for 10 minutes to remove residual plasma and collect the red blood cell precipitate. The red blood cell precipitate was washed 3 times with PBS buffer. The washed red blood cell precipitate was mixed with PBS buffer at a mass ratio of 1:1, and after incubation at 4°C for 1 hour, it was centrifuged at 8000g for 10 minutes to collect the red blood cell membrane. The collected red blood cell membrane was added to PBS buffer, and the red blood cell membrane was mixed and dispersed with PBS buffer to obtain a red blood cell membrane dispersion with a pH of 7.5-8, which was recorded as Rm dispersion. The concentration of red blood cell membrane in the red blood cell membrane dispersion was 6 mg / mL.

[0077] The Rm dispersion was mixed with the PCL / SF / ACR vascular scaffold in a mass ratio of 1:2 and incubated at 4°C for 12 h to obtain the Rm@PCL / SF / ACR vascular scaffold.

[0078] Example 6

[0079] A method for preparing a small-caliber tissue engineering vascular scaffold is different from Example 5 in that the concentration of the erythrocyte membrane in the erythrocyte membrane dispersion is 7 mg / mL.

[0080] Example 7

[0081] A method for preparing a small-caliber tissue engineering vascular scaffold is different from Example 5 in that the concentration of the red blood cell membrane in the red blood cell membrane dispersion is 8 mg / mL.

[0082] Example 8

[0083] A method for preparing a small-caliber tissue engineering vascular scaffold, which is different from Example 5 in that the mass ratio of the Rm dispersion to the PCL / SF / ACR vascular scaffold is 1:1.

[0084] Example 9

[0085] A method for preparing a small-caliber tissue engineering vascular scaffold, which differs from Example 5 in that the mass ratio of the Rm dispersion to the PCL / SF / ACR vascular scaffold is 2:1.

[0086] Example 10

[0087] A method for preparing a small-caliber tissue engineering vascular scaffold, which differs from Example 5 in that the mass ratio of the Rm dispersion to the PCL / SF / ACR vascular scaffold is 1.5:2.

[0088] In order to explore the optimal mass ratio of Rm dispersion and PCL / SF / ACR vascular stent, the Rm dispersion and PCL / SF / ACR vascular stent were first mixed at a mass ratio of 1:1 and 2:1. However, it was found that due to the excessive proportion of Rm dispersion, the Rm coating was too thick on the fiber surface, and the red blood cell membrane that was not modified into the vascular stent was very easy to fall off, which affected the performance of the vascular stent and subsequent experiments. Therefore, the mass of Rm dispersion was reduced, and Rm dispersion was mixed with PCL / SF / ACR vascular stent at a mass ratio of 1 to 1.5:2. The results showed that the anticoagulant performance of the vascular stent was not reduced, and there was no situation where the Rm coating was too thick. After further experimental verification, the Rm@PCL / SF / ACR vascular stent was optimal when the Rm dispersion was mixed with the PCL / SF / ACR vascular stent at a mass ratio of 1:2.

[0089] In order to better modify Rm and Gly, the pH was adjusted to 7.5-8 using PBS buffer. Since proline in silk fibroin is an acidic amino acid and is negatively charged in an alkaline environment, the proline in silk fibroin can combine with the positively charged head of Rm.

[0090] Since the isoelectric point of glycine is 5.97, when pH>5.97, glycine is negatively charged and can bind to the positively charged head of Rm, thereby loading glycine onto the vascular stent; and when pH>10, the silk protein will be denatured. Therefore, before adding glycine, the pH of the glycine solution is adjusted to 7-10 so that the negatively charged glycine can bind to the positively charged head of Rm.

[0091] Based on this, the following examples of the present invention further illustrate the modification method of Rm and Gly.

[0092] Embodiment 11

[0093] A method for preparing a small-caliber tissue engineering vascular stent comprises the following steps:

[0094] Step 1, preparation of PCL / SF / ACR vascular stent:

[0095] ACR was dissolved in dimethyl sulfoxide to form an ACR solution with a concentration of 4 mmol / L.

[0096] 8 g of polycaprolactone and 2 g of silk fibroin were dissolved in 100 g of hexafluoroisopropanol to obtain a polycaprolactone-silk fibroin mixed solution with a mass percentage of 9%.

[0097] The polycaprolactone-silk fibroin mixed solution and the ACR solution were mixed at a volume ratio of 100:1 to form a spinning solution. The spinning solution was then fixed on the propeller, and the electrospinning parameters were adjusted to: voltage 20kV, rotation speed 150rpm / min, extrusion speed 10mm / s, and curing distance 14cm. The solution was cured into fibers using a high-voltage electric field to obtain a small-caliber tissue engineering vascular scaffold, which was recorded as a PCL / SF / ACR vascular scaffold.

[0098] Step 2, preparation of Rm@PCL / SF / ACR vascular stent:

[0099] Whole blood from the ear vein of SD rats was placed in a sodium citrate anticoagulation tube. The whole blood was centrifuged at 200g for 10 minutes to remove platelet-rich plasma, and then centrifuged at 1000g for 10 minutes to remove residual plasma and collect the red blood cell precipitate. The red blood cell precipitate was washed 3 times with PBS buffer. The washed red blood cell precipitate was mixed with PBS buffer at a mass ratio of 1:1, and after incubation at 4°C for 1 hour, it was centrifuged at 8000g for 10 minutes to collect the red blood cell membrane. The collected red blood cell membrane was added to PBS buffer at a mass ratio of 6:1 to PBS buffer, and the mixture was dispersed to obtain a red blood cell membrane dispersion with a pH of 7.5-8, which was recorded as Rm dispersion.

[0100] The Rm dispersion was mixed with the PCL / SF / ACR vascular scaffold in a mass ratio of 1:2 and incubated at 4°C for 12 h to obtain the Rm@PCL / SF / ACR vascular scaffold.

[0101] Step 3, preparation of Gly-(Rm@PCL / SF / ACR) vascular stent:

[0102] The Rm@PCL / SF / ACR vascular stent was placed in a 0.1 mol / L glycine solution; wherein the pH of the glycine solution was 7.5, and the mass ratio of the Rm@PCL / SF / ACR vascular stent to the glycine solution was 1:2. After treatment at 37°C for 48 hours, the Gly-(Rm@PCL / SF / ACR) vascular stent was obtained. The Gly-(Rm@PCL / SF / ACR) vascular stent was placed in a ventilated cabinet for ventilation and drying, and then stored in a clean sealed bag for later use.

[0103] Example 12

[0104] A method for preparing a small-caliber tissue engineering vascular scaffold, which is different from Example 11 in that the mass ratio of the Rm@PCL / SF / ACR vascular scaffold to the glycine solution is 1:1.

[0105] Embodiment 13

[0106] A method for preparing a small-caliber tissue engineering vascular scaffold, which is different from Example 11 in that the mass ratio of the Rm@PCL / SF / ACR vascular scaffold to the glycine solution is 2:3.

[0107] Embodiment 14

[0108] A method for preparing a small-caliber tissue engineering vascular scaffold, which differs from Example 11 in that the mass ratio of the Rm@PCL / SF / ACR vascular scaffold to the glycine solution is 1:3.

[0109] In order to explore the optimal mass ratio of Rm@PCL / SF / ACR vascular scaffold and glycine solution, the mass ratio of Rm@PCL / SF / ACR vascular scaffold and glycine solution was first set to 1:1 and 2:3. It was found that due to the high mass of Rm@PCL / SF / ACR vascular scaffold, glycine solution was not enough to completely modify Rm@PCL / SF / ACR vascular scaffold. Then the mass of Rm@PCL / SF / ACR vascular scaffold was reduced, and the mass ratio of Rm@PCL / SF / ACR vascular scaffold and glycine solution was set to 1:2 and 1:3. It was found that glycine could be modified into Rm@PCL / SF / ACR vascular scaffold and could improve the anti-calcification performance of Gly-(Rm@PCL / SF / ACR) vascular scaffold.

[0110] Further experiments proved that setting the mass ratio of Rm@PCL / SF / ACR vascular stent and glycine solution to 1:2 would result in the optimal Gly-(Rm@PCL / SF / ACR) vascular stent.

[0111] Four types of vascular stents were prepared in the above examples: PCL / SF vascular stent prepared in Example 1, PCL / SF / ACR vascular stent prepared in Example 4, Rm@PCL / SF / ACR vascular stent prepared in Example 5, and Gly-(Rm@PCL / SF / ACR) vascular stent prepared in Example 11. The following four types of vascular stents were used as samples to conduct corresponding tests. In the following tests, the samples of these four types of vascular stents are referred to as PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group.

[0112] Test 1: Scanning electron microscope test.

[0113] Scanning electron microscopy (SEM) was used to observe the microstructures of the PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group, and to evaluate the micromorphology of the four vascular stents.

[0114] Four groups of vascular stents were cut axially and cut into 2mm×2mm films. The vascular stents were sputtered with gold. The microstructure of the vascular stents was observed using SEM at an accelerating voltage of 10kV / cm and a working distance of 9mm, and images were taken. Figure 2 shown.

[0115] Figure 2The scanning electron microscope images are shown in Figure 1. A is the microscopic morphology of the PCL / SF vascular stent prepared in Example 1; B is the microscopic morphology of the PCL / SF / ACR vascular stent prepared in Example 4; C is the microscopic morphology of the Rm@PCL / SF / ACR vascular stent prepared in Example 5; and D is the microscopic morphology of the Gly-(Rm@PCL / SF / ACR) vascular stent prepared in Example 11.

[0116] Depend on Figure 2 As can be seen in Figure A, the scaffold fibers are smooth and uniform, the structure is complete and there are no liquid beads.

[0117] Figure 2 As can be seen from Figures B to D, the scaffold fibers did not change much compared with the control group, which confirmed that the addition of ACR, erythrocyte membrane and glycine did not affect the microscopic morphology of the vascular scaffold.

[0118] Test 2: ACR release test.

[0119] The Gly-(Rm@PCL / SF / ACR) vascular scaffold was placed in 2 mL PBS buffer and stored at 37°C. At different time points: 1, 4, 6, 9, 12, 15, and 18 days, 100 μL of supernatant was collected and the absorbance was measured using a NanoDrop spectrophotometer.

[0120] ACR was dissolved in PBS buffer to form a standard dilution with a concentration of 0.0195 mM to 5 mM. The absorbance value of the standard dilution was measured, and a standard curve was prepared. The ACR concentration at different time points was measured based on the standard curve.

[0121] Figure 3 This is a graph showing the ACR release results of the Gly-(Rm@PCL / SF / ACR) vascular stent prepared in Example 11.

[0122] Depend on Figure 3 The release test results showed that the ACR concentration was accelerated in 1 to 9 days, about 50% of ACR was released in the first 4 days, and the release concentration reached 0.6mM on the 9th day. The ACR-modified Gly-(Rm@PCL / SF / ACR) vascular stent could continuously release ACR for nearly 9 days.

[0123] Test 3: Blood compatibility test.

[0124] 3.1. Activated partial thromboplastin time and plasma thrombin time test.

[0125] The activated partial thromboplastin time is abbreviated as APTT; the thrombin time is called TT.

[0126] Experimental group: The four groups of vascular stent materials were cut into 5 mm × 5 mm samples, recorded as PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group, and then placed in PBS buffer at 37°C for 1 hour. The samples were taken out and incubated with 1 mL of platelet-poor plasma at 37°C for 1 hour.

[0127] A blank tissue culture plate without specimens was used as the blank control group.

[0128] PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, Gly-

[0129] The (Rm@PCL / SF / ACR) group and the blank control group were subjected to APTT and TT tests using a coagulation analysis system, respectively. The results are shown in Figure 4 Figure A and Figure B of the diagram.

[0130] 3.2. Hemolysis rate, referred to as HR.

[0131] Blood was collected from the abdominal aorta of SD rats and injected into a centrifuge tube containing heparin. The tube was centrifuged at 2000 rpm for 15 min, the supernatant was discarded, and then physiological saline and precipitated red blood cells were diluted into a red blood cell suspension of appropriate concentration.

[0132] Experimental group: The four groups of vascular stent materials were cut into samples of 5 mm × 5 mm in size, recorded as PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group, and then 200 μL of the prepared red blood cell suspension was added respectively.

[0133] The positive control group was added with 5 mL of sterile distilled water; the negative control group was added with 5 mL of normal saline.

[0134] At the same time, all test tubes were incubated in a water bath at 37°C for 1.5 h. The samples to be tested were centrifuged at 2000 rpm for 10 min, and after removing the culture medium, they were added to a 96-well plate and the absorbance value was detected at 540 nm using an ELISA reader. The calculation formula for HR is as follows:

[0135] Among them, A0 represents the absorbance of the test group; A1 represents the absorbance of the positive control group; A2 represents the absorbance of the negative control group. The HR value was calculated 3 times for each group.

[0136] 3.3. Plasma recalcification time test, referred to as PRT test.

[0137] Experimental group: The four groups of vascular stent materials were cut into 5 mm × 5 mm samples, recorded as PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group, and then placed in sterile centrifuge tubes. PBS buffer was added to the centrifuge tubes and stored at 37°C for 1 h. After removing the PBS buffer, 200 μL of platelet-poor plasma was added and incubated at 37°C for 1 h, and then 200 μL of 25 mM CaCl2 solution was added to each test tube.

[0138] A blank tissue culture plate without specimens was used as the blank control group.

[0139] Then a stainless steel hook was placed into each test tube, and the earliest time of fibrin formation was observed, which was recorded as the plasma recalcification time.

[0140] 3.4. Blood compatibility test results.

[0141] Figure 4 The graphs are the results of blood compatibility tests. A is the result of APTT test; B is the result of TT test; C is the result of HR test; and D is the result of PRT test.

[0142] Figure 4 In the test, APTT stands for Activated Partial Thromboplastin Time. TT stands for Thrombin Time. HR stands for Hemolysis Ratio. PRT stands for Plasma Recalcification Time. APTT is a coagulation function test indicator that reflects the endogenous coagulation pathway, especially the comprehensive activity of the first-stage coagulation factors. The higher the APTT value, the less likely it is that the blood vessels will be blocked.

[0143] Figure 4 Figures A and B in the figure show that after gradual modification, the APTT and TT values ​​of the four groups of vascular stents gradually increased, with similar trends.

[0144] The APTT value of the (Rm@PCL / SF / ACR) group was significantly higher than that of the other two groups and the blank control group, indicating that the artificial blood vessels modified by Rm have better patency.

[0145] TT as an indicator parameter has a similar meaning to APTT, which also shows that the Rm-modified artificial blood vessel has a good anticoagulant effect. HR mainly reflects the degree of red blood cell rupture after the vascular graft comes into contact with peripheral blood. The higher the HR value, the more serious the red blood cell damage. The rupture of red blood cells can easily cause platelet deformation and coagulation.

[0146] from Figure 4 As can be seen in Figure C, the HR value of the gradually modified vascular stent gradually decreases, which shows that the less peripheral red blood cell rupture, the better the blood compatibility of the artificial blood vessel. This also indirectly shows that the Gly-(Rm@PCL / SF / ACR) vascular stent has good blood compatibility.

[0147] like Figure 4 As shown in Figure D, the plasma recalcification time of the experimental group was longer than that of the blank control group without any sample, and the recalcification time of the Rm@PCL / SF / ACR group and the Gly-(Rm@PCL / SF / ACR) group was significantly longer than that of the experimental group, and fell within the normal range of 2.8±0.5min.

[0148] Test 4: In vitro inflammation study.

[0149] 4.1. Collect and culture macrophages.

[0150] Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation. The specific method is as follows: (1) Take fresh anticoagulated whole blood, add it to PBS at a ratio of 1:1, dilute the blood (to reduce blood viscosity), mix it gently, and set aside. (2) Add an appropriate amount of mononuclear cell separation solution to a sterile centrifuge tube, spread the diluted blood sample on the surface of the separation solution (separation solution: diluted whole blood = 1:2), and keep the interface between the two liquid surfaces clear. (3) Centrifuge at 800g, room temperature for 20min-30min. (4) After centrifugation, discard the plasma layer, carefully absorb the PBMCs layer (i.e., the white film layer) and transfer it to a 15mL centrifuge tube. (5) Add 10mL PBS to the centrifuge tube to resuspend the cells, centrifuge at 250g, room temperature for 10min, and discard the supernatant. Repeat this step 1 to 2 times and it can be used for subsequent experiments.

[0151] RPMI-1640 culture medium was purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0152] Preparation of mixed culture medium: Add 9-10 mL of 10% FBS buffer, 1 mL of 100 U / mL penicillin and 1 mL of 0.1 mg / mL streptomycin solution to each 1 L of RPMI-1640 culture medium to prepare a mixed culture medium. FBS is the full name of Fetal Bovine Serum in English and the Chinese name is fetal bovine serum.

[0153] PBMCs and mixed culture medium were placed in a culture flask and cultured in a 37°C cell culture incubator in a 5% carbon dioxide atmosphere for 3 hours, then washed twice with PBS buffer to remove excess non-adherent cells. The remaining adherent cells are macrophages, which were collected with a sterile cell scraper for later use.

[0154] Experimental group: Four groups of vascular stents were cut into 5 mm × 5 mm samples, named PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group, and then placed in a 24-well plate. The obtained macrophages were cultured at 1 × 10 6 The cells were inoculated into the inner wall of the sample tube and cultured in a cell culture incubator for 6 days. The mixed culture medium was replaced every two days.

[0155] Macrophages cultured in plates without material served as a control group.

[0156] 4.2. Real-time fluorescence quantitative PCR detection of gene expression.

[0157] Real-time fluorescence quantitative PCR was used to detect the gene expression of macrophage markers CD163 and CCR7 and the expression of IL-1α and IL-10 cytokines.

[0158] With Trieasy TM Total RNA kit was used to separate total RNA from samples of the experimental group and the control group according to the instructions and measure the concentration.

[0159] All RNA was then reverse transcribed into cDNA. Universal Blue qPCR Green Master Mix, primers, and sterile distilled water were mixed and measured using a Lightcycler 96 real-time fluorescence quantitative PCR instrument. The real-time PCR primer sequences are shown in Table 2.

[0160] Table 2 Real-time PCR primer sequences

[0161]

[0162]

[0163] Among them, Trieasy TM The total RNA kit is a total RNA extraction reagent suitable for various animals, plants, bacterial tissues, and cells. It has a strong lysis ability and can lyse cell and tissue samples in a short time, and effectively inhibit the degradation of RNA in the sample to maintain the integrity of RNA.

[0164] Hieff Universal Blue qPCR Green Master Mix is ​​a premixed solution for real-time fluorescence quantitative PCR amplification, produced by Yisheng Biotechnology (Shanghai) Co., Ltd. PCR is the abbreviation of polymerase chain reaction.

[0165] Lightcycler 96 is a real-time fluorescence quantitative PCR instrument launched by Roche, Switzerland.

[0166] Figures 5 to 9 This is the expression diagram of inflammatory factors. Figure 5 This is a graph showing the gene expression results of the macrophage marker CCR7. Figure 6 This is a graph showing the gene expression results of the macrophage marker CD163. Figure 7 This is a graph showing the expression ratio of CCR7 / CD163. Figure 8 The figure shows the expression results of IL-10 cytokine. Fig. 9 The graph shows the expression results of IL-1α cytokine.

[0167] Figure 5 The results showed that the expression rate of macrophage pro-inflammatory gene CCR7 in the PCL / SF group was significantly higher than that in the other three groups on the 8th day.

[0168] Figure 6 The results showed that the expression rate of macrophage anti-inflammatory gene CD163 in the three modified groups with ACR was significantly higher than that in the control group on the 16th day.

[0169] Figure 7 It was shown that the expression ratio of CCR7 / CD163 in the other three groups increased significantly faster than that in the PCL / SF group.

[0170] Figure 8 The figure shows that the expression levels of IL-10 related genes in the other three groups were higher than that in the PCL / SF group.

[0171] Fig. 9 The results showed that the expression of IL-1α related genes in the PCL / SF group was higher than that in the other three groups on the 4th day.

[0172] Test 5: Determination of calcium content trend.

[0173] The experimental mice were SD rats weighing about 100 g, purchased from the Animal Experiment Center of Xinxiang Medical College.

[0174] Test group: Four groups of vascular stents were cut into 5mm×5mm samples, recorded as PCL / SF group, PCL / SF / ACR group, Rm@PCL / SF / ACR group, and Gly-(Rm@PCL / SF / ACR) group, and then embedded in the subcutaneous tissue of experimental mice. The subcutaneously embedded vascular stents in the experimental mice were obtained at four different time points, namely the 3rd day, the 7th day, the 15th day, and the 30th day, to obtain four groups of vascular stent samples. Five sections were randomly selected from each of the four groups of vascular stent samples, and each section weighed about 0.1g. They were repeatedly rinsed with sterilized PBS buffer. Then the obtained samples were placed in a polytetrafluoroethylene crucible, 5mL of nitric acid and 1mL of perchloric acid were added, and heated to a slight boil for 2 hours. After the digestion was completed, it was allowed to cool to room temperature, transferred to a 50mL volumetric flask with test water, fixed to volume, and shaken well as the test sample to be tested. The calcium content of the test sample was determined by atomic absorption spectrophotometry.

[0175] A blank sample without any sample added was used as a control sample, and the calcium content of the control sample was determined by atomic absorption spectrophotometry.

[0176] Pipette appropriate amounts of calcium standard solutions into 100mL volumetric flasks, add 1% nitric acid to the scale, and shake well to make the mass concentration of calcium element 0mg / L, 1mg / L, 2mg / L, 5mg / L, 8mg / L, and 10mg / L. Measure the absorbance of the standard solutions from low concentration to high concentration, and establish a standard curve.

[0177] At the same time, the absorbance of the test sample and the blank sample without any sample added is measured. According to the established standard curve and the absorbance of the test sample and the blank sample without any sample added, the mass concentration of calcium in the test sample is obtained, and then the content of calcium in the test sample is calculated according to the calculation formula of the content of calcium in the test sample.

[0178] The calculation formula for the calcium content in the test sample is:

[0179] Wherein, w is the content of calcium in the test sample, mg / kg; P is the mass concentration of calcium in the test sample calculated by the standard curve, mg / L; P0 is the mass concentration of calcium and magnesium in the control sample, mg / L; V is the fixed volume of the test sample, mL; D is the dilution factor of the test sample; m is the weight of the test sample, g.

[0180] Fig.10 This is a graph showing the changing trend of calcium content in four groups of vascular stent samples.

[0181] Fig.10 The results showed that the calcium content of the four groups of vascular stents increased with the increase of subcutaneous embedding time in mice, but the calcium content of the vascular stent containing glycine was lower than that of the other three groups.

[0182] In summary, the Gly-(Rm@PCL / SF / ACR) vascular scaffold loaded with ACR, red blood cell membrane and glycine has good blood compatibility, and has anti-inflammatory, anti-coagulation and anti-calcification functions. It can ensure the long-term patency of artificial blood vessel transplantation, which is beneficial to vascular regeneration and reconstruction; it can also achieve long-term slow release of drugs under normal pH of human blood, induce directional growth of endothelial cells, inhibit the adhesion of blood components on the surface of artificial blood vessels, prevent thrombosis and intimal hyperplasia, and achieve anti-inflammatory effects by stimulating macrophages to polarize to the M2 phenotype, relieve the excessive state of intracellular calcium ion concentration, and then inhibit its downstream pathways, so that artificial blood vessels are not easy to form thrombosis, inflammation and calcification, and have clinical application potential.

[0183] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a small-caliber tissue engineering vascular stent, characterized in that: The following steps are involved: The polycaprolactone-silk fibroin mixed solution was mixed with the 5-amino-4-imidazolecarboxamide nucleoside solution to prepare a spinning solution; the spinning solution was solidified into fibers using high-voltage electrospinning technology to prepare a PCL / SF / ACR vascular stent; Mixing a red blood cell membrane dispersion with a pH of 7.5 to 8 with a PCL / SF / ACR vascular scaffold, and incubating at 2°C to 6°C to prepare a Rm@PCL / SF / ACR vascular scaffold; The Rm@PCL / SF / ACR vascular scaffold was placed in a glycine solution with a pH of 7 to 10 and incubated at 30° C. to 40° C. to prepare a small-caliber tissue engineering vascular scaffold.

2. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: The mass ratio of polycaprolactone to silk fibroin is 8-8.5:2-1.

5.

3. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: The polycaprolactone-silk fibroin mixed solution is obtained by dissolving polycaprolactone and silk fibroin in hexafluoroisopropanol; The total mass of polycaprolactone and silk fibroin accounts for 8% to 10% of the mass percentage of the polycaprolactone-silk fibroin mixed solution.

4. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: The 5-amino-4-imidazolecarboxamide nucleoside solution is obtained by dissolving 5-amino-4-imidazolecarboxamide nucleoside in dimethyl sulfoxide; The concentration of the 5-amino-4-imidazolecarboxamide nucleoside solution is 3 mmol / L to 5 mmol / L.

5. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: The volume ratio of the polycaprolactone-silk fibroin mixed solution to the 5-amino-4-imidazolecarboxamide nucleoside solution is 100:

1.

6. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: In the red blood cell membrane dispersion, the concentration of red blood cell membrane is 6 mg / mL to 8 mg / mL; The mass ratio of red blood cell membrane dispersion to PCL / SF / ACR vascular scaffold was 1-1.5:2; The mass ratio of Rm@PCL / SF / ACR vascular stent to glycine solution is 1:2-3.

7. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: The electrospinning parameters of high voltage electrospinning technology are: Voltage 16kV~20kV, rotation speed 150rpm / min~200rpm / min, extrusion speed 10mm / s~12mm / s, curing distance 14cm~16cm.

8. The method for preparing a small-caliber tissue engineering vascular stent according to claim 1, characterized in that: The preparation process of red blood cell membrane dispersion is as follows: Whole blood is centrifuged to remove plasma and obtain a red blood cell pellet; The erythrocyte sediment was mixed with PBS buffer, incubated at 2°C to 6°C, centrifuged, the erythrocyte membranes were collected, and mixed and dispersed with PBS buffer to obtain an erythrocyte membrane dispersion with a pH of 7.5 to 8.

9. The method for preparing a small-caliber tissue engineering vascular stent according to claim 8, characterized in that: The mass ratio of the red blood cell membrane precipitate to the PBS buffer is 1:1-2; the pH value of the PBS buffer is 7.5-8.

10. A small-caliber tissue engineering vascular stent prepared by the preparation method according to any one of claims 1 to 9.

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