WO2022028396A1 - 一种蚕丝抗凝血管支架覆膜及其制备方法 - Google Patents

一种蚕丝抗凝血管支架覆膜及其制备方法 Download PDF

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WO2022028396A1
WO2022028396A1 PCT/CN2021/110228 CN2021110228W WO2022028396A1 WO 2022028396 A1 WO2022028396 A1 WO 2022028396A1 CN 2021110228 W CN2021110228 W CN 2021110228W WO 2022028396 A1 WO2022028396 A1 WO 2022028396A1
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silk
silk fibroin
solution
fibroin
polyethylene glycol
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PCT/CN2021/110228
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English (en)
French (fr)
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宋广州
王建南
裔洪根
许建梅
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/148—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/606—Coatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/06—Coatings containing a mixture of two or more compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/08—Coatings comprising two or more layers

Definitions

  • the invention relates to the technical field of preparation of vascular stent coating, in particular to a silk anticoagulant tube stent coating and a preparation method thereof.
  • Cardiovascular and cerebrovascular diseases such as embolism, vascular stenosis, and vascular aneurysms are a serious threat to human health, and have become the death rate of human beings. highest cause.
  • minimally invasive endovascular isolation has gradually become the main treatment method for various arterial diseases due to its small surgical trauma.
  • Vascular stent-graft is the main body of endovascular isolation, and its performance directly affects the clinical treatment effect.
  • vascular stent system including stents, coverings and delivery systems.
  • the covered stents used in clinical practice in my country rely on imports and are expensive.
  • the membrane is an important undertaker of intraluminal isolation, and its biological properties are extremely important.
  • the currently used clinical coating materials do not have excellent histocompatibility and endothelialization function, and a high proportion of thrombosis and restenosis still occur in the long-term after surgery, especially for small-caliber vascular stents, and complications such as slippage also occur.
  • the existing publicly reported covering materials for vascular stents are mainly synthetic polymers such as polytetrafluoroethylene, polyester, polyamide, polyethylene, polypropylene, polyurethane, etc. into a film.
  • Synthetic polymers lack histocompatibility and hemocompatibility, which is an important cause of postoperative complications.
  • the inner surface of the membrane is difficult to endothelialize, and immune rejection will occur after being implanted in the body for a period of time. Long-term medication will induce other more serious disease.
  • synthetic polymers have better biocompatibility. Among them, silk fibroin has been proved to support the growth of a variety of cells, and its application in tissue engineering materials has received great attention.
  • Preferred materials for absorbable coatings are mainly synthetic polymers such as polytetrafluoroethylene, polyester, polyamide, polyethylene, polypropylene, polyurethane, etc. into a film.
  • Synthetic polymers lack histocompatibility and hemocompatibility, which is an important cause of
  • the anticoagulant modification of silk fibroin The prior art mainly reports the grafting of macromolecules with anticoagulant effect and the method of sulfation or heparinization. Heparin belongs to an indirect inhibitor of thrombin. Not necessarily anticoagulant. Therefore, it is necessary to develop a silk anticoagulant stent covering in view of the bottleneck problem of existing clinical applications and the incidence of younger and younger (even live-born babies).
  • the purpose of the present invention is to provide a silk anticoagulant tube stent coating and a preparation method thereof, which are used to produce a vascular stent with sustainable anticoagulation, absorbability, rapid endothelialization in situ, and inducing the reconstruction of healthy vascular tissue.
  • a technical scheme of the present invention is:
  • the leakage is less than 7ml/min.cm 2 under the water pressure of 120mmHg, the hemolysis rate is less than 0.1%, and the continuous anticoagulation of the silk anticoagulation tube stent coating is more than 6 months.
  • the preparation method of the above-mentioned silk anticoagulant tube stent coating comprises the following steps:
  • Bombyx mori silk fibroin fiber and cooked silk thread after preparation degumming choose 40 ⁇ 160D silk thread and silk monofilament, described silk thread is obtained by twisting and merging of Bombyx mori raw silk, and described silk monofilament and silk thread are obtained by 1:50 (g) /mL) in deionized water, boiled for 7 hours at a temperature of 98-100 ° C, and replaced the deionized water several times during the boiling period, until the deionized water made the silk single The sericin contained in the silk and the silk thread is fully removed, and then the silk monofilament and the silk thread are dried in an oven with a temperature of 60° C. to obtain the degummed silk fibroin fiber and cooked silk thread;
  • the cooked silk thread is braided into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 2 to 20 mm on a stainless steel rod by braiding technology, and the tubular structure is placed in the modified Immerse in the sex silk fibroin solution for 30 ⁇ 5 seconds, take it out, place it in a blast drying oven and rotate it in the circumferential direction to air dry under the condition that the temperature is less than 37°C, rinse and then air dry to obtain a silk fibroin tubular coating;
  • the silk thread in step (1) is 80D-120D.
  • the molecular weight cut-off of the dialysis bag in step (2) is 50 kDa or 14-16 kDa.
  • a mixed solution of silk fibroin and polyethylene glycol diamine is prepared in a mass ratio of 100:5 to 10, and a mass ratio of 1 to 20% of silk fibroin is added to the mixed solution.
  • -(3-Dimethylaminopropyl)-3-ethylcarbodiimide stir evenly, then add N-hydroxysuccinimide with a mass ratio of 10% of silk fibroin and a mass ratio of 20% of silk fibroin % 2-morpholineethanesulfonic acid, reacted for 20 minutes and dialyzed with deionized water for 12 to 48 hours to obtain a cationized silk fibroin solution of polyethylene glycol diamine, which was concentrated, adjusted and measured by a rotary evaporator again.
  • the mass fraction of the polyethylene glycol diamine cationized silk fibroin solution is 1 to 10%, and replaced by: adding the mass ratio of the silk fibroin 0.6 polyethylene glycol diglycidyl ether, stir well and degas.
  • step (4) the inner temperature of the blast drying oven is rotated and air-dried along the circumferential direction under the condition that the temperature is less than 37 °C, and the obtained silk fibroin tubular coating is replaced by: the temperature in the hot air drying oven is less than 65 °C.
  • the tubular stent-graft was obtained by rotating and air-drying in the circumferential direction under the condition of
  • the method further includes: preparing a cationized silk fibroin tubular coating; configuring and measuring the concentration of a polyethylene glycol diamine solution, the polyethylene glycol diamine
  • the solution includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and 2-morpholinoethanesulfonic acid containing 1.5-fold molar concentration of polyethylene glycol diamine
  • the tubular covered stent is immersed in the polyethylene glycol diamine solution for 20 minutes of reaction, then taken out to air dry at room temperature, rinsed and then air dried to obtain a cationized silk fibroin tubular covering.
  • Another technical scheme of the present invention is:
  • a preparation method of silk anticoagulant tube stent covering comprising the following steps:
  • Bombyx mori silk fibroin fiber and cooked silk thread after preparation degumming choose 40 ⁇ 160D silk thread and silk monofilament, described silk thread is obtained by twisting and merging of Bombyx mori raw silk, and described silk monofilament and silk thread are obtained by 1:50 (g) /mL) in deionized water, boiled for 7 hours at a temperature of 98-100 ° C, and replaced the deionized water several times during the boiling period, until the deionized water made the silk single The sericin contained in the silk and the silk thread is fully removed, and then the silk monofilament and the silk thread are dried in an oven with a temperature of 60° C. to obtain the degummed silk fibroin fiber and cooked silk thread;
  • the dialysis bag of the described Bombyx mori fibroin solution is placed in a container filled with deionized water, and the liquid in the container is replaced with new deionized water every 2 hours, and the dialysis is continued for 3 days to obtain purified Bombyx mori fibroin.
  • protein aqueous solution
  • the cooked silk thread is braided into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 2 to 20 mm on a stainless steel rod by braiding technology, and the tubular structure is placed in the modified Immerse in the sex silk fibroin solution for 30 ⁇ 5 seconds, take it out, place it in a blast drying oven and rotate it in the circumferential direction to air dry under the condition that the temperature is less than 37°C, repeat the dipping coating 2 to 10 times to obtain a silk anticoagulant tube stent coating .
  • the silk thread in step (1) is 80D-120D.
  • the molecular weight cut-off of the dialysis bag in step (2) is 50 kDa or 14-16 kDa.
  • the invention provides a silk anticoagulant tube stent coating and a preparation method thereof.
  • the prepared coating has excellent biomechanical properties and lasting anticoagulation activity, and can induce in situ vascular intima regeneration that regulates the balance of the blood system.
  • the film can be prepared into various shapes and applied to the treatment of cardiovascular diseases, such as heart patch, vascular stent, in vivo graft of artificial heart valve, and can also be extended to other medical devices such as tissue engineering stents.
  • the purpose of the present invention is to address the serious clinical problems such as long-term restenosis, thrombosis, slippage and long-term fatigue damage of vascular stent-grafts after endovascular isolation for the treatment of cardiovascular and cerebrovascular diseases.
  • Preparation technology of vascular stent coating with anticoagulant factor natural polymer silk fibroin material the coating axial tensile strength> 1.8MPa, elongation at break> 45%, circumferential tensile strength> 7.0MPa , the elongation at break is >130%, the overall water leakage is less than 7ml/min.cm 2 under 120mmHg water pressure, and the hemolysis rate is ⁇ 0.1%.
  • Continuous anticoagulation effect the continuous anticoagulation of silk anticoagulant tube stent coating is more than 6 months, and has the function of inducing tissue regeneration of lesions and defective blood vessels and restoring blood balance system.
  • the invention provides a method for preparing a silk anticoagulant tube stent coating, comprising:
  • Step 1 Preparation of degummed Bombyx mori silk fibroin and cooked silk thread:
  • silk thread and silk monofilament are obtained by twisting and merging Bombyx mori raw silk, described silk monofilament and silk thread are put into at the liquor ratio of 1:50 (g/mL) In deionized water, boil for 7 hours at a temperature of 98-100°C, and change deionized water several times during the boiling period, until the deionized water fully removes the sericin contained in the silk monofilament and silk thread After cleaning, the silk monofilament and silk thread are dried in an oven at a temperature of 60° C. to obtain degummed Bombyx mori silk fibroin and cooked silk thread.
  • Step 2 prepare the silk fibroin aqueous solution of Bombyx mori:
  • the degummed Bombyx mori silk fibroin fibers are dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL), and treated at a temperature of 65 ⁇ 10° C. until the Bombyx mori silk fibroin fibers are completely dissolved, The Bombyx mori silk fibroin dissolving solution is obtained, and the Bombyx mori The dialysis bag was placed in a container filled with deionized water, and the liquid in the container was replaced with new deionized water every 2 hours, and the dialysis was continued for 3 days to obtain a purified silk fibroin aqueous solution.
  • Method 1 use a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified silk fibroin aqueous solution, prepare a mixed solution of silk fibroin and polyethylene glycol diamine in a mass ratio of 100:5 to 10, and add it to the solution.
  • 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a mass ratio of 20% of silk fibroin is added to the mixed solution, stirred evenly, and then added with a mass ratio of 10% of silk fibroin
  • the N-hydroxysuccinimide and 2-morpholineethanesulfonic acid with a mass ratio of 20% silk fibroin were reacted for 20 minutes and dialyzed with deionized water for 12 to 48 hours to obtain polyethylene glycol bisamine cationized Silk fibroin solution, again using a rotary evaporator to concentrate, adjust and measure the polyethylene glycol diamine cationized silk fibroin solution to make the mass fraction of the polyethylene glycol diamine cationized silk fibroin solution 1-10% to obtain a modified silk fibroin solution.
  • Method 2 use a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified silk fibroin aqueous solution, add polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to the silk fibroin (referring to the purified The mass ratio of the silk fibroin in the silk fibroin aqueous solution to polyethylene glycol diglycidyl ether is 10:6), stirring evenly and debubbling to obtain a modified silk fibroin solution.
  • Method 3 use a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified silk fibroin aqueous solution, prepare a mixed solution of silk fibroin and polyethylene glycol diamine in a mass ratio of 100:5 to 10, and add it to the solution.
  • 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with a mass ratio of 20% of silk fibroin is added to the mixed solution, stirred evenly, and then added with a mass ratio of 10% of silk fibroin
  • the N-hydroxysuccinimide and 2-morpholineethanesulfonic acid with a mass ratio of 20% silk fibroin were reacted for 20 minutes and dialyzed with deionized water for 12 to 48 hours to obtain polyethylene glycol bisamine cationized Silk fibroin solution, again using a rotary evaporator to concentrate, adjust and measure the polyethylene glycol diamine cationized silk fibroin solution to make the mass fraction of the polyethylene glycol diamine cationized silk fibroin solution 0 to 500 U/mL hirudin, a direct inhibitor of thrombin, is added to the polyethylene glycol diamine cationized silk fibroin solution at a concentration of 1 to 10%, stirred evenly, and debubble to obtain
  • the cooked silk thread is braided on a stainless steel rod into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 2 to 20 mm, and the tubular structure is immersed in the modified silk fibroin solution for 30 ⁇ 5 seconds. Take it out, put it in a blast drying oven and rotate it in the circumferential direction under the condition that the temperature is less than 37°C, rinse and then air-dry it, or place it in a hot-air drying oven and rotate it in the circumferential direction under the condition that the temperature is less than 65°C to obtain silk fibroin.
  • Tubular film Take it out, put it in a blast drying oven and rotate it in the circumferential direction under the condition that the temperature is less than 37°C, rinse and then air-dry it, or place it in a hot-air drying oven and rotate it in the circumferential direction under the condition that the temperature is less than 65°C to obtain silk fibroin.
  • Tubular film Take it out, put it in a blast drying oven and rotate it in the circumfer
  • step 4 If the above step 4 is using the method 2 of the step 3 as the step 3, then add the steps: prepare a cationized silk fibroin tubular coating: configure a polyethylene glycol diamine solution of a certain concentration, the polyethylene glycol
  • the bisamine solution includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and 2-morpholinide containing 1.5-fold molar concentration of polyethylene glycol bisamine sulfonic acid, immersing the tubular covered stent in the polyethylene glycol diamine solution to react for 20 minutes, then taking it out to air-dry at room temperature, rinsing and then air-drying to obtain a cationized silk fibroin tubular covering.
  • Step 5 preparing the silk anticoagulant tube stent covering.
  • hirudin aqueous solution 0 to 500 U/mL
  • the number of coatings in the above steps is related to the concentration of silk fibroin. If the concentration is thin, the number of coatings needs to be increased; if the concentration is thick, the number of coatings can be reduced accordingly.
  • step 4 is using the method 3 of the step 3 as the step 3, then the step 5 is not required.
  • Each layer coating of the silk anticoagulant tube stent prepared by the above method is almost a molecular layer, and the silk fibroin macromolecular chain is fully self-assembled into the most stable molecular conformation during the air-drying process, and the silk is degummed with deionized water.
  • the silk fibroin fiber and the regenerated silk fibroin macromolecular chain are kept from being damaged, so that the film has excellent tensile properties, burst strength, compliance and fatigue resistance.
  • the membrane does not have endoleak, and is tightly combined with the lesion tissue without slipping after implantation.
  • the stent coating provided by the present invention has excellent cytocompatibility, blood compatibility and tissue compatibility.
  • the layer-by-layer assembly method greatly increases the loading of anticoagulant factors, and continues to exert anticoagulant function with the gradient degradation and absorption of the coating; the regulation of the large molecular weight of silk fibroin also endows the controlled release of hirudin, preventing long-term thrombus and restenosis; and the inner surface of the membrane can obtain rapid endothelialization, in situ repair of the intimal tissue of the blood vessel, and will not trouble the device failure due to long-term fatigue damage after implantation in the human body. Rapid endothelialization is the fundamental factor to inhibit thrombosis and restenosis, and the rapid formation of intimal tissue will completely restore the regulation function of vascular coagulation system balance.
  • one embodiment or “an embodiment” refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention.
  • the appearances of "in one embodiment” in various places in this specification are not all referring to the same embodiment, nor are they separate or selectively mutually exclusive from other embodiments.
  • This example shows a preparation method for a silk anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 80-120D silk thread group, and the other group is a silk monofilament group. Boiling at ⁇ 100°C for 7 hours, changing deionized water several times during this period, and then fully washing the silk with deionized water, drying it in an oven at 60°C, and obtaining degummed silk fibroin fibers and cooked silk threads.
  • the Bombyx mori silk fibroin dissolving solution is perfused into a dialysis bag, the wall of the dialysis bag is a semi-permeable membrane, and the molecular weight cut-off is in the range of 14-16 kDa, and the dialysis bag perfused with the Bombyx mori silk fibroin dissolving solution is placed in a container filled with deionized water, The water in the container was replaced with new deionized water every 2 hours, and the dialysis was continued for 3 days to obtain a purified Bombyx mori silk fibroin aqueous solution.
  • the cooked silk thread is braided on the stainless steel rod into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 30 to 90°, immersed in the modified silk fibroin solution in the above step 3 for about 30 seconds and taken out, and placed in a tube. Rotate and air dry in the inner circumferential direction of the hot air drying box below 65°C.
  • Steps 4 to 6 are alternately repeated 5 times to obtain a silk fibroin composite vascular stent covering, namely a silk anticoagulant tube stent covering.
  • the above-mentioned silk anticoagulant tube stent coating has excellent mechanical properties.
  • the axial tensile strength is >1.8MPa
  • the elongation at break is >45%
  • the circumferential tensile strength is >7.0MPa.
  • the elongation at break is >130%
  • the overall water leakage is less than 5ml/min.cm 2 under 120mmHg water pressure.
  • the hemolysis rate is less than 0.1% by coating the silk anticoagulant tube stent according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the silk anticoagulant tube stent coating has no sensitization through animal experiments, and the cytotoxicity is less than or equal to 1 according to the national standard.
  • the silk anticoagulant tube stent coating has significant anticoagulant properties and has a sustained anticoagulant effect.
  • This example shows a preparation method for a silk anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 80-120D silk thread group, and the other group is a silk monofilament group. Boiling at ⁇ 100°C for 7 hours, changing deionized water several times during this period, and then fully washing the silk with deionized water, drying it in an oven at 60°C, and obtaining degummed silk fibroin fibers and cooked silk threads.
  • the cooked silk thread is braided on a stainless steel rod into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 2 to 20 mm, and immersed in the cationized silk fibroin solution of the third step above. Take it out for about 30 seconds, and place it in a blast drying oven below 37°C to rotate and air dry in the circumferential direction to obtain a silk fibroin tubular coating.
  • the above-mentioned silk anticoagulant tube stent coating has excellent mechanical properties.
  • the axial tensile strength is >2.5MPa
  • the elongation at break is >70%
  • the circumferential tensile strength is >8.0MPa.
  • the elongation at break is >150%
  • the overall water leakage is less than 2ml/min.cm 2 under 120mmHg water pressure.
  • the hemolysis rate is less than 0.1% by coating the silk anticoagulant tube stent according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the silk anticoagulant tube stent coating has no sensitization through animal experiments, and the cytotoxicity is less than or equal to 1 according to the national standard.
  • the silk anticoagulant tube stent coating has significant anticoagulation performance and has a continuous anticoagulation effect, and the continuous anticoagulation effect is significantly better than that of Examples 1 and 4.
  • This example shows a preparation method for a silk anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 80-120D silk thread group, and the other group is a silk monofilament group. Boiling at ⁇ 100°C for 7 hours, changing deionized water several times during this period, and then fully washing the silk with deionized water, drying it in an oven at 60°C, and obtaining degummed silk fibroin fibers and cooked silk threads.
  • the cooked silk thread is braided on the stainless steel rod into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 30 to 90°, immersed in the modified silk fibroin solution in the above step 3 for about 30 seconds and taken out, and placed in a tube. Rotate and air dry in the inner circumferential direction of the blast drying oven below 37°C. The dipping coating was repeated 5 times to obtain a silk-silk fibroin composite vascular stent coating, that is, a silk anticoagulant tube stent coating.
  • the above-mentioned silk anticoagulant tube stent coating has excellent mechanical properties.
  • the axial tensile strength is >2.8MPa
  • the elongation at break is >70%
  • the circumferential tensile strength is >10.0MPa.
  • the elongation at break is >150%
  • the overall water leakage is less than 2ml/min.cm 2 under 120mmHg water pressure.
  • the hemolysis rate is less than 0.1% by coating the silk anticoagulant tube stent according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the silk anticoagulant tube stent coating has no sensitization through animal experiments, and the cytotoxicity is less than or equal to 1 according to the national standard.
  • the silk anticoagulant tube stent coating has significant anticoagulation performance and has a continuous anticoagulation effect, and the continuous anticoagulation effect is significantly better than that of Examples 1 and 4.
  • This example shows a preparation method for a silk anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 80-120D silk thread group, and the other group is a silk monofilament group. Boiling at ⁇ 100°C for 7 hours, changing deionized water several times during this period, and then fully washing the silk with deionized water, drying it in an oven at 60°C, and obtaining degummed silk fibroin fibers and cooked silk threads.
  • the Bombyx mori silk fibroin dissolving solution is perfused into a dialysis bag, the wall of the dialysis bag is a semi-permeable membrane, and the molecular weight cut-off is in the range of 14-16 kDa, and the dialysis bag perfused with the Bombyx mori silk fibroin dissolving solution is placed in a container filled with deionized water, The water in the container was replaced with new deionized water every 2 hours, and the dialysis was continued for 3 days to obtain a purified Bombyx mori silk fibroin aqueous solution.
  • the cooked silk thread is braided on the stainless steel rod into a tubular structure with an inner diameter of 2 to 20 mm and an inner diameter of 30 to 90°, immersed in the modified silk fibroin solution in the above step 3 for about 30 seconds and taken out, and placed in a tube. Rotate and air dry in the inner circumferential direction of the blast drying oven below 37°C. The dipping coating was repeated 5 times to obtain a silk-silk fibroin composite vascular stent coating, that is, a silk anticoagulant tube stent coating.
  • the above-mentioned silk anticoagulant tube stent coating has excellent mechanical properties.
  • the axial tensile strength is >2.0MPa
  • the elongation at break is >45%
  • the circumferential tensile strength is >7.0MPa.
  • the elongation at break is >130%
  • the overall water leakage is less than 7ml/min.cm 2 under 120mmHg water pressure.
  • the hemolysis rate is less than 0.1% by coating the silk anticoagulant tube stent according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the silk anticoagulant tube stent coating has no sensitization through animal experiments, and the cytotoxicity is less than or equal to 1 according to the national standard.
  • the stent covering has significant anticoagulant properties and has a sustained anticoagulant effect.

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Abstract

本发明公开了一种蚕丝抗凝血管支架覆膜及其制备方法，将家蚕蚕丝分为两组，其中一组通过加捻合并获得丝线组，另一组为单丝组，然后均采用去离子水煮沸进行脱胶。丝线组采用编结机编结成管状结构，单丝组蚕丝溶解于溴化锂中性盐溶液制备丝素蛋白溶解液，然后将丝素溶解液灌注于透析袋内，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，持续透析，得到纯化后的家蚕丝素蛋白水溶液，然后采用旋转蒸发器浓缩丝素蛋白水溶液，然后加入适量的交联剂反应并层层浸渍涂层丝素管状编结物，其间多次交替或混合浸渍涂层抗凝药物水蛭素，获得蚕丝抗凝血管支架覆膜。该覆膜具有卓越的生物力学性能和持久的抗凝活性。

Description

一种蚕丝抗凝血管支架覆膜及其制备方法
本申请要求于2020年08月06日提交中国专利局、申请号为202010781130.9、发明名称为“一种蚕丝抗凝血管支架覆膜及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及血管支架覆膜制备技术领域，具体涉及一种蚕丝抗凝血管支架覆膜及其制备方法。
背景技术
随着生活水平的提高和老龄化进程的加快，心脑血管疾病的发病率正逐年显著升高，栓塞、血管狭窄、血管动脉瘤等心脑血管疾病严重威胁着人类健康，已成为人类死亡率最高的病因。随着医学的发展，微创的腔内隔绝术由于手术创伤小逐渐成为动脉多种病变的主要治疗方式，血管覆膜支架是腔内隔绝术治疗的主体，其性能直接影响着临床治疗效果，但术后中、远期仍然会出现如血管覆膜支架的滑移、内漏和支架源性新破口等并发症，以及仍然会出现血栓和再狭窄，这些临床问题值得密切关注。血管覆膜支架本身的生物相容性(尤其是组织相容性和血液相容性)差、生物力学性能不相配及不降解材料长期被血液里的成分包裹是诱发术后并发症的主要原因，因此迫切需要开发出与宿主血管间生物学性能匹配的血管覆膜支架。
腔内修复术的关键在于血管支架系统的研制，其中包括支架、覆膜和输送系统，目前我国临床运用的覆膜支架依赖进口，价格昂贵。覆膜是腔内隔绝的重要承担者，其生物学性能极其重要。现在临床应用的覆膜材料没有优异的组织相容性和内皮化功能，术后远期仍发生较高比例的血栓和再狭窄，尤其是小口径血管支架，也发生滑移等并发症。对于发病率越来越高的中青年人群、甚至婴幼儿来说，长期靠药物来维持抗血栓是极不合适的选择。因此，设计和制备新型的具有抗凝功能血管支架、且能彻底恢复血管凝血系统平衡功能对解决临床不断出现的新问题具有重要研究和 应用价值。
现有公开报导的血管支架的覆膜材料主要是聚四氟乙烯、聚酯、聚酰胺、聚乙烯、聚丙烯、聚氨酯等合成高分子，还有采用涤纶丝与镍钛合金丝编织或交替编织成覆膜。合成高分子缺乏组织相容性和血液相容性，是引起术后并发症的重要原因，覆膜内表面难以内皮化，置入体内一段时间后会产生免疫排异，长期服药，会诱发其它更为严重疾病的发生。相比于合成高分子，天然高分子具有较好的生物相容性，其中蚕丝丝素蛋白已证明能够支持多种细胞的生长，在组织工程材料方面的应用研究受到极大的关注，是作为可吸收覆膜的优选材料。丝素蛋白的抗凝血改性现有技术主要报导了接枝具有抗凝作用的高分子及硫酸化或肝素化方法，肝素属于一种凝血酶间接抑制剂，材料中共混或键合的肝素不一定都能发挥抗凝作用。因此，针对现有临床应用瓶颈问题和越来越年轻化(甚至活产婴儿)的发病现象，有必要研发一种蚕丝抗凝血管支架覆膜。
发明内容
有鉴于此，本发明的目的在于提供一种蚕丝抗凝血管支架覆膜及其制备方法，用于生产可持续抗凝、可吸收、原位快速内皮化、诱导健康血管组织重建的血管支架。
本发明的一种技术方案是：
提供一种蚕丝抗凝血管支架覆膜，轴向抗拉伸强度>1.8MPa，断裂伸长率>45％，圆周向抗拉伸强度>7.0MPa，断裂伸长率>130％，整体水渗漏在120mmHg水压下小于7ml/min.cm 2，溶血率<0.1％，所述蚕丝抗凝血管支架覆膜的持续抗凝大于6个月。
上述蚕丝抗凝血管支架覆膜的制备方法，包括如下步骤：
(1)制备脱胶后的家蚕丝素纤维和熟丝线：选取40～160D丝线和蚕丝单丝，所述丝线由家蚕生丝加捻合并获得，将所述蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，在温度为98～100℃的条件下沸煮7小时，并且在沸煮期间多次更换去离子水，直至所述去离子水将所述蚕丝单丝和丝线含有的丝胶充分去除干净，然后将所述蚕丝单丝和丝线置于温度为60℃烘箱内干燥，获得脱胶后的家蚕丝素纤维和熟丝线；
(2)制备家蚕丝素蛋白水溶液：将所述脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，在温度为65±10℃的条件下处理直至家蚕丝素纤维完全溶解，得到家蚕丝素溶解液，将所述家蚕丝素溶解液灌注于透析袋内，所述透析袋的材质为半透膜，截留分子量为10～100kDa，将灌注了所述家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的液体，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
(3)制备改性丝素蛋白溶液：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋白溶液的质量分数为1～10％，得到改性丝素蛋白溶液；
(4)制备丝素蛋白管状覆膜：将所述熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，将所述管状结构放入所述改性丝素蛋白溶液中浸渍30±5秒取出，置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，冲洗再风干，获得丝素蛋白管状覆膜；
(5)配置水蛭素水溶液0～500U/mL，将所述丝素蛋白管状覆膜放置于所述水蛭素水溶液中浸渍20±5分钟取出，置于鼓风干燥箱内，在温度为小于37℃的条件下沿圆周方向旋转风干，重复步骤(4)～(5)2～10次，获得蚕丝抗凝血管支架覆膜。
进一步的，步骤(1)中所述丝线为80D～120D。
进一步的，步骤(2)中所述透析袋的截留分子量为50kDa或14～16kDa。
进一步的，步骤(3)中所述按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的 1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋白溶液的质量分数为1～10％，替换为：添加与丝素蛋白质量比为0.6的聚乙二醇二缩水甘油醚，搅拌均匀并脱气泡。
进一步的，步骤(4)中所述置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，获得丝素蛋白管状覆膜替换为：置于热风干燥箱内在温度小于65℃的条件下沿圆周方向旋转风干，获得管状覆膜支架。
进一步的，在步骤(4)之后，步骤(5)之前还包括：制备阳离子化的丝素蛋白管状覆膜：配置并测定聚乙二醇双胺溶液的浓度，所述聚乙二醇双胺溶液包括含有聚乙二醇双胺1.5倍摩尔浓度的1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基琥珀酰亚胺和2-吗啉乙磺酸，将所述管状覆膜支架浸渍于所述聚乙二醇双胺溶液中反应20分钟后取出室温风干、冲洗再风干，得到阳离子化的丝素蛋白管状覆膜。
本发明的另一种技术方案是：
提供一种蚕丝抗凝血管支架覆膜的制备方法，该方法包括如下步骤：
(1)制备脱胶后的家蚕丝素纤维和熟丝线：选取40～160D丝线和蚕丝单丝，所述丝线由家蚕生丝加捻合并获得，将所述蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，在温度为98～100℃的条件下沸煮7小时，并且在沸煮期间多次更换去离子水，直至所述去离子水将所述蚕丝单丝和丝线含有的丝胶充分去除干净，然后将所述蚕丝单丝和丝线置于温度为60℃烘箱内干燥，获得脱胶后的家蚕丝素纤维和熟丝线；
(2)制备家蚕丝素蛋白水溶液：将所述脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，在温度为65±10℃的条件下处理直至丝素纤维完全溶解，得到家蚕丝素溶解液，将所述家蚕丝素溶解液灌注于透析袋内，所述透析袋的材质为半透膜，截留分子量为10～100kDa，将灌注了所述家蚕丝素溶解液的透析袋置于盛有去离子水的 容器内，每隔2小时用新的去离子水更换容器内的液体，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
(3)制备改性丝素蛋白溶液：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋白溶液的质量分数为1～10％，向所述聚乙二醇双胺阳离子化的丝素蛋白溶液中加入0～500U/mL凝血酶直接抑制剂水蛭素搅拌均匀并脱气泡，得到改性丝素蛋白溶液；
(4)制备丝素蛋白管状覆膜：将所述熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，将所述管状结构放入所述改性丝素蛋白溶液中浸渍30±5秒取出，置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，重复浸渍涂层2～10次，获得蚕丝抗凝血管支架覆膜。
进一步的，步骤(1)中所述丝线为80D～120D。
进一步的，步骤(2)中所述透析袋的截留分子量为50kDa或14～16kDa。
本发明提供了一种蚕丝抗凝血管支架覆膜及其制备方法，所制备的覆膜具有卓越的生物力学性能和持久的抗凝活性，且原位诱导调节血液系统平衡的血管内膜再生，该覆膜可以用于制备成各种形貌应用于心血管疾病治疗，如心脏修补片、血管支架、人工心脏瓣膜的体内移植物，也可以拓展用于其它一些组织工程支架等医疗器械。
具体实施方式
本发明的目的是针对目前心脑血管疾病腔内隔绝术治疗术后出现的血管覆膜支架长期再狭窄、血栓再发生、滑移及长期疲劳损坏等严峻的临 床问题，而开发的一种载抗凝因子的天然高分子丝素蛋白材料的血管支架覆膜的制备技术，该覆膜轴向抗拉伸强度>1.8MPa，断裂伸长率>45％，圆周向抗拉伸强度>7.0MPa，断裂伸长率>130％，整体水渗漏在120mmHg水压下小于7ml/min.cm 2，溶血率<0.1％。持续发挥抗凝功效，蚕丝抗凝血管支架覆膜的持续抗凝大于6个月，并具有诱导病变和缺损血管的组织再生与恢复血液平衡系统的调节功能。
本发明提供一种蚕丝抗凝血管支架覆膜的制备方法，包括：
步骤一、制备脱胶后的家蚕丝素纤维和熟丝线：
选取40～160D(优选80D～120D)丝线和蚕丝单丝，所述丝线由家蚕生丝加捻合并获得，将所述蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，在温度为98～100℃的条件下沸煮7小时，并且在沸煮期间多次更换去离子水，直至所述去离子水将所述蚕丝单丝和丝线含有的丝胶充分去除干净，然后将所述蚕丝单丝和丝线置于温度为60℃烘箱内干燥，获得脱胶后的家蚕丝素纤维和熟丝线。
步骤二、制备家蚕丝素蛋白水溶液：
将所述脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，在温度为65±10℃的条件下处理直至家蚕丝素纤维完全溶解，得到家蚕丝素溶解液，将所述家蚕丝素溶解液灌注于透析袋内，所述透析袋的材质为半透膜，截留分子量为10～100kDa，将灌注了所述家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的液体，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液。
步骤三、制备改性丝素蛋白溶液：
方法一：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶 液，再次采用旋转蒸发器浓缩、调整并测定所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋白溶液的质量分数为1～10％，得到改性丝素蛋白溶液。
方法二：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，添加与丝素蛋白质量比为0.6的聚乙二醇二缩水甘油醚(指纯化后的家蚕丝素蛋白水溶液中的丝素蛋白与聚乙二醇二缩水甘油醚的质量比是10：6)，搅拌均匀并脱气泡，得到改性丝素蛋白溶液。
方法三：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋白溶液的质量分数为1～10％，向所述聚乙二醇双胺阳离子化的丝素蛋白溶液中加入0～500U/mL凝血酶直接抑制剂水蛭素搅拌均匀并脱气泡，得到改性丝素蛋白溶液。
步骤四、制备丝素蛋白管状覆膜：
将所述熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，将所述管状结构放入所述改性丝素蛋白溶液中浸渍30±5秒取出，置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，冲洗再风干，或者置于热风干燥箱内在温度小于65℃的条件下沿圆周方向旋转风干，获得丝素蛋白管状覆膜。
如果上述步骤四是在利用步骤三的方法二作为步骤三的话，那么增加步骤：制备阳离子化的丝素蛋白管状覆膜：配置一定浓度的聚乙二醇双胺溶液，所述聚乙二醇双胺溶液包括含有聚乙二醇双胺1.5倍摩尔浓度的1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基琥珀酰亚胺和2-吗啉乙磺酸，将所述管状覆膜支架浸渍于所述聚乙二醇双胺溶液中反应20分钟后取出 室温风干、冲洗再风干，得到阳离子化的丝素蛋白管状覆膜。
步骤五、制备蚕丝抗凝血管支架覆膜。
配置水蛭素水溶液0～500U/mL，将所述丝素蛋白管状覆膜放置于所述水蛭素水溶液中浸渍20±5分钟取出，置于鼓风干燥箱内，在温度为小于37℃的条件下沿圆周方向旋转风干，重复步骤(4)～(5)2～10次，获得蚕丝抗凝血管支架覆膜。
上述步骤中的涂层次数与丝素蛋白浓度相关，如果浓度稀则需要增加涂层次数；如果浓度稠可以相应减少涂层次数，通过综合丝素蛋白浓度和涂层次数来调节覆膜性能和功能。
如果上述步骤四是在利用步骤三的方法三作为步骤三的话，那么不需要本步骤五。
上述方法所制备的蚕丝抗凝血管支架覆膜每一层涂层近乎为分子层，丝素蛋白大分子链在风干的过程中充分自组装为最稳定的分子构象，以及蚕丝采用去离子水脱胶保持了丝素纤维及再生丝素蛋白大分子链不被破坏，使覆膜具有优越的拉伸性能、爆破强度、顺应性和耐疲劳性，即使拉伸断裂时也不滑移，卓越地承担血流剪切和血管收缩扩张的作用。覆膜不发生内漏，植入后与病灶组织结合紧密不滑移。通过调节丝素蛋白浓度、管状纺织结构几何参数、自组装层数等制备参数获得生物体不同部位物理尺寸和物理性能要求的血管支架覆膜。另一方面，本发明提供的支架覆膜具有优异的细胞相容性、血液相容性和组织相容性。层层组装方式高倍数地提高了抗凝因子的加载，并随着覆膜梯度降解吸收同时持续发挥抗凝功能；丝素蛋白大分子量的调控也同时赋予了水蛭素的控释能力，长期阻止血栓和再狭窄；且覆膜内表面能获得快速内皮化，原位修复血管内膜组织，不会困扰于植入人体后因长期的疲劳损坏而导致装置失效。快速内皮化是抑制血栓形成和再狭窄的根本因素，内膜组织快速形成将彻底恢复血管凝血系统平衡的调节功能。
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例，还应包括在本发明所要求的权利范围内其他任何公知的改变。
首先，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1
本实施案例展示一种蚕丝抗凝血管支架覆膜的制备方法，包括：
1.将家蚕生丝加捻合并得80～120D丝线组，另一组为蚕丝单丝组，将蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，于98～100℃沸煮7小时，期间多次更换去离子水，然后用去离子水将丝充分清洗干净，置于60℃烘箱内干燥，得到脱胶后的家蚕丝素纤维和熟丝线。
2.称取脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至家蚕丝素纤维完全溶解，得家蚕丝素溶解液。将家蚕丝素溶解液灌注于透析袋内，透析袋壁是半透膜，截留分子量为14～16kDa范围，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液。
3.采用旋转蒸发器浓缩、调整并测定纯化后的家蚕丝素蛋白水溶液质量分数为5％，添加与丝素蛋白质量比为0.6的聚乙二醇二缩水甘油醚，搅拌均匀并脱气泡，得到改性丝素蛋白溶液。
4.将熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，于上述第3步的改性丝素蛋白溶液中浸渍30秒左右取出，置于65℃以下的热风干燥箱内圆周方向旋转风干。
5.配置一定浓度聚乙二醇双胺的水溶液，其中含有聚乙二醇双胺1.5倍摩尔浓度的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，少量的N-羟基琥珀酰亚胺和2-吗啉乙磺酸，搅拌均匀。将第4步的管状覆膜支架浸渍于聚乙二醇双胺溶液中反应20分钟后取出室温风干、冲洗再风干，得阳离子化的丝素蛋白管状覆膜。
6.配置水蛭素水溶液(100U/mL)，将丝素蛋白管状覆膜于水蛭素水溶液中浸渍20分钟左右取出，置于37℃以下的鼓风干燥箱内圆周方向旋 转风干。交替重复4～6步骤5次，获得蚕丝丝素复合血管支架覆膜，即蚕丝抗凝血管支架覆膜。
经检测，上述蚕丝抗凝血管支架覆膜具有卓越的力学性能，按照国标检测方法测定轴向抗拉伸强度>1.8MPa，断裂伸长率>45％，圆周向抗拉伸强度>7.0MPa，断裂伸长率>130％，整体水渗漏在120mmHg水压下小于5ml/min.cm 2。
通过对蚕丝抗凝血管支架覆膜按照溶血率测试方法测定溶血率<0.1％，完全符合非溶血性材料的标准(0～2％)。蚕丝抗凝血管支架覆膜通过动物实验无致敏性，按照国标检测细胞毒性≤1。同时，蚕丝抗凝血管支架覆膜有显著的抗凝性能，并有持续抗凝效果。
实施例2
本实施案例展示一种蚕丝抗凝血管支架覆膜的制备方法，包括：
1.将家蚕生丝加捻合并得80～120D丝线组，另一组为蚕丝单丝组，将蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，于98～100℃沸煮7小时，期间多次更换去离子水，然后用去离子水将丝充分清洗干净，置于60℃烘箱内干燥，得到脱胶后的家蚕丝素纤维和熟丝线。
2.称取脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至家蚕丝素纤维完全溶解，得家蚕丝素溶解液。将家蚕丝素溶解液灌注于透析袋内，透析袋壁是半透膜，截留分子量为50kDa范围，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的液体，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液。
3.采用旋转蒸发器浓缩、调整并测定纯化后的家蚕丝素蛋白水溶液质量分数为5～10％，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向上述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺搅拌均匀、10％的N-羟基琥珀酰亚胺和20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定聚乙二醇双胺阳离子化的丝素蛋白溶液质量分数为5％。
4.将熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，于上述第3步的聚乙二醇双胺阳离子化的丝素蛋白溶液中浸渍30秒左右取出，置于37℃以下的鼓风干燥箱内圆周方向旋转风干，得丝素蛋白管状覆膜。
5.配置水蛭素水溶液(100U/mL)，将丝素蛋白管状覆膜于水蛭素水溶液中浸渍20分钟左右取出，置于37℃以下的鼓风干燥箱内圆周方向旋转风干。交替重复4和5两步涂层各5次，获得蚕丝丝素复合血管支架覆膜，即蚕丝抗凝血管支架覆膜。
经检测，上述蚕丝抗凝血管支架覆膜具有卓越的力学性能，按照国标检测方法测定轴向抗拉伸强度>2.5MPa，断裂伸长率>70％，圆周向抗拉伸强度>8.0MPa，断裂伸长率>150％，整体水渗漏在120mmHg水压下小于2ml/min.cm 2。
通过对蚕丝抗凝血管支架覆膜按照溶血率测试方法测定溶血率<0.1％，完全符合非溶血性材料的标准(0～2％)。蚕丝抗凝血管支架覆膜通过动物实验无致敏性，按照国标检测细胞毒性≤1。同时，蚕丝抗凝血管支架覆膜有显著的抗凝性能，并有持续抗凝效果，持续抗凝效果显著优于实施例1和4。
实施例3
本实施案例展示一种蚕丝抗凝血管支架覆膜的制备方法，包括：
1.将家蚕生丝加捻合并得80～120D丝线组，另一组为蚕丝单丝组，将蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，于98～100℃沸煮7小时，期间多次更换去离子水，然后用去离子水将丝充分清洗干净，置于60℃烘箱内干燥，得到脱胶后的家蚕丝素纤维和熟丝线。
2.称取脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，65℃处理直至家蚕丝素纤维完全溶解，得家蚕丝素溶解液。将家蚕丝素溶解液灌注于透析袋内，透析袋壁是半透膜，截留分子量为50kDa范围，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液。
3.采用旋转蒸发器浓缩、调整并测定纯化后的家蚕丝素蛋白水溶液质量分数为5～10％，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向上述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺搅拌均匀、10％的N-羟基琥珀酰亚胺和20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定阳离子化的丝素蛋白溶液质量分数为5％。再向阳离子化的丝素蛋白溶液中加入100U/mL凝血酶直接抑制剂水蛭素搅拌均匀并脱气泡，得到改性丝素蛋白溶液。
4.将熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，于上述第3步的改性丝素蛋白溶液中浸渍30秒左右取出，置于37℃以下的鼓风干燥箱内圆周方向旋转风干。重复浸渍涂层5次，获得蚕丝丝素复合血管支架覆膜，即蚕丝抗凝血管支架覆膜。
经检测，上述蚕丝抗凝血管支架覆膜具有卓越的力学性能，按照国标检测方法测定轴向抗拉伸强度>2.8MPa，断裂伸长率>70％，圆周向抗拉伸强度>10.0MPa，断裂伸长率>150％，整体水渗漏在120mmHg水压下小于2ml/min.cm 2。
通过对蚕丝抗凝血管支架覆膜按照溶血率测试方法测定溶血率<0.1％，完全符合非溶血性材料的标准(0～2％)。蚕丝抗凝血管支架覆膜通过动物实验无致敏性，按照国标检测细胞毒性≤1。同时，蚕丝抗凝血管支架覆膜有显著的抗凝性能，并有持续抗凝效果，持续抗凝效果显著优于实施例1和4。
实施例4
本实施案例展示一种蚕丝抗凝血管支架覆膜的制备方法，包括：
1.将家蚕生丝加捻合并得80～120D丝线组，另一组为蚕丝单丝组，将蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，于98～100℃沸煮7小时，期间多次更换去离子水，然后用去离子水将丝充分清洗干净，置于60℃烘箱内干燥，得到脱胶后的家蚕丝素纤维和熟丝线。
2.称取脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的 溴化锂水溶液中，65℃处理直至家蚕丝素纤维完全溶解，得家蚕丝素溶解液。将家蚕丝素溶解液灌注于透析袋内，透析袋壁是半透膜，截留分子量为14～16kDa范围，将灌注了家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的水，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液。
3.采用旋转蒸发器浓缩、调整并测定透析后的丝素蛋白水溶液质量分数为5～10％，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向上述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺搅拌均匀、10％的N-羟基琥珀酰亚胺和20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定阳离子化的丝素蛋白溶液质量分数为5％。再向阳离子化的丝素蛋白溶液中加入100U/mL凝血酶直接抑制剂水蛭素搅拌均匀并脱气泡，得到改性丝素蛋白溶液。
4.将熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，于上述第3步的改性丝素蛋白溶液中浸渍30秒左右取出，置于37℃以下的鼓风干燥箱内圆周方向旋转风干。重复浸渍涂层5次，获得蚕丝丝素复合血管支架覆膜，即蚕丝抗凝血管支架覆膜。
经检测，上述蚕丝抗凝血管支架覆膜具有卓越的力学性能，按照国标检测方法测定轴向抗拉伸强度>2.0MPa，断裂伸长率>45％，圆周向抗拉伸强度>7.0MPa，断裂伸长率>130％，整体水渗漏在120mmHg水压下小于7ml/min.cm 2。
通过对蚕丝抗凝血管支架覆膜按照溶血率测试方法测定溶血率<0.1％，完全符合非溶血性材料的标准(0-2％)。蚕丝抗凝血管支架覆膜通过动物实验无致敏性，按照国标检测细胞毒性≤1。同时，支架覆膜有显著的抗凝性能，并有持续抗凝效果。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精 神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. 一种蚕丝抗凝血管支架覆膜，其特征在于：轴向抗拉伸强度>1.8MPa，断裂伸长率>45％，圆周向抗拉伸强度>7.0MPa，断裂伸长率>130％，整体水渗漏在120mmHg水压下小于7ml/min.cm 2，溶血率<0.1％，所述蚕丝抗凝血管支架覆膜的持续抗凝大于6个月。
  2. 一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于，该方法包括如下步骤：
    (1)制备脱胶后的家蚕丝素纤维和熟丝线：选取40～160D丝线和蚕丝单丝，所述丝线由家蚕生丝加捻合并获得，将所述蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，在温度为98～100℃的条件下沸煮7小时，并且在沸煮期间多次更换去离子水，直至所述去离子水将所述蚕丝单丝和丝线含有的丝胶充分去除干净，然后将所述蚕丝单丝和丝线置于温度为60℃烘箱内干燥，获得脱胶后的家蚕丝素纤维和熟丝线；
    (2)制备家蚕丝素蛋白水溶液：将所述脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，在温度为65±10℃的条件下处理直至家蚕丝素纤维完全溶解，得到家蚕丝素溶解液，将所述家蚕丝素溶解液灌注于透析袋内，所述透析袋的材质为半透膜，截留分子量为10-100kDa，将灌注了所述家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的液体，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
    (3)制备改性丝素蛋白溶液：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋 白溶液的质量分数为1～10％，得到改性丝素蛋白溶液；
    (4)制备丝素蛋白管状覆膜：将所述熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，将所述管状结构放入所述改性丝素蛋白溶液中浸渍30±5秒取出，置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，冲洗再风干，获得丝素蛋白管状覆膜；
    (5)配置水蛭素水溶液0～500U/mL，将所述丝素蛋白管状覆膜放置于所述水蛭素水溶液中浸渍20±5分钟取出，置于鼓风干燥箱内，在温度为小于37℃的条件下沿圆周方向旋转风干，重复步骤(4)～(5)2～10次，获得蚕丝抗凝血管支架覆膜。
  3. 根据权利要求2所述的一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于：步骤(1)中所述丝线为80D～120D。
  4. 根据权利要求2所述的一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于：步骤(2)中所述透析袋的截留分子量为50kDa或14～16kDa。
  5. 根据权利要求4所述的一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于，步骤(3)中所述按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的丝素蛋白溶液的质量分数为1～10％，替换为：添加与丝素蛋白质量比为0.6的聚乙二醇二缩水甘油醚，搅拌均匀并脱气泡。
  6. 根据权利要求5所述的一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于，步骤(4)中所述置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，获得丝素蛋白管状覆膜替换为：置于热风干燥箱内在温度小于65℃的条件下沿圆周方向旋转风干，获得管状覆膜支架。
  7. 根据权利要求6所述的一种蚕丝抗凝血管支架覆膜的制备方法， 其特征在于，在步骤(4)之后，步骤(5)之前还包括：制备阳离子化的丝素蛋白管状覆膜：配置并测定聚乙二醇双胺溶液的浓度，所述聚乙二醇双胺溶液包括含有聚乙二醇双胺1.5倍摩尔浓度的1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基琥珀酰亚胺和2-吗啉乙磺酸，将所述管状覆膜支架浸渍于所述聚乙二醇双胺溶液中反应20分钟后取出室温风干、冲洗再风干，得到阳离子化的丝素蛋白管状覆膜。
  8. 一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于，该方法包括如下步骤：
    (1)制备脱胶后的家蚕丝素纤维和熟丝线：选取40～160D丝线和蚕丝单丝，所述丝线由家蚕生丝加捻合并获得，将所述蚕丝单丝和丝线按1:50(g/mL)的浴比放入去离子水中，在温度为98～100℃的条件下沸煮7小时，并且在沸煮期间多次更换去离子水，直至所述去离子水将所述蚕丝单丝和丝线含有的丝胶充分去除干净，然后将所述蚕丝单丝和丝线置于温度为60℃烘箱内干燥，获得脱胶后的家蚕丝素纤维和熟丝线；
    (2)制备家蚕丝素蛋白水溶液：将所述脱胶后的家蚕丝素纤维按1:10(g/mL)的浴比溶解于9.3M的溴化锂水溶液中，在温度为65±10℃的条件下处理直至丝素纤维完全溶解，得到家蚕丝素溶解液，将所述家蚕丝素溶解液灌注于透析袋内，所述透析袋的材质为半透膜，截留分子量为10～100kDa，将灌注了所述家蚕丝素溶解液的透析袋置于盛有去离子水的容器内，每隔2小时用新的去离子水更换容器内的液体，持续透析3天，得到纯化后的家蚕丝素蛋白水溶液；
    (3)制备改性丝素蛋白溶液：采用旋转蒸发器浓缩、调整并测定所述纯化后的家蚕丝素蛋白水溶液的质量分数，按质量比100:5～10配置丝素蛋白与聚乙二醇双胺的混合溶液，向所述混合溶液中添加质量比为丝素蛋白20％的1-(3-二甲氨基丙基)-3-乙基碳二亚胺，并搅拌均匀，再添加质量比为丝素蛋白10％的N-羟基琥珀酰亚胺和质量比为丝素蛋白20％的2-吗啉乙磺酸，反应20分钟后用去离子水透析12～48小时，得到聚乙二醇双胺阳离子化的丝素蛋白溶液，再次采用旋转蒸发器浓缩、调整并测定所述聚乙二醇双胺阳离子化的丝素蛋白溶液，使所述聚乙二醇双胺阳离子化的 丝素蛋白溶液的质量分数为1～10％，向所述聚乙二醇双胺阳离子化的丝素蛋白溶液中加入0～500U/mL凝血酶直接抑制剂水蛭素搅拌均匀并脱气泡，得到改性丝素蛋白溶液；
    (4)制备丝素蛋白管状覆膜：将所述熟丝线采用编结技术在不锈钢棒上编成30～90°交织、内径为2～20mm的管状结构，将所述管状结构放入所述改性丝素蛋白溶液中浸渍30±5秒取出，置于鼓风干燥箱内在温度小于37℃的条件下沿圆周方向旋转风干，重复浸渍涂层2～10次，获得蚕丝抗凝血管支架覆膜。
  9. 根据权利要求8所述的一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于：步骤(1)中所述丝线为80D～120D。
  10. 根据权利要求8所述的一种蚕丝抗凝血管支架覆膜的制备方法，其特征在于：步骤(2)中所述透析袋的截留分子量为50kDa或14～16kDa。
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