WO2022028395A1 - Anticoagulant intravascular stent cover film and preparation method therefor - Google Patents

Anticoagulant intravascular stent cover film and preparation method therefor Download PDF

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Publication number
WO2022028395A1
WO2022028395A1 PCT/CN2021/110227 CN2021110227W WO2022028395A1 WO 2022028395 A1 WO2022028395 A1 WO 2022028395A1 CN 2021110227 W CN2021110227 W CN 2021110227W WO 2022028395 A1 WO2022028395 A1 WO 2022028395A1
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Prior art keywords
silk fibroin
silk
solution
tubular
aqueous solution
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PCT/CN2021/110227
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French (fr)
Chinese (zh)
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王建南
戴梦男
宋广州
裔洪根
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苏州大学
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Publication of WO2022028395A1 publication Critical patent/WO2022028395A1/en

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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

  • 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. Although it has been found that the improvement of anticoagulant performance has been achieved, the content of stably bound hirudin is still very low. Therefore, in view of the bottleneck problem of existing clinical applications and the incidence of younger and younger (even live-born babies), it is necessary. An anticoagulant stent covering was developed.
  • a technical scheme of the present invention is:
  • the content is less than 9 ml/min.cm 2
  • the hemolysis rate is less than 0.1%
  • the continuous anticoagulation of the silk anticoagulant tube stent coating is more than 6 months.
  • the preparation method of the above-mentioned anticoagulation 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 silk and the silk thread are fully cleaned, and then the silk monofilament and the silk thread are placed in an oven with a temperature of 60 ° C to dry to obtain the degummed Bombyx mori fibroin and cooked silk thread;
  • modified silk fibroin solution using a rotary evaporator to concentrate, adjust and measure the purified silk fibroin aqueous solution so that the mass fraction of the purified silk fibroin aqueous solution is 1-10% , adding polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stirring evenly and defoaming to obtain a modified silk fibroin solution, which is loaded into the first syringe;
  • the cooked silk thread is braided on a stainless steel rod with a knitting technique to form a tubular structure with an inner diameter of 2-20 mm and an inner diameter of 30-90°, and is installed in a rotatable and capable of advancing or retreating Connect one end of the silicone tube to the first syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the tubular structure, and comprehensively adjust the modified silk fibroin solution in the first syringe.
  • the flow rate, the rotation and the running speed of the shaft, the surface of the tubular structure is coated with a continuous thin layer of modified silk fibroin solution, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air with a temperature less than 37 ° C to obtain silk fibroin. tubular covering;
  • hirudin aqueous solution put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular coating, the silk
  • the fibroin tubular membrane keeps rotating while moving forward, the surface of the silk fibroin tubular membrane is coated with the hirudin aqueous solution, the flow rate of the second syringe is adjusted, and the hirudin is controlled in the silk fibroin tubular membrane.
  • the coating amount on the surface is 0-50 U/cm 2 , and at the same time it is air-dried at room temperature, and steps (4) to (5) are alternately repeated 5 times to obtain an anticoagulant tube stent coating.
  • step (1) is 120D.
  • the molecular weight cut-off of the dialysis bag in step (2) is 50 kDa or 14-16 kDa.
  • step (3) polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin is added, stirred evenly and debubbled to obtain a modified silk fibroin solution, and the modified silk fibroin solution is Fill the first syringe and replace it with: adding polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stirring evenly and defoaming to obtain a modified silk fibroin solution.
  • step (4) 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 2 to 20 mm by knitting technology, and is installed on a rotatable electric shaft that can advance or retreat.
  • One end of the silicone tube is connected to the syringe, the other end is connected to a section of bundled cooked silk thread and attached to the surface of the tubular structure, and the certain flow rate of the modified silk fibroin solution in the syringe, the rotation of the shaft and the running speed are comprehensively adjusted.
  • the method further includes: preparing a cationized silk fibroin tubular coating; configuring and measuring the mass fraction of the polyethylene glycol diamine solution, the polyethylene glycol diamine
  • the amine solution includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and 2-morpholineethanesulfonate containing 1.5-fold molar concentration of polyethylene glycol bisamine acid, the silk fibroin tubular coating was immersed in the polyethylene glycol diamine solution to react for 20 minutes, then taken out and air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular coating.
  • the mass fraction of the purified Bombyx mori silk fibroin aqueous solution in step (3) is 4-5%.
  • step (5) the coating amount of hirudin on the surface of the silk fibroin tubular film is controlled to be 20 U/cm 2 .
  • the invention provides an anticoagulant tube stent coating and a preparation method thereof.
  • the deionized water degumming silk fibroin fiber textile structure core layer, layer by layer self-assembled macromolecular silk fibroin, and layer by layer loading anticoagulation factors, etc. The prepared film has excellent biomechanical properties and long-lasting anticoagulant activity, and in situ induces vascular intimal regeneration that regulates the balance of the blood system.
  • the film can be prepared into various shapes and applied to cardiovascular diseases. Treatment, such as heart patch, vascular stent, artificial heart valve and other in vivo grafts, 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>35%, circumferential tensile strength>4.0MPa , the elongation at break>100%, the overall water leakage is less than 9ml/min.cm 2 under 120mmHg water pressure, and the hemolysis rate is ⁇ 0.1%.
  • the present invention provides a method for preparing an anticoagulant tube stent coating, comprising:
  • Step 1 Preparation of degummed Bombyx mori silk fibroin and cooked silk thread:
  • the silk thread is obtained by twisting and combining the silkworm raw silk, the silk monofilament and silk thread are put into deionization at the liquor ratio of 1:50 (g/mL).
  • the silk monofilament and silk thread are placed in an oven at a temperature of 60° C. to be dried to obtain degummed silk fibroin fibers and cooked silk threads.
  • 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 Concentrate, adjust and measure the purified silk fibroin aqueous solution by using a rotary evaporator, so that the mass fraction of the purified silk fibroin aqueous solution is 1-10%, and add the amount of silk fibroin in a ratio of 1 to 10%.
  • Be 0.6 polyethylene glycol diglycidyl ether referring to the mass ratio of silk fibroin and polyethylene glycol diglycidyl ether in the purified silk fibroin aqueous solution is 10:6), stir evenly and debubble, A modified silk fibroin solution is obtained, and the modified silk fibroin solution is loaded into the first syringe.
  • Method 2 use a rotary evaporator to concentrate, adjust and measure the purified silk fibroin aqueous solution, so that the mass fraction of the purified silk fibroin aqueous solution is 1-10% and the ratio of the addition to the silk fibroin is 0.6% of polyethylene glycol diglycidyl ether, stir evenly and degassing to obtain a modified silk fibroin solution.
  • step 3 If method 1 is adopted in step 3, method 1 is also adopted in this step: the cooked silk thread is braided on the stainless steel rod into a 30-90° interlaced tubular structure with an inner diameter of 2-20 mm, and is installed on a rotatable And on the electric shaft that can advance or retreat, take one end of the silicone tube to connect the first syringe, and the other end to connect a section of bundled cooked silk thread and stick it on the surface of the tubular structure, and comprehensively adjust the modification in the first syringe.
  • the flow rate of the silk fibroin solution, the rotation of the shaft and the running speed, the surface of the tubular structure is coated with a continuous thin layer of the modified silk fibroin solution, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air with a temperature of less than 37° C. , to obtain a silk fibroin tubular membrane.
  • the second method is also adopted in this step: the cooked silk thread is braided on the stainless steel rod into a 30-90° interlaced tubular structure with an inner diameter of 2-20 mm, and the tubular structure is woven into the stainless steel rod. Put it into the modified silk fibroin solution and immerse it for 30 ⁇ 5 seconds, take it out, and place it in a hot-air drying box to rotate and air-dry in the circumferential direction under the condition that the temperature is less than 65°C.
  • any one of the above-mentioned steps and four methods can add steps: preparing cationized silk fibroin tubular coating: configuring and measuring the mass fraction of polyethylene glycol diamine solution, the polyethylene glycol diamine solution including poly 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and 2-morpholineethanesulfonic acid in 1.5 times molar concentration of ethylene glycol diamine, the The silk fibroin tubular membrane was immersed in the polyethylene glycol diamine solution for 20 minutes of reaction, then taken out to be air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular membrane.
  • Step 5 preparing the anticoagulant tube stent coating.
  • Method 1 Prepare an aqueous solution of hirudin, put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular coating.
  • the fibroin tubular membrane keeps rotating while moving forward, the surface of the silk fibroin tubular membrane is coated with the hirudin aqueous solution, the flow rate of the second syringe is adjusted, and the hirudin is controlled in the silk fibroin tubular membrane.
  • the coating amount on the surface is 0-50 U/cm 2 (preferably 20 U/cm 2 ), and at the same time it is air-dried at room temperature, and steps (4) to (5) are alternately repeated 5 times to obtain an anticoagulant tube stent coating.
  • a hirudin aqueous solution put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked
  • the silk thread is attached to the surface of the cationized silk fibroin tubular membrane, the cationized silk fibroin tubular membrane keeps rotating while advancing, and is coated on the surface of the cationized silk fibroin tubular membrane
  • the flow rate of the second syringe is adjusted, and the coating amount of hirudin on the surface of the cationized silk fibroin tubular film is controlled to be 0-50 U/cm 2 (preferably 20U/cm 2 ), At the same time, it was air-dried at room temperature, and steps (4) to (5) were alternately repeated 5 times to obtain an anticoagulant tube stent coating.
  • Each coating of the 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 kept by deionized water.
  • the silk fibroin fiber and the regenerated silk fibroin macromolecular chain are not damaged, so that the film has excellent tensile properties, burst strength, compliance and fatigue resistance.
  • the role of flow shear and vasoconstriction in dilation The membrane does not have endoleak, and is tightly combined with the lesion tissue without slipping after implantation.
  • the vascular stent coverings required by the physical size and physical properties of different parts of the organism are obtained.
  • the stent coating provided by the present invention has excellent cytocompatibility, blood compatibility and tissue compatibility.
  • 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 method for preparing an anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at °C for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 °C to obtain 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 2 to 20 mm, immersed in the modified silk fibroin solution obtained in the third step above for 30 ⁇ 5 seconds and taken out, It was placed in a hot-air drying box below 65°C and air-dried by rotating in the circumferential direction to obtain a silk fibroin tubular coating.
  • Step 6 Prepare an aqueous solution of hirudin, put it into a syringe, connect one end of the silicone tube to the syringe, and connect the other end with a bunch of cooked silk thread and stick it on the surface of the cationized silk fibroin tubular film in step 5, adjust the flow rate of the syringe, cationize
  • the silk fibroin tubular membrane kept rotating and moved forward, and the surface of the cationized silk fibroin tubular membrane was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature.
  • Steps 4 to 6 are alternately repeated for 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
  • the above-mentioned 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 fracture is broken.
  • the elongation is >130%
  • the overall water leakage is less than 8ml/min.cm 2 under 120mmHg water pressure.
  • the hemolysis rate is less than 0.1% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the 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 anticoagulation tube stent coating has significant anticoagulation performance, and has a continuous anticoagulation effect, and the anticoagulation performance is better than that of Example 2.
  • This example shows a method for preparing an anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at °C for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 °C to obtain 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 2 to 20 mm on a stainless steel rod, and is installed on an electric shaft that can rotate and advance/retract.
  • One end of the silicone tube is connected to the first syringe, the other end is connected to a bundle of cooked silk threads and attached to the surface of the tubular structure, and the flow rate, rotation and running speed of the modified silk fibroin solution in the first
  • the structure is coated with a continuous thin layer, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air below 37°C to obtain a silk fibroin tubular coating.
  • Step 5 Prepare the hirudin aqueous solution, put it into the second syringe, connect one end of the silicone tube to the second syringe, and connect the other end with a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular film in step 4, and adjust the second syringe
  • the silk fibroin tubular membrane kept rotating and moved forward, and the surface of the silk fibroin tubular membrane was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature.
  • Steps 4 to 5 are alternately repeated 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
  • the above-mentioned anticoagulant tube stent coating has excellent mechanical properties.
  • the axial tensile strength is >2.0MPa
  • the elongation at break is >35%
  • the circumferential tensile strength is >4.0MPa
  • the fracture is broken. Elongation>100%
  • the overall water leakage is less than 9ml/min.cm 2 under 120mmHg water pressure.
  • the hemolysis rate is less than 0.1% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the 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 anticoagulant tube stent coating has significant anticoagulant properties and has a sustained anticoagulant effect.
  • This example shows a method for preparing an anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at °C for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 °C to obtain 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 2 to 20 mm on a stainless steel rod, and is installed on an electric shaft that can rotate and advance/retract. Take one end of the silicone tube and connect it to the syringe, and the other end to connect a bunch of cooked silk thread and stick it on the surface of the tubular structure. Comprehensively adjust the flow rate of the modified silk fibroin solution in the first syringe, the rotation of the shaft and the running speed, and coat the tubular structure. A thin layer of continuous modified silk fibroin solution is covered, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air below 37° C. to obtain a tubular film of silk fibroin.
  • Step 6 Prepare an aqueous solution of hirudin, put it into the second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the cationized silk fibroin tubular film that was air-dried in step 5. Adjust At the flow rate of the second syringe, the cationized silk fibroin tubular film was kept rotating while advancing, and the surface of the cationized silk fibroin tubular film was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature. Steps 4 to 6 are alternately repeated for 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
  • the above-mentioned 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 >5.0MPa
  • the fracture is broken.
  • the elongation is >100%
  • the overall water leakage is less than 8ml/min.cm 2 under 120mmHg water pressure.
  • This example shows a method for preparing an anticoagulant tube stent covering, including:
  • the silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at °C for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 °C to obtain degummed silk fibroin fibers and cooked silk threads.
  • Step 6 Prepare an aqueous solution of hirudin, put it into the second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the cationized silk fibroin tubular film that was air-dried in step 5. Adjust At the flow rate of the second syringe, the cationized silk fibroin tubular film was kept rotating while advancing, and the surface of the cationized silk fibroin tubular film was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature. Steps 4 to 6 are alternately repeated for 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
  • the above-mentioned anticoagulant tube stent coating has excellent mechanical properties.
  • the axial tensile strength is >2.5MPa
  • the elongation at break is >50%
  • the circumferential tensile strength is >8.0MPa
  • the fracture is broken.
  • the elongation 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% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%).
  • the 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 coating has significant anticoagulation performance, and the continuous anticoagulation effect is significantly better than that of Examples 1-3.

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Abstract

Disclosed are an anticoagulant intravascular stent cover film and a preparation method therefor. Silkworm silk is divided into two groups, wherein one of the groups is twisted and combined to obtain a silk thread group, and the other group is a monofilament group; both groups are then boiled with deionized water for degumming; the silk thread group is braided into a tubular structure by using a braiding machine, while the silk of the monofilament group is dissolved in a lithium bromide neutral salt solution to prepare a dissolved silk fibroin solution, the dissolved silk fibroin solution is then poured into a dialysis bag, and the dialysis bag is placed in a container containing deionized water for continuous dialysis to obtain a purified silkworm silk fibroin aqueous solution; the silk fibroin aqueous solution is then concentrated by means of a rotary evaporator; an appropriate amount of a cross-linking agent is added to the silk fibroin aqueous solution for a reaction, and the solution is then coated or spin-coated on the tubular structure; and after air drying with hot air in a rotary manner, same is further spin-coated with hirudin and then air-dried in a rotary manner at room temperature to obtain the anticoagulant intravascular stent cover film. The cover film has excellent biomechanical properties and a long-lasting anticoagulant activity.

Description

一种抗凝血管支架覆膜及其制备方法A kind of anticoagulant tube stent coating and preparation method thereof
本申请要求于2020年08月06日提交中国专利局、申请号为202010781141.7、发明名称为“一种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。This application claims the priority of the Chinese patent application with the application number 202010781141.7 and the invention titled "An anticoagulant tube stent coating and its preparation method", which was submitted to the China Patent Office on August 6, 2020, the entire contents of which are by reference Incorporated in this application.
技术领域technical field
本发明涉及血管支架覆膜制备技术领域，具体涉及一种抗凝血管支架覆膜及其的制备方法。The invention relates to the technical field of preparation of vascular stent coating, in particular to an anticoagulant tube stent coating and a preparation method thereof.
背景技术Background technique
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。With the improvement of living standards and the acceleration of the aging process, the incidence of cardiovascular and cerebrovascular diseases is increasing significantly year by year. 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. With the development of medicine, 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. However, complications such as stent-graft slippage, endoleak, and stent-derived new breaks, as well as thrombosis and restenosis still occur in the mid- and long-term postoperatively. These clinical problems deserve close attention. The poor biocompatibility (especially histocompatibility and hemocompatibility) of the stent-graft itself, the incompatibility of biomechanical properties, and the long-term encapsulation of non-degradable materials by blood components are the main reasons for postoperative complications. Therefore, there is an urgent need to develop vascular stent-grafts that match the biological properties of host vessels.
腔内修复术的关键在于血管支架***的研制，其中包括支架、覆膜和输送***，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***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。The key to endovascular repair lies in the development of a vascular stent system, including stents, coverings and delivery systems. Currently, 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. For the young and middle-aged population, and even infants and young children with an increasing incidence, it is extremely inappropriate to rely on drugs to maintain antithrombotics for a long time. Therefore, the design and preparation of a new type of vascular stent with anticoagulation function, which can completely restore the balance function of the vascular coagulation system, has important research and application value for solving new clinical problems.
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。虽已发现实现了抗凝血性能的提高，但稳定结合的水蛭素含量还是很低，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，有必要研发一种抗凝血管支架覆膜。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. Compared with synthetic polymers, natural 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. 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. Although it has been found that the improvement of anticoagulant performance has been achieved, the content of stably bound hirudin is still very low. Therefore, in view of the bottleneck problem of existing clinical applications and the incidence of younger and younger (even live-born babies), it is necessary. An anticoagulant stent covering was developed.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明的目的在于提供一种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。In view of this, the purpose of the present invention is to provide an 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.
本发明的一种技术方案是：A technical scheme of the present invention is:
提供一种抗凝血管支架覆膜，轴向抗拉伸强度>1.8MPa，断裂伸长率>35％，圆周向抗拉伸强度>4.0MPa，断裂伸长率>100％，整体水渗漏在120mmHg水压下小于9ml/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。 Provided is an anticoagulant tube stent covering with axial tensile strength>1.8MPa, elongation at break>35%, circumferential tensile strength>4.0MPa, elongation at break>100%, and overall water leakage Under the water pressure of 120 mmHg, the content is less than 9 ml/min.cm 2 , the hemolysis rate is less than 0.1%, and the continuous anticoagulation of the silk anticoagulant tube stent coating is more than 6 months.
上述抗凝血管支架覆膜的制备方法，该方法包括如下步骤：The preparation method of the above-mentioned anticoagulation tube stent coating, the method comprises the following steps:
(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充分清洗干净，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；(1) 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 silk and the silk thread are fully cleaned, and then the silk monofilament and the silk thread are placed in an oven with a temperature of 60 ° C to dry to obtain the degummed Bombyx mori fibroin and cooked silk thread;
(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；(2) Preparation of Bombyx mori silk fibroin aqueous solution: the degummed Bombyx mori silk fibroin fiber was dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL) at a temperature of 65±10°C Lower treatment until the Bombyx mori silk fibroin fibers are completely dissolved to obtain a Bombyx mori silk fibroin dissolving solution, and the Bombyx mori silk fibroin dissolving solution is perfused in a dialysis bag, the material of the dialysis bag is a semi-permeable membrane, and the molecular weight cut-off is 10-100 kDa, and the The dialysis bag perfused with the 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 Vegetarian protein aqueous solution;
(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，使所述纯化后的家蚕丝素蛋白水溶液的质量分数为1～10％，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，将所述改性丝素蛋白溶液装入第一注射器；(3) Preparation of modified silk fibroin solution: using a rotary evaporator to concentrate, adjust and measure the purified silk fibroin aqueous solution so that the mass fraction of the purified silk fibroin aqueous solution is 1-10% , adding polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stirring evenly and defoaming to obtain a modified silk fibroin solution, which is loaded into the first syringe;
(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，并安装于可旋转且能够前进或后退的电动轴上，取硅胶管一端连接所述第一注射器，另一端连接一段束状熟丝线并贴在所述管状结构的表面，综合调节所述第一注射器内所述改性丝素蛋白溶液的流速、轴的旋转和运行速度，给所述管状结构表面涂覆一层连续的改性丝素蛋白溶液薄层，同时在温度小于37℃的热风中沿圆周方向旋转风干，获得丝素蛋白管状覆膜；(4) Preparation of silk fibroin tubular coating: the cooked silk thread is braided on a stainless steel rod with a knitting technique to form a tubular structure with an inner diameter of 2-20 mm and an inner diameter of 30-90°, and is installed in a rotatable and capable of advancing or retreating Connect one end of the silicone tube to the first syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the tubular structure, and comprehensively adjust the modified silk fibroin solution in the first syringe. The flow rate, the rotation and the running speed of the shaft, the surface of the tubular structure is coated with a continuous thin layer of modified silk fibroin solution, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air with a temperature less than 37 ° C to obtain silk fibroin. tubular covering;
(5)配置水蛭素水溶液，装入第二注射器，将硅胶管一端连接所述第二注射器，另一端连接一段束状熟丝线并贴在所述丝素蛋白管状覆膜的表面，所述丝素蛋白管状覆膜保持一边旋转一边前进，在所述丝素蛋白管状覆膜表面涂覆所述水蛭素水溶液，调节所述第二注射器的流速，控制水蛭素在所述丝素蛋白管状覆膜表面的涂覆量为0～50U/cm2，同时室温风干，交替重复(4)～(5)步骤5次，获得抗凝血管支架覆膜。(5) Prepare a hirudin aqueous solution, put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular coating, the silk The fibroin tubular membrane keeps rotating while moving forward, the surface of the silk fibroin tubular membrane is coated with the hirudin aqueous solution, the flow rate of the second syringe is adjusted, and the hirudin is controlled in the silk fibroin tubular membrane. The coating amount on the surface is 0-50 U/cm 2 , and at the same time it is air-dried at room temperature, and steps (4) to (5) are alternately repeated 5 times to obtain an anticoagulant tube stent coating.
进一步的，步骤(1)中所述丝线为120D。Further, the silk thread in step (1) is 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。Further, the molecular weight cut-off of the dialysis bag in step (2) is 50 kDa or 14-16 kDa.
进一步的，步骤(3)中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，将所述改性丝素蛋白溶液装入第一注射器替换为：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，得到改性丝素蛋白溶液。Further, in step (3), polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin is added, stirred evenly and debubbled to obtain a modified silk fibroin solution, and the modified silk fibroin solution is Fill the first syringe and replace it with: adding polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stirring evenly and defoaming to obtain a modified silk fibroin solution.
进一步的，步骤(4)中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，并安装于可旋转且能够前进或后退的电动轴上，取硅胶管一端连接所述注射器，另一端连接一段束状熟丝线并贴在所述管状结构表面，综合调节注射器内所述改性丝素蛋白溶液一定的流速、轴的旋转和运行速度，给所述管状结构表面涂覆一层连续的改性丝素蛋白溶液薄层，同时在温度小于37℃的热风中沿圆周方向旋转风干替换为：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。Further, in step (4), 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 2 to 20 mm by knitting technology, and is installed on a rotatable electric shaft that can advance or retreat. One end of the silicone tube is connected to the syringe, the other end is connected to a section of bundled cooked silk thread and attached to the surface of the tubular structure, and the certain flow rate of the modified silk fibroin solution in the syringe, the rotation of the shaft and the running speed are comprehensively adjusted. , coat a continuous thin layer of modified silk fibroin solution on the surface of the tubular structure, and rotate and air-dry along the circumferential direction in a hot air with a temperature less than 37°C. The top is braided into a tubular structure with an inner diameter of 2 to 20 mm, 30-90° interlacing, put the tubular structure into the modified silk fibroin solution and immerse it for 30 ± 5 seconds, take it out, and place it in a hot-air drying oven at a temperature of less than 65 Rotate in the circumferential direction to air dry under the condition of ℃.
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质量分数，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，将所述丝素蛋白管状覆膜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，得阳离子化的丝素蛋白管状覆膜。Further, after step (4) and before step (5), the method further includes: preparing a cationized silk fibroin tubular coating; configuring and measuring the mass fraction of the polyethylene glycol diamine solution, the polyethylene glycol diamine The amine solution includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and 2-morpholineethanesulfonate containing 1.5-fold molar concentration of polyethylene glycol bisamine acid, the silk fibroin tubular coating was immersed in the polyethylene glycol diamine solution to react for 20 minutes, then taken out and air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular coating.
进一步的，步骤(3)中所述纯化后的家蚕丝素蛋白水溶液的质量分数为4～5％。Further, the mass fraction of the purified Bombyx mori silk fibroin aqueous solution in step (3) is 4-5%.
进一步的，步骤(5)中，控制水蛭素在所述丝素蛋白管状覆膜表面的涂覆量为20U/cm 2。 Further, in step (5), the coating amount of hirudin on the surface of the silk fibroin tubular film is controlled to be 20 U/cm 2 .
本发明提供了一种抗凝血管支架覆膜及其制备方法，通过去离子水脱胶丝素纤维纺织结构芯层、层层自组装大分子丝素蛋白和层层载入抗凝因子等过程，使得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***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、人工心脏瓣膜等体内移植物，也可以拓展用于其它一些组织工程支架等医疗器械。The invention provides an anticoagulant tube stent coating and a preparation method thereof. The deionized water degumming silk fibroin fiber textile structure core layer, layer by layer self-assembled macromolecular silk fibroin, and layer by layer loading anticoagulation factors, etc., The prepared film has excellent biomechanical properties and long-lasting anticoagulant activity, and in situ induces vascular intimal regeneration that regulates the balance of the blood system. The film can be prepared into various shapes and applied to cardiovascular diseases. Treatment, such as heart patch, vascular stent, artificial heart valve and other in vivo grafts, can also be extended to other medical devices such as tissue engineering stents.
具体实施方式detailed description
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>35％，圆周向抗拉伸强度>4.0MPa，断裂伸长率>100％，整体水渗漏在120mmHg水压下小于9ml/min.cm 2，溶血率<0.1％。持续发挥抗凝功效，并具有诱导病变和缺损血管的组织再生与恢复血液平衡***的调节功能。 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>35%, circumferential tensile strength>4.0MPa , the elongation at break>100%, the overall water leakage is less than 9ml/min.cm 2 under 120mmHg water pressure, and the hemolysis rate is <0.1%. Continue to exert anticoagulant effect, and has the function of inducing tissue regeneration of diseased and defective blood vessels and restoring blood balance system.
本发明提供一种抗凝血管支架覆膜的制备方法，包括：The present invention provides a method for preparing an anticoagulant tube stent coating, comprising:
步骤一、制备脱胶后的家蚕丝素纤维和熟丝线：Step 1. Preparation of degummed Bombyx mori silk fibroin and cooked silk thread:
选取40～160D(优选的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充分清洗干净，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。Choose 40～160D (preferred 120D) silk thread and silk monofilament, the silk thread is obtained by twisting and combining the silkworm raw silk, the silk monofilament and silk thread are put into deionization at the liquor ratio of 1:50 (g/mL). In 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 cleans the silk monofilament and silk thread, and then The silk monofilament and silk thread are placed in an oven at a temperature of 60° C. to be dried to obtain degummed silk fibroin fibers and cooked silk threads.
步骤二、制备家蚕丝素蛋白水溶液：Step 2, prepare the silk fibroin aqueous solution of Bombyx mori:
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。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.
步骤三、制备改性丝素蛋白溶液：Step 3. Prepare modified silk fibroin solution:
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，使所述纯化后的家蚕丝素蛋白水溶液的质量分数为1～10％，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，将所述改性丝素蛋白溶液装入第一注射器。Method 1: Concentrate, adjust and measure the purified silk fibroin aqueous solution by using a rotary evaporator, so that the mass fraction of the purified silk fibroin aqueous solution is 1-10%, and add the amount of silk fibroin in a ratio of 1 to 10%. Be 0.6 polyethylene glycol diglycidyl ether (referring to the mass ratio of silk fibroin and polyethylene glycol diglycidyl ether in the purified silk fibroin aqueous solution is 10:6), stir evenly and debubble, A modified silk fibroin solution is obtained, and the modified silk fibroin solution is loaded into the first syringe.
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，使所述纯化后的家蚕丝素蛋白水溶液的质量分数为1～10％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。Method 2: use a rotary evaporator to concentrate, adjust and measure the purified silk fibroin aqueous solution, so that the mass fraction of the purified silk fibroin aqueous solution is 1-10% and the ratio of the addition to the silk fibroin is 0.6% of polyethylene glycol diglycidyl ether, stir evenly and degassing to obtain a modified silk fibroin solution.
步骤四、制备丝素蛋白管状覆膜：Step 4. Preparation of silk fibroin tubular coating:
若步骤三采用方法一的话，那么本步骤也采用方法一：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，并安装于可旋转且能够前进或后退的电动轴上，取硅胶管一端连接所述第一注射器，另一端连接一段束状熟丝线并贴在所述管状结构的表面，综合调节所述第一注射器内所述改性丝素蛋白溶液的流速、轴的旋转和运行速度，给所述管状结构表面涂覆一层连续的改性丝素蛋白溶液薄层，同时在温度小于37℃的热风中沿圆周方向旋转风干，获得丝素蛋白管状覆膜。If method 1 is adopted in step 3, method 1 is also adopted in this step: the cooked silk thread is braided on the stainless steel rod into a 30-90° interlaced tubular structure with an inner diameter of 2-20 mm, and is installed on a rotatable And on the electric shaft that can advance or retreat, take one end of the silicone tube to connect the first syringe, and the other end to connect a section of bundled cooked silk thread and stick it on the surface of the tubular structure, and comprehensively adjust the modification in the first syringe. The flow rate of the silk fibroin solution, the rotation of the shaft and the running speed, the surface of the tubular structure is coated with a continuous thin layer of the modified silk fibroin solution, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air with a temperature of less than 37° C. , to obtain a silk fibroin tubular membrane.
若步骤三采用方法二的话，那么本步骤也采用方法二：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。If the second method is adopted in the third step, then the second method is also adopted in this step: the cooked silk thread is braided on the stainless steel rod into a 30-90° interlaced tubular structure with an inner diameter of 2-20 mm, and the tubular structure is woven into the stainless steel rod. Put it into the modified silk fibroin solution and immerse it for 30±5 seconds, take it out, and place it in a hot-air drying box to rotate and air-dry in the circumferential direction under the condition that the temperature is less than 65°C.
上述步骤四方法的任意一种均可以增加步骤：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质量分数，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，将所述丝素蛋白管状覆膜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，得阳离子化的丝素蛋白管状覆膜。Any one of the above-mentioned steps and four methods can add steps: preparing cationized silk fibroin tubular coating: configuring and measuring the mass fraction of polyethylene glycol diamine solution, the polyethylene glycol diamine solution including poly 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and 2-morpholineethanesulfonic acid in 1.5 times molar concentration of ethylene glycol diamine, the The silk fibroin tubular membrane was immersed in the polyethylene glycol diamine solution for 20 minutes of reaction, then taken out to be air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular membrane.
步骤五、制备抗凝血管支架覆膜。Step 5, preparing the anticoagulant tube stent coating.
方法一：配置水蛭素水溶液，装入第二注射器，将硅胶管一端连接所述第二注射器，另一端连接一段束状熟丝线并贴在所述丝素蛋白管状覆膜 的表面，所述丝素蛋白管状覆膜保持一边旋转一边前进，在所述丝素蛋白管状覆膜表面涂覆所述水蛭素水溶液，调节所述第二注射器的流速，控制水蛭素在所述丝素蛋白管状覆膜表面的涂覆量0～50U/cm 2(优选20U/cm 2)，同时室温风干，交替重复(4)～(5)步骤5次，获得抗凝血管支架覆膜。 Method 1: Prepare an aqueous solution of hirudin, put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular coating. The fibroin tubular membrane keeps rotating while moving forward, the surface of the silk fibroin tubular membrane is coated with the hirudin aqueous solution, the flow rate of the second syringe is adjusted, and the hirudin is controlled in the silk fibroin tubular membrane. The coating amount on the surface is 0-50 U/cm 2 (preferably 20 U/cm 2 ), and at the same time it is air-dried at room temperature, and steps (4) to (5) are alternately repeated 5 times to obtain an anticoagulant tube stent coating.
如果上一步增加制备阳离子化的丝素蛋白管状覆膜的话，那么运用方法二：配置水蛭素水溶液，装入第二注射器，将硅胶管一端连接所述第二注射器，另一端连接一段束状熟丝线并贴在所述阳离子化的丝素蛋白管状覆膜的表面，所述阳离子化的丝素蛋白管状覆膜保持一边旋转一边前进，在所述阳离子化的丝素蛋白管状覆膜表面涂覆所述水蛭素水溶液，调节所述第二注射器的流速，控制水蛭素在所述阳离子化的丝素蛋白管状覆膜表面的涂覆量为0～50U/cm 2(优选20U/cm 2)，同时室温风干，交替重复(4)～(5)步骤5次，获得抗凝血管支架覆膜。 If the preparation of cationized silk fibroin tubular film was added in the previous step, then use method 2: prepare a hirudin aqueous solution, put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked The silk thread is attached to the surface of the cationized silk fibroin tubular membrane, the cationized silk fibroin tubular membrane keeps rotating while advancing, and is coated on the surface of the cationized silk fibroin tubular membrane For the hirudin aqueous solution, the flow rate of the second syringe is adjusted, and the coating amount of hirudin on the surface of the cationized silk fibroin tubular film is controlled to be 0-50 U/cm 2 (preferably 20U/cm 2 ), At the same time, it was air-dried at room temperature, and steps (4) to (5) were alternately repeated 5 times to obtain an anticoagulant tube stent coating.
上述方法所制备的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，使覆膜具有卓越的拉伸性能、***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、注射器流速、涂层时的旋转速度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***平衡的调节功能。Each coating of the 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 kept by deionized water. The silk fibroin fiber and the regenerated silk fibroin macromolecular chain are not damaged, so that the film has excellent tensile properties, burst strength, compliance and fatigue resistance. The role of flow shear and vasoconstriction in dilation. The membrane does not have endoleak, and is tightly combined with the lesion tissue without slipping after implantation. By adjusting the preparation parameters such as silk fibroin concentration, geometrical parameters of tubular textile structure, number of self-assembled layers, injector flow rate, rotation speed during coating, etc., the vascular stent coverings required by the physical size and physical properties of different parts of the organism are obtained. On the other hand, 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 Thrombosis and restenosis; and the inner surface of the covering can obtain rapid endothelialization, in situ repair of vascular intimal tissue, and will not be troubled by 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.
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。In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the technical solutions of the present invention are further described below in conjunction with the embodiments. However, the present invention is not limited to the listed embodiments, but also includes any other known modifications within the scope of the claimed rights of the present invention.
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。First, reference herein to "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.
实施例1Example 1
本实施案例展示一种抗凝血管支架覆膜的制备方法，包括：This example shows a method for preparing an anticoagulant tube stent covering, including:
1.将家蚕生丝加捻合并得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。1. The silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at ℃ for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 ℃ to obtain degummed silk fibroin fibers and cooked silk threads.
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。2. Weigh the degummed Bombyx mori silk fibroin and dissolve it in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL), and treat at 65°C until the Bombyx mori silk fibroin fiber is completely dissolved to obtain a Bombyx mori fibroin solution. 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.
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。3. Using a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified Bombyx mori silk fibroin aqueous solution to be 5%, add polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stir evenly and debubble, A modified silk fibroin solution was obtained.
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步的得到改性丝素蛋白溶液中浸渍30±5秒取出，置于65℃以下的热风干燥箱内圆周方向旋转风干，得丝素蛋白管状覆膜。4. 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 2 to 20 mm, immersed in the modified silk fibroin solution obtained in the third step above for 30 ± 5 seconds and taken out, It was placed in a hot-air drying box below 65°C and air-dried by rotating in the circumferential direction to obtain a silk fibroin tubular coating.
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。5. Prepare an aqueous solution of polyethylene glycol bisamine with a certain concentration, which contains 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with 1.5 times the molar concentration of polyethylene glycol bisamine, and a small amount of N-Hydroxysuccinimide and 2-morpholineethanesulfonic acid, stir well. The tubular film of the fourth step was immersed in the polyethylene glycol diamine solution to react for 20 minutes, then taken out and air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular film.
6.配置水蛭素水溶液，装入注射器，将硅胶管一端接于注射器，另一端接上一束熟丝线并贴在第5步阳离子化的丝素蛋白管状覆膜表面，调节注射器流速，阳离子化的丝素蛋白管状覆膜保持一边旋转一边前进，在阳离子化的丝素蛋白管状覆膜表面涂覆水蛭素(20U/cm 2)，同时室温风干。交替重复4～6步骤5次，获得蚕丝丝素复合血管支架覆膜，即抗凝血管支架覆膜。 6. Prepare an aqueous solution of hirudin, put it into a syringe, connect one end of the silicone tube to the syringe, and connect the other end with a bunch of cooked silk thread and stick it on the surface of the cationized silk fibroin tubular film in step 5, adjust the flow rate of the syringe, cationize The silk fibroin tubular membrane kept rotating and moved forward, and the surface of the cationized silk fibroin tubular membrane was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature. Steps 4 to 6 are alternately repeated for 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
经检测，上述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8ml/min.cm 2。 After testing, the above-mentioned anticoagulant tube stent coating has excellent mechanical properties. According to the national standard testing method, the axial tensile strength is >1.8MPa, the elongation at break is >45%, the circumferential tensile strength is >7.0MPa, and the fracture is broken. The elongation is >130%, and the overall water leakage is less than 8ml/min.cm 2 under 120mmHg water pressure.
通过对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％)。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，抗凝血管支架覆膜有显著的抗凝性能，并有持续抗凝效果，抗凝性能优于实施例2。The hemolysis rate is less than 0.1% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%). The 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. At the same time, the anticoagulation tube stent coating has significant anticoagulation performance, and has a continuous anticoagulation effect, and the anticoagulation performance is better than that of Example 2.
实施例2Example 2
本实施案例展示一种抗凝血管支架覆膜的制备方法，包括：This example shows a method for preparing an anticoagulant tube stent covering, including:
1.将家蚕生丝加捻合并得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。1. The silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at ℃ for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 ℃ to obtain degummed silk fibroin fibers and cooked silk threads.
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。2. Weigh the degummed Bombyx mori silk fibroin and dissolve it in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL), and treat at 65°C until the Bombyx mori silk fibroin fiber is completely dissolved to obtain a Bombyx mori fibroin solution. 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.
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，搅拌均匀并脱气泡后装入第一注射器，得到改性丝素蛋白溶液。3. Use a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified silk fibroin aqueous solution to be 5%, add polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stir evenly and debubble Load the first syringe to obtain the modified silk fibroin solution.
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，并安装于可旋转和前进/后退的电动轴上。取硅胶管一端接于第一注射器，另一端接上一段束状熟丝线并贴在管状结构表面，综合调节第一注射器内改性丝素蛋白溶液的流速、轴的旋转和运行速度，给管状结构涂覆一层连续的薄层，同时37℃以下的热风中圆周方向旋转风干，获得丝素蛋白管状覆膜。4. 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 2 to 20 mm on a stainless steel rod, and is installed on an electric shaft that can rotate and advance/retract. One end of the silicone tube is connected to the first syringe, the other end is connected to a bundle of cooked silk threads and attached to the surface of the tubular structure, and the flow rate, rotation and running speed of the modified silk fibroin solution in the first The structure is coated with a continuous thin layer, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air below 37°C to obtain a silk fibroin tubular coating.
5.配置水蛭素水溶液，装入第二注射器，取硅胶管一端接于第二注射器，另一端接上一段束状熟丝线并贴在第4步丝素蛋白管状覆膜表面，调节第二注射器一定的流速，丝素蛋白管状覆膜保持一边旋转一边前进，在丝素蛋白管状覆膜表面涂覆水蛭素(20U/cm 2)，同时室温风干。交替重复4～5步骤5次，获得蚕丝丝素复合血管支架覆膜，即抗凝血管支架覆膜。 5. Prepare the hirudin aqueous solution, put it into the second syringe, connect one end of the silicone tube to the second syringe, and connect the other end with a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular film in step 4, and adjust the second syringe At a certain flow rate, the silk fibroin tubular membrane kept rotating and moved forward, and the surface of the silk fibroin tubular membrane was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature. Steps 4 to 5 are alternately repeated 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
经检测，上述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>35％，圆周向抗拉伸强度>4.0MPa，断裂伸长率>100％，整体水渗漏在120mmHg水压下小于9ml/min.cm 2。 After testing, the above-mentioned anticoagulant tube stent coating has excellent mechanical properties. According to the national standard testing method, the axial tensile strength is >2.0MPa, the elongation at break is >35%, the circumferential tensile strength is >4.0MPa, and the fracture is broken. Elongation>100%, the overall water leakage is less than 9ml/min.cm 2 under 120mmHg water pressure.
通过对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％)。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，抗凝血管支架覆膜有显著的抗凝性能，并有持续抗凝效果。The hemolysis rate is less than 0.1% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%). The 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. At the same time, the anticoagulant tube stent coating has significant anticoagulant properties and has a sustained anticoagulant effect.
实施例3Example 3
本实施案例展示一种抗凝血管支架覆膜的制备方法，包括：This example shows a method for preparing an anticoagulant tube stent covering, including:
1.将家蚕生丝加捻合并得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。1. The silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at ℃ for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 ℃ to obtain degummed silk fibroin fibers and cooked silk threads.
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。2. Weigh the degummed Bombyx mori silk fibroin and dissolve it in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL), and treat at 65°C until the Bombyx mori silk fibroin is completely dissolved to obtain a Bombyx mori fibroin solution. 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.
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，搅拌均匀并脱气泡后，得到改性丝素蛋白溶液，装入第一注射器。3. Use a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified silk fibroin aqueous solution to be 5%, add polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stir evenly and debubble , to obtain a modified silk fibroin solution, which is loaded into the first syringe.
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，并安装于可旋转和前进/后退的电动轴上。取硅胶管一端接于注射器，另一端接上一段束状熟丝线并贴在管状结构表面，综合调节第一注射器内改性丝素蛋白溶液的流速、轴的旋转和运行速度，给管状结构涂覆一层连续改性丝素蛋白溶液薄层，同时37℃以下的热风中圆周方向旋转风干，得丝素蛋白管状覆膜。4. 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 2 to 20 mm on a stainless steel rod, and is installed on an electric shaft that can rotate and advance/retract. Take one end of the silicone tube and connect it to the syringe, and the other end to connect a bunch of cooked silk thread and stick it on the surface of the tubular structure. Comprehensively adjust the flow rate of the modified silk fibroin solution in the first syringe, the rotation of the shaft and the running speed, and coat the tubular structure. A thin layer of continuous modified silk fibroin solution is covered, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air below 37° C. to obtain a tubular film of silk fibroin.
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步的丝素蛋白管状覆膜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。5. Prepare an aqueous solution of polyethylene glycol bisamine with a certain concentration, which contains 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with 1.5 times the molar concentration of polyethylene glycol bisamine, and a small amount of N-Hydroxysuccinimide and 2-morpholineethanesulfonic acid, stir well. The silk fibroin tubular covering in the fourth step was immersed in a polyethylene glycol diamine solution to react for 20 minutes, then taken out to be air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular covering.
6.配置水蛭素水溶液，装入第二注射器，将硅胶管一端连接第二注射器，另一端连接一段束状熟丝线并贴在第5步风干的阳离子化的丝素蛋白管状覆膜表面，调节第二注射器的流速，阳离子化的丝素蛋白管状覆膜保持一边旋转一边前进，在阳离子化的丝素蛋白管状覆膜表面涂覆水蛭素(20U/cm 2)，同时室温风干。交替重复4～6步骤5次，获得蚕丝丝素复合血管支架覆膜，即抗凝血管支架覆膜。 6. Prepare an aqueous solution of hirudin, put it into the second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the cationized silk fibroin tubular film that was air-dried in step 5. Adjust At the flow rate of the second syringe, the cationized silk fibroin tubular film was kept rotating while advancing, and the surface of the cationized silk fibroin tubular film was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature. Steps 4 to 6 are alternately repeated for 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
经检测，上述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>5.0MPa，断裂伸长率>100％，整体水渗漏在120mmHg水压下小于8ml/min.cm 2。 After testing, the above-mentioned anticoagulant tube stent coating has excellent mechanical properties. According to the national standard testing method, the axial tensile strength is >2.0MPa, the elongation at break is >45%, the circumferential tensile strength is >5.0MPa, and the fracture is broken. The elongation is >100%, and the overall water leakage is less than 8ml/min.cm 2 under 120mmHg water pressure.
通过对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％)。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，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2。The hemolysis rate is less than 0.1% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%). The 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. At the same time, the 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 Example 2.
实施例4Example 4
本实施案例展示一种抗凝血管支架覆膜的制备方法，包括：This example shows a method for preparing an anticoagulant tube stent covering, including:
1.将家蚕生丝加捻合并得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。1. The silkworm raw silk is twisted and merged to obtain a 120D silk thread group, and the other group is a silk monofilament group. Boil at ℃ for 7 hours, during which the deionized water was replaced several times, and then the silk was fully cleaned with deionized water, and dried in an oven at 60 ℃ to obtain degummed silk fibroin fibers and cooked silk threads.
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。2. Weigh the degummed Bombyx mori silk fibroin and dissolve it in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL), and treat at 65°C until the Bombyx mori silk fibroin fiber is completely dissolved to obtain a Bombyx mori fibroin solution. The Bombyx mori fibroin solution was poured into a dialysis bag, the wall of the dialysis bag was a semi-permeable membrane, and the molecular weight cut-off was in the range of 50kDa. The water in the container was replaced with new deionized water for 2 hours, and the dialysis was continued for 3 days to obtain the purified silk fibroin aqueous solution.
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4％，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，装入第一注射器。3. Using a rotary evaporator to concentrate, adjust and measure the mass fraction of the purified Bombyx mori silk fibroin aqueous solution to be 4%, add polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stir evenly and debubble, The modified silk fibroin solution was obtained and charged into the first syringe.
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，并安装于可旋转和前进/后退的电动轴上。取硅胶管一端连接第一注射器，另一端连接一段束状熟丝线并贴在管状结构表面，综合调节第一注射器内改性丝素蛋白溶液的流速、轴的旋转和运行速度，给管状结构涂覆一层连续的改性丝素蛋白溶液薄层，同时37℃以下的热风中圆周方向旋转风干，得丝素蛋白管状覆膜。4. 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 2 to 20 mm on a stainless steel rod, and is installed on an electric shaft that can rotate and advance/retract. One end of the silicone tube is connected to the first syringe, and the other end is connected to a bundle of cooked silk threads and attached to the surface of the tubular structure. A continuous thin layer of modified silk fibroin solution is covered, and at the same time, it is rotated and air-dried in a circumferential direction in a hot air below 37° C. to obtain a tubular film of silk fibroin.
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步的丝素蛋白管状覆膜浸渍于聚 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。5. Prepare an aqueous solution of polyethylene glycol bisamine with a certain concentration, which contains 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with 1.5 times the molar concentration of polyethylene glycol bisamine, and a small amount of N-Hydroxysuccinimide and 2-morpholineethanesulfonic acid, stir well. The silk fibroin tubular covering in the fourth step was immersed in the polyethylene glycol diamine solution to react for 20 minutes, then taken out to be air-dried at room temperature, rinsed and then air-dried to obtain a cationized silk fibroin tubular covering.
6.配置水蛭素水溶液，装入第二注射器，将硅胶管一端连接第二注射器，另一端连接一段束状熟丝线并贴在第5步风干的阳离子化的丝素蛋白管状覆膜表面，调节第二注射器的流速，阳离子化的丝素蛋白管状覆膜保持一边旋转一边前进，在阳离子化的丝素蛋白管状覆膜表面涂覆水蛭素(20U/cm 2)，同时室温风干。交替重复4～6步骤5次，获得蚕丝丝素复合血管支架覆膜，即抗凝血管支架覆膜。 6. Prepare an aqueous solution of hirudin, put it into the second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the cationized silk fibroin tubular film that was air-dried in step 5. Adjust At the flow rate of the second syringe, the cationized silk fibroin tubular film was kept rotating while advancing, and the surface of the cationized silk fibroin tubular film was coated with hirudin (20 U/cm 2 ) and air-dried at room temperature. Steps 4 to 6 are alternately repeated for 5 times to obtain a silk fibroin composite vascular stent coating, that is, an anticoagulant tube stent coating.
经检测，上述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>50％，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。 After testing, the above-mentioned anticoagulant tube stent coating has excellent mechanical properties. According to the national standard testing method, the axial tensile strength is >2.5MPa, the elongation at break is >50%, the circumferential tensile strength is >8.0MPa, and the fracture is broken. The elongation is >150%, and the overall water leakage is less than 2ml/min.cm 2 under 120mmHg water pressure.
通过对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％)。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，持续抗凝效果显著优于实施例1～3。The hemolysis rate is less than 0.1% as determined by the anticoagulation tube stent coating according to the hemolysis rate test method, which fully meets the standard of non-hemolytic material (0-2%). The 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. At the same time, the stent coating has significant anticoagulation performance, and the continuous anticoagulation effect is significantly better than that of Examples 1-3.
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。It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent substitutions without departing from the spirit and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims (9)

  1. 一种抗凝血管支架覆膜，其特征在于：轴向抗拉伸强度>1.8MPa，断裂伸长率>35％，圆周向抗拉伸强度>4.0MPa，断裂伸长率>100％，整体水渗漏在120mmHg水压下小于9ml/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。 An anticoagulant tube stent coating, characterized in that: axial tensile strength>1.8MPa, elongation at break>35%, circumferential tensile strength>4.0MPa, elongation at break>100%, the overall The water leakage is less than 9ml/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.
  2. 一种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：A method for preparing an anticoagulant tube stent coating, characterized in that the method comprises the following steps:
    (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充分清洗干净，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；(1) 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 silk and the silk thread are fully cleaned, and then the silk monofilament and the silk thread are placed in an oven with a temperature of 60 ° C to dry to obtain the degummed Bombyx mori fibroin and cooked silk thread;
    (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；(2) Preparation of Bombyx mori silk fibroin aqueous solution: the degummed Bombyx mori silk fibroin fiber was dissolved in a 9.3M lithium bromide aqueous solution at a bath ratio of 1:10 (g/mL) at a temperature of 65±10°C Lower treatment until the Bombyx mori silk fibroin fibers are completely dissolved to obtain a Bombyx mori silk fibroin dissolving solution, and the Bombyx mori silk fibroin dissolving solution is perfused in a dialysis bag, the material of the dialysis bag is a semi-permeable membrane, and the molecular weight cut-off is 10-100 kDa, and the The dialysis bag perfused with the 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 Vegetarian protein aqueous solution;
    (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，使所述纯化后的家蚕丝素蛋白水溶液的质量分数为1～10％，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，将所述改性丝素蛋白溶液装入第一注射器；(3) Preparation of modified silk fibroin solution: using a rotary evaporator to concentrate, adjust and measure the purified silk fibroin aqueous solution so that the mass fraction of the purified silk fibroin aqueous solution is 1-10% , adding polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stirring evenly and defoaming to obtain a modified silk fibroin solution, which is loaded into the first syringe;
    (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，并安装于可旋转且能够前进或后退的电动轴上，取硅胶管一端连接所述第一注射器，另一端 连接一段束状熟丝线并贴在所述管状结构的表面，综合调节所述第一注射器内所述改性丝素蛋白溶液的流速、轴的旋转和运行速度，给所述管状结构表面涂覆一层连续的改性丝素蛋白溶液薄层，同时在温度小于37℃的热风中沿圆周方向旋转风干，获得丝素蛋白管状覆膜；(4) Preparation of silk fibroin tubular coating: the cooked silk thread is braided on a stainless steel rod with a knitting technique to form a tubular structure with an inner diameter of 2-20 mm and an inner diameter of 30-90°, and is installed in a rotatable and capable of advancing or retreating Connect one end of the silicone tube to the first syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the tubular structure, and comprehensively adjust the modified silk fibroin solution in the first syringe. The flow rate, the rotation and the running speed of the shaft, the surface of the tubular structure is coated with a continuous thin layer of modified silk fibroin solution, and at the same time, it is rotated and air-dried in the circumferential direction in a hot air with a temperature less than 37 ° C to obtain silk fibroin. tubular covering;
    (5)配置水蛭素水溶液，装入第二注射器，将硅胶管一端连接所述第二注射器，另一端连接一段束状熟丝线并贴在所述丝素蛋白管状覆膜的表面，所述丝素蛋白管状覆膜保持一边旋转一边前进，在所述丝素蛋白管状覆膜表面涂覆所述水蛭素水溶液，调节所述第二注射器的流速，控制水蛭素在所述丝素蛋白管状覆膜表面的涂覆量为0～50U/cm 2，同时室温风干，交替重复(4)～(5)步骤5次，获得抗凝血管支架覆膜。 (5) Prepare a hirudin aqueous solution, put it into a second syringe, connect one end of the silicone tube to the second syringe, and connect the other end to a bundle of cooked silk thread and stick it on the surface of the silk fibroin tubular coating, the silk The fibroin tubular membrane keeps rotating while moving forward, the surface of the silk fibroin tubular membrane is coated with the hirudin aqueous solution, the flow rate of the second syringe is adjusted, and the hirudin is controlled on the silk fibroin tubular membrane. The coating amount of the surface is 0-50 U/cm 2 , and at the same time air-drying at room temperature, steps (4) to (5) are alternately repeated 5 times to obtain an anticoagulant tube stent coating.
  3. 根据权利要求2所述的一种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120D。The method for preparing an anticoagulant tube stent coating according to claim 2, wherein the silk thread in step (1) is 120D.
  4. 根据权利要求2所述的一种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。The method for preparing an anticoagulant tube stent coating according to claim 2, wherein the molecular weight cut-off of the dialysis bag in step (2) is 50 kDa or 14-16 kDa.
  5. 根据权利要求4所述的一种抗凝血管支架覆膜的制备方法，其特征在于，步骤(3)中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，将所述改性丝素蛋白溶液装入第一注射器替换为：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，得到改性丝素蛋白溶液。The method for preparing an anticoagulant tube stent coating according to claim 4, wherein in step (3), polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin is added, stirred evenly and Debubble to obtain a modified silk fibroin solution. The modified silk fibroin solution is loaded into the first syringe and replaced by: adding polyethylene glycol diglycidyl ether with a mass ratio of 0.6 to silk fibroin, stirring evenly and mixing. Debubble to obtain a modified silk fibroin solution.
  6. 根据权利要求5所述的一种抗凝血管支架覆膜的制备方法，其特征在于，步骤(4)中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，并安装于可旋转且能够前进或后退的电动轴上，取硅胶管一端连接所述注射器，另一端连接一段束状熟丝线并贴在所述管状结构表面，综合调节注射器内所述改性丝素蛋白溶液的流速、轴的旋转和运行速度，给所述管状结构表面涂覆一层连续的改性丝素蛋白溶液薄层，同时在温度小于37℃的热风中沿圆周方向旋转风干替换为：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。The method for preparing an anticoagulant tube stent coating according to claim 5, wherein in step (4), the cooked silk thread is braided on a stainless steel rod into a 30-90° interlace with an inner diameter of 2-20mm tubular structure, and installed on a rotatable electric shaft that can move forward or backward, take one end of the silicone tube to connect the syringe, and the other end to connect a bundle of cooked silk thread and stick it on the surface of the tubular structure, comprehensive adjustment The flow rate of the modified silk fibroin solution in the syringe, the rotation of the shaft and the running speed, the surface of the tubular structure is coated with a continuous thin layer of the modified silk fibroin solution, while the temperature is less than 37 ℃ in hot air Rotating and air-drying in the circumferential direction is replaced by: knitting the cooked silk thread on a stainless steel rod into a tubular structure with an inner diameter of 2-20 mm and a 30-90° interlacing on a stainless steel rod, and placing the tubular structure into the modified silk fibroin Immerse in the protein solution for 30±5 seconds, take it out, and place it in a hot-air drying oven to rotate and air-dry in the circumferential direction under the condition that the temperature is less than 65°C.
  7. 根据权利要求2或6所述的一种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质量分数，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，将所述丝素蛋白管状覆膜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，得阳离子化的丝素蛋白管状覆膜。The method for preparing an anticoagulant tube stent coating according to claim 2 or 6, characterized in that, after step (4) and before step (5), the method further comprises: preparing a cationized silk fibroin tubular coating : Configure and measure the mass fraction of polyethylene glycol diamine solution, which includes 1-(3-dimethylaminopropyl)-3 containing 1.5 times the molar concentration of polyethylene glycol diamine -Ethylcarbodiimide, N-hydroxysuccinimide and 2-morpholineethanesulfonic acid, the silk fibroin tubular film was immersed in the polyethylene glycol diamine solution and reacted for 20 minutes and then taken out Air-dried at room temperature, rinsed and air-dried to obtain a cationized silk fibroin tubular coating.
  8. 根据权利要求7所述的一种抗凝血管支架覆膜的制备方法，其特征在于：步骤(3)中所述纯化后的家蚕丝素蛋白水溶液的质量分数为4～5％。The method for preparing an anticoagulant tube stent coating according to claim 7, wherein the mass fraction of the purified Bombyx mori silk fibroin aqueous solution in step (3) is 4-5%.
  9. 根据权利要求2所述的一种抗凝血管支架覆膜的制备方法，其特征在于：步骤(5)中控制水蛭素在所述丝素蛋白管状覆膜表面的涂覆量为20U/cm 2。 The method for preparing an anticoagulant tube stent coating according to claim 2, wherein in step (5), the coating amount of hirudin on the surface of the silk fibroin tubular coating is controlled to be 20U/cm 2 .
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