WO2022083758A1 - 一种抗凝血的人工心脏瓣膜材料及其制备方法与应用 - Google Patents

一种抗凝血的人工心脏瓣膜材料及其制备方法与应用 Download PDF

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WO2022083758A1
WO2022083758A1 PCT/CN2021/125823 CN2021125823W WO2022083758A1 WO 2022083758 A1 WO2022083758 A1 WO 2022083758A1 CN 2021125823 W CN2021125823 W CN 2021125823W WO 2022083758 A1 WO2022083758 A1 WO 2022083758A1
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heart valve
valve material
fluoride
biological
preparation
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PCT/CN2021/125823
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English (en)
French (fr)
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王云兵
雷洋
杨立
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吉林启明皓月生物科技有限公司
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Publication of WO2022083758A1 publication Critical patent/WO2022083758A1/zh

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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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • 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
    • 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
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • 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
    • A61L33/0076—Chemical modification of the substrate
    • 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
    • A61L33/04—Use of organic materials, e.g. acetylsalicylic acid
    • 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
    • A61L2300/202—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with halogen atoms, e.g. triclosan, povidone-iodine
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves

Definitions

  • the invention belongs to the technical field of biomedical materials, and in particular relates to an anticoagulant artificial heart valve material and a preparation method and application thereof.
  • Prosthetic valves are vascular implants and therefore need to have good blood compatibility.
  • the blood flow in the heart is fast, and the blood compatibility of the biological valve is relatively good, so the probability of thrombosis is small, and the interventional biological valve generally does not require long-term use of anticoagulant drugs.
  • bioprosthetic valves have traditionally been considered a good option for anticoagulation, data analysis in recent years has shown a higher incidence of thrombosis associated with prosthetic bioprostheses, especially with the advent of transcatheter aortic valve replacement.
  • Bioprosthetic valve thrombosis is the main cause of acute or chronic bioprosthetic valve degeneration.
  • the present invention develops an anticoagulant artificial heart valve material.
  • a fluoride superlubricant anticoagulant coating on the surface of the biological heart valve material, the adhesion of platelets and whole blood can be significantly reduced, and the material can be greatly improved. anticoagulant properties.
  • An anticoagulant artificial heart valve material comprising a biological heart valve material, a fluoride superlubricant anticoagulant coating chemically combined with the biological heart valve material, and a lubricating agent maintained on the fluoride superlubricant anticoagulation coating
  • the fluoride superlubricant anticoagulant coating is formed by chemically combining trichlorosilane fluoride and biological heart valve materials by chemical vapor deposition or liquid deposition; or by combining double bond fluoride with biological heart valve materials Materials are chemically combined.
  • the lubricant may remain on the surface of the fluoride superlubricating anticoagulation coating and/or within the coating structure.
  • the thickness of the fluoride superlubricating anticoagulation coating is 0.01-1 ⁇ m. Coating thickness can be determined using cross-sectional biological scanning electron microscopy or elemental analysis.
  • the biological heart valve material can be a biological valve material commonly used in the prior art, and can be selected from the group consisting of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, Skin or ligament, preferably porcine or bovine pericardium.
  • Trichlorosilane fluoride is easy to chemically combine with biological heart valve materials due to its good hydroxyl reactivity. Further, the trichlorosilane fluoride is specifically: trichloro(1H,1H,2H,2H-perfluorooctyl)silane (CAS number: 78560-45-9).
  • the fluoride superlubricant anticoagulant coating is formed by chemically combining double-bond fluoride with the biological heart valve material.
  • the double bond fluoride is specifically: allyl pentafluorobenzene (CAS number: 1736-60-3), 1H, 1H, 2H-perfluoro-1-octene (CAS number: 25291-17- 2), 1H, 1H, 2H-perfluoro-1-dodecene (CAS number: 30389-25-4), 2-(perfluorooctyl)ethyl methacrylate (CAS number: 1996-88- 9) one or more of them.
  • the double bond fluoride is easily reacted with the active group on the surface of the biological heart valve material through the double bond to form a stable chemical bond.
  • the lubricant is perfluorodecalin or pentafluorophenol diethyl trimethenate.
  • the lubricant has good biosafety and biocompatibility.
  • the present invention also provides a preparation method of the above-mentioned artificial heart valve material, characterized in that it comprises the following steps:
  • the glutaraldehyde-crosslinked biological heart valve material is immersed in an oxygen free radical activator solution for group activation;
  • the biological heart valve material is contacted with trichlorosilane fluoride or double bond fluoride, so that the fluoride is chemically combined with the biological heart valve material through the activated group in step a, and a fluoride superoxide is formed on the surface of the biological heart valve material.
  • Lubricating anti-coagulation coating
  • step b Add lubricant to the system of step b to soak the biological valve material or take out the biological heart valve material and completely immerse it in the lubricant, so that the lubricant is kept on the fluoride superlubricant anticoagulant coating.
  • the glutaraldehyde cross-linking is specifically immersing the biological heart valve material in a 0.2-2.5 vt% glutaraldehyde solution for 1-7 days.
  • step a the glutaraldehyde-crosslinked biological heart valve material is soaked in 5-500mM oxygen radical activator solution for 1-24h.
  • the oxygen radical activator includes: ammonium persulfate (CAS number: 7727-54-0)/sodium bisulfite (CAS number: 7631-90-5), ceric ammonium nitrate (CAS number: 7631-90-5) 16774-21-3).
  • ammonium persulfate can generate reactive oxygen radicals, and sodium bisulfite further enhances the quantity and efficiency of generating reactive oxygen radicals.
  • step b the trichlorosilane fluoride is chemically combined with the biological heart valve material by vapor deposition or liquid deposition, or double bond fluoride is added to chemically combine with the biological heart valve material.
  • the "carbon-carbon double bond" contained in the double bond fluoride exists in the main chain, which is a non-polar bond, and its electron cloud is evenly distributed, which is easily attacked by a nucleophile and undergoes an electrophilic addition reaction. Therefore, It can undergo polymerization reaction with the activated free radicals in step a to form covalent bonding with the biological heart valve material.
  • the liquid deposition includes adding a trichlorosilane fluoride solution or a double bond fluoride solution to the system in step a, and continuing to soak the biological heart valve material; or immersing the biological heart valve material processed in step a Take it out, and then completely immerse it in trichlorosilane fluoride solution or double bond fluoride solution.
  • the concentration of the trichlorosilane fluoride solution and the double bond fluoride solution are both 5-20 vt%.
  • step c the soaking or immersion time is 1-24h.
  • the preparation method further includes that after the biological heart valve material processed in step c is rinsed, it is preserved by using a bacteriostatic solvent or by dehydrating and drying in an alcohol solution.
  • physiological saline or phosphate buffered saline (PBS) rinsing is more preferred.
  • the pH of the phosphate buffer is 7-7.4.
  • Physiological saline and phosphate buffer are water-soluble solvents typically used in the processing of biological materials, which can ensure that the material maintains an osmotic pressure compatible with the human body, and will not cause osmotic pressure imbalance after the material is implanted into the human body.
  • the biological heart valve material can be rinsed multiple times, and the same or different rinse solutions can be used for two consecutive times.
  • the preservation of the bacteriostatic solvent is specifically: soaking the valve material in 20-100vt% isopropanol or 70-100vt% ethanol aqueous solution for preservation.
  • the preservation of the alcohol solution after dehydration and drying is specifically: soaking the biological valve material in a mixed solution of 10-30vt% glycerol and 70-90vt% ethanol in equal volumes or 10-30vt% glycerol, 35-45vt% Dehydrate in a mixed solution of ethanol and 35-45vt% isopropanol in equal volume for 4-24h, and then dry.
  • the drying step is preferably natural air drying.
  • the choice of the solvent in all the above solutions mainly considers the solubility of each fluoride, and also needs to consider the side effects of its potential damage to the pericardium of animals; generally, if it is a water-soluble reagent, PBS, physiological saline or acid solution can be selected. , If the water solubility is poor, ethanol is generally used as the solvent.
  • the present invention also provides an application of the above-mentioned artificial heart valve material for making artificial aortic valve, pulmonary valve, venous valve, mitral valve or tricuspid valve.
  • the anticoagulant artificial heart valve material of the present invention can significantly reduce platelet adhesion and whole blood adhesion by constructing a fluoride superlubricant anticoagulation coating on the surface of the biological heart valve material, and greatly improve the anticoagulation of the material. blood performance.
  • the fluoride selected in the present invention has been approved by the US FDA for use in blood contact materials, and has good biological safety.
  • the preparation method of the fluoride superlubricant anticoagulant coating in the present invention is simple, and only needs to be simply immersed in solution or vapor deposition, the damage to the biological heart valve material is small, and the introduction of condensation reagents is avoided in the preparation process to reduce Additional introduced or residual chemical reagents are beneficial to maintain good biocompatibility of the material.
  • the fluoride super-lubricating anticoagulation coating structure constructed by the present invention has good repelling and anti-adhesion effect on various simple and complex liquids, and has good and stable anti-coagulation effect.
  • Fig. 1 is the schematic diagram of the technical route 1 described in the embodiment of the present invention.
  • Fig. 2 is the schematic diagram of the technical route 2 described in the embodiment of the present invention.
  • FIG. 3 is a photo of whole blood adhesion corresponding to the material described in the experimental example of the present invention.
  • the present invention adopts two technical routes: (1) chemical modification of trichlorosilane fluoride after activation of the pericardium surface by an oxygen radical activator (Fig. 1). (2) Double bond fluoride chemically modifies the pericardium after the oxygen radical activator activates the pericardium surface (Fig. 2).
  • the present invention does not limit the base material. Since the pericardium is an artificial heart valve material that has been widely verified and has a good effect, the following examples mainly take the pericardium as an example to demonstrate the present invention. Those skilled in the art can easily Replace it with other biological tissues such as valves, intestinal membranes, meninges, lung membranes, blood vessels, skin or ligaments.
  • control group and the materials prepared in the examples were set to conduct platelet and whole blood adhesion tests respectively.
  • Control group The porcine pericardium was soaked in 0.625vt% glutaraldehyde solution for cross-linking for 1 day. After rinsing, use 25vt% isopropanol solution for preservation.
  • Rabbit blood was collected using a sodium citrate blood collection tube, the samples (12 mm in diameter) were immersed in 1 mL of whole blood, incubated at 37°C for 2 hours, then the whole blood was aspirated and washed three times with 1 mL of PBS solution for 5 minutes each time , and then photographed and analyzed the adhesion of whole blood.
  • Fresh sodium citrate anticoagulated rabbit blood was centrifuged at 1500 rpm for 15 min to obtain platelet rich supernatant (PRP). After the experimental samples were cut into 12mm disks and washed three times with PBS solution, they were placed in a 24-well plate with the smooth side up, and 1 mL of fresh platelet-rich plasma was added to each well and incubated at 37°C for 1 hour (pure PRP was set as positive). control group). After incubation, the samples were washed three times with PBS to remove unadhered platelets in the system, and the LDH released by the cells was measured using the LDH cytotoxicity kit to indirectly quantify the number of platelets adhered to the surface of the material.
  • PRP platelet rich supernatant
  • the relative adhesion amount of platelets (absorbance at 490nm of test sample - absorbance at 490nm of negative control)/(absorbance at 490nm of positive control - absorbance at 490nm of negative control).
  • Table 1 The relative content of platelet adhesion.

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Abstract

本发明公开了一种抗凝血的人工心脏瓣膜材料及其制备方法与应用，属于生物医学材料技术领域。所述抗凝血的人工心脏瓣膜材料，包括生物心脏瓣膜材料、与所述生物心脏瓣膜材料化学结合的氟化物超润滑抗凝涂层、保持在所述氟化物超润滑抗凝涂层的润滑剂；所述氟化物超润滑抗凝涂层是通过化学气相沉积或液相沉积将三氯硅烷氟化物与生物心脏瓣膜材料进行化学结合而成；或者是通过将双键氟化物与生物心脏瓣膜材料进行化学结合而成。通过在生物心脏瓣膜材料表面构建氟化物超润滑抗凝涂层，能够显著减小血小板黏附以及全血黏附，大幅提高材料的抗凝血性能，有效克服了生物瓣膜的血栓原性。所述人工心脏瓣膜材料的制备方法简单，只需要简单的溶液浸泡或气相沉积，对生物心脏瓣膜材料的损伤较小，额外引入或残留的化学试剂少，有利于保持材料良好的生物相容性。

Description

一种抗凝血的人工心脏瓣膜材料及其制备方法与应用 技术领域
本发明属于生物医学材料技术领域，具体涉及一种抗凝血的人工心脏瓣膜材料及其制备方法与应用。
 背景技术
人工瓣膜是血管植入物，因此需要具有良好的血液相容性。对于介入生物心脏瓣膜，心脏内血液流速快，生物瓣膜的血液相容性相对较好，因此形成血栓的概率小，介入生物瓣膜一般无需长期服用抗凝药。尽管传统上生物瓣膜被认为是抗凝的一个很好选择，但近年来的数据分析显示，与人工生物瓣膜相关的血栓形成发生率较高，尤其是随着经导管主动脉瓣置换术的出现，生物瓣膜血栓形成是导致急性或慢性生物瓣膜变性的主要原因。
最近，关于生物瓣膜置换术后的患者的亚临床小叶血栓形成的报道进一步加剧了这一争论。传统上，采用超声心动图诊断瓣膜血栓形成，但随着电子计算机断层扫描（CT）的广泛应用，越来越多的病例出现在人们的视野。利用四维容积CT研究发现，在一个生物瓣膜临床试验中40%的患者和在两个注册中心接受瓣膜置换治疗13%的患者中发现瓣叶运动减少与瓣叶血栓形成一致（Cardiol Res, 2018. 9(6): p. 335-342；Curr Treat Options Cardiovasc Med, 2018. 20(5): p. 42； Heart, 2017. 103(24): p. 1934-1941.）。
综上所述，生物瓣膜的血栓原性仍然是需要解决的问题。瓣膜的凝血问题，是除去钙化、免疫排斥反应之外，导致生物瓣膜失效的重要因素。然而，目前尚没有一种能够很好地克服生物瓣膜的血栓原性的人工心脏瓣膜材料问世。
 技术问题
本发明针对上述问题，开发了一种抗凝血的人工心脏瓣膜材料，通过在生物心脏瓣膜材料表面构建氟化物超润滑抗凝涂层，来显著减小血小板黏附以及全血黏附，大幅提高材料的抗凝血性能。
 技术解决方案
本发明包含以下技术方案：
一种抗凝血的人工心脏瓣膜材料，包括生物心脏瓣膜材料、与所述生物心脏瓣膜材料化学结合的氟化物超润滑抗凝涂层、保持在所述氟化物超润滑抗凝涂层的润滑剂；所述氟化物超润滑抗凝涂层是通过化学气相沉积或液相沉积将三氯硅烷氟化物与生物心脏瓣膜材料进行化学结合而成；或者是通过将双键氟化物与生物心脏瓣膜材料进行化学结合而成。其中，润滑剂可保持在氟化物超润滑抗凝涂层的表面和/或保持在涂层结构的内部。
作为可选方式，所述氟化物超润滑抗凝涂层的厚度为0.01-1μm。可采用截面生物扫描电镜或元素分析方法确定涂层厚度。
作为可选方式，在上述人工心脏瓣膜材料中，所述生物心脏瓣膜材料可以是现有技术中常用的生物瓣膜材料，可以选自心包膜、瓣膜、肠膜、脑膜、肺膜、血管、皮肤或韧带，优选为猪或牛心包膜。
三氯硅烷氟化物由于具有良好的羟基反应活性，容易与生物心脏瓣膜材料进行化学结合。进一步的，所述三氯硅烷氟化物具体为：三氯(1H,1H,2H,2H-全氟辛基)硅烷（CAS号：78560-45-9）。
作为可选方式，在上述人工心脏瓣膜材料中，所述氟化物超润滑抗凝涂层是通过将双键氟化物与生物心脏瓣膜材料进行化学结合而成。进一步的，所述双键氟化物具体为：烯丙基五氟苯（CAS号：1736-60-3），1H,1H,2H-全氟-1-辛烯（CAS号：25291-17-2），1H,1H,2H-全氟-1-十二烯（CAS号：30389-25-4），2-(全氟辛基)乙基甲基丙烯酸酯（CAS号：1996-88-9）中的一种或几种。所述双键氟化物容易通过双键与生物心脏瓣膜材料表面的活性基团反应形成稳定的化学结合。
作为可选方式，在上述人工心脏瓣膜材料中，所述润滑剂为全氟萘烷或五氟苯酚二乙基三甲基烯酯。所述润滑剂具有良好的生物安全性和生物相容性。
本发明还提供了一种上述的人工心脏瓣膜材料的制备方法，其特征在于，包括以下步骤：
a．将戊二醛交联的生物心脏瓣膜材料，浸入氧自由基活化剂溶液中，进行基团活化；
b．将生物心脏瓣膜材料与三氯硅烷氟化物或双键氟化物接触，使氟化物通过步骤a中的被活化的基团与生物心脏瓣膜材料进行化学结合，在生物心脏瓣膜材料表面形成氟化物超润滑抗凝涂层；
c. 向步骤b体系中加入润滑剂浸泡生物瓣膜材料或取出生物心脏瓣膜材料并将其完全浸没于润滑剂中，使润滑剂保持在所述氟化物超润滑抗凝涂层。
作为可选方式，在上述制备方法中，在步骤a中，所述戊二醛交联具体为将生物心脏瓣膜材料浸泡于0.2-2.5vt%的戊二醛溶液中1-7天。
作为可选方式，在步骤a中，将戊二醛交联的生物心脏瓣膜材料浸泡在5-500mM氧自由基活化剂溶液中1-24h。
作为可选方式，所述氧自由基活化剂包括：过硫酸铵（ CAS号：7727-54-0 ）/亚硫酸氢钠（CAS号：7631-90-5），硝酸铈铵（CAS号：16774-21-3）。所述过硫酸铵可以产生活性氧自由基，亚硫酸氢钠进一步增强产生活性氧自由基的数量及效率。
作为可选方式，在步骤b中，通过气相沉积或液相沉积将三氯硅烷氟化物与生物心脏瓣膜材料进行化学结合，或加入双键氟化物与生物心脏瓣膜材料进行化学结合。其中，双键氟化物中含有的“碳碳双键”存在于主链中，为非极性键，其电子云分布均匀，容易受到亲核试剂的进攻而发生亲电加成反应，因此，能够与步骤a中被活化的自由基发生聚合反应而与生物心脏瓣膜材料形成共价结合。
作为可选方式，所述液相沉积包括向步骤a的体系中加入三氯硅烷氟化物溶液或双键氟化物溶液，继续浸泡生物心脏瓣膜材料；或将经步骤a处理后的生物心脏瓣膜材料取出，再完全浸泡于三氯硅烷氟化物溶液或双键氟化物溶液。
作为可选方式，所述三氯硅烷氟化物溶液和双键氟化物溶液的浓度均为5-20vt%。
作为可选方式，在步骤c中，浸泡或浸没的时间为1-24h。
作为可选方式，所述制备方法还包括步骤c处理后的生物心脏瓣膜材料经漂洗后，采用抑菌溶剂保存或采用醇溶液脱水、干燥后保存。
作为可选方式，可选用去离子水等常用的漂洗液漂洗，更优选为采用生理盐水或磷酸盐缓冲液（PBS）漂洗。其中磷酸盐缓冲液的pH为7-7.4。生理盐水和磷酸盐缓冲液是生物材料处理中典型使用的水溶性溶剂，可以确保材料保持与人体兼容的渗透压，不至于材料植入人体后发生渗透压失衡。
作为可选方式，可对生物心脏瓣膜材料进行多次漂洗，且相邻两次可采用相同或不同的漂洗液。
作为可选方式，所述抑菌溶剂保存具体为：将瓣膜材料浸泡于20-100vt%的异丙醇或70-100vt%的乙醇水溶液中保存。
作为可选方式，所述醇溶液脱水干燥后保存具体为：将生物瓣膜材料浸泡于10-30vt%甘油与70-90vt%乙醇等体积混合的混合溶液或10-30vt%甘油、35-45vt%乙醇与35-45vt%异丙醇等体积混合的混合溶液中脱水4-24h，再进行干燥。所述干燥步骤优选为自然风干。
上述所有溶液中溶剂的选择主要考虑各氟化物的溶解度，还需考虑其对动物心包膜的潜在产生破坏的副作用；一般地，如果是水溶性试剂，选用PBS、生理盐水或酸溶液就可以，如果水溶性较差，一般选用乙醇作为溶剂。
本发明还提供了一种上述的人工心脏瓣膜材料的应用，将其用于制作人工主动脉瓣膜、肺动脉瓣膜、静脉瓣膜、二尖瓣膜或三尖瓣膜。
本说明书中公开的所有特征，或公开的所有方法或过程中的步骤，除了互相排斥的特征和/或步骤以外，均可以以任何方式组合。
 有益效果
本发明的有益效果：
（1）本发明所述抗凝血的人工心脏瓣膜材料，通过在生物心脏瓣膜材料表面构建氟化物超润滑抗凝涂层，能够显著减小血小板黏附以及全血黏附，大幅提高材料的抗凝血性能。
（2）本发明选用的氟化物已经被美国FDA批准用于血液接触材料，具有较好的生物安全性。
（3）本发明中氟化物超润滑抗凝涂层制备方法简单，，只需要简单地溶液浸泡或气相沉积，对生物心脏瓣膜材料的损伤较小，且制备过程中避免引入缩合试剂，以减少额外引入或残留的化学试剂，有利于保持材料良好的生物相容性。
（4）本发明构建的氟化物超润滑抗凝涂层结构对各种简单和复杂的液体都具有良好排斥抗黏附作用，具有良好的、稳定的抗凝作用。
 附图说明
图1为本发明实施例所述技术路线1的示意图；
图2为本发明实施例所述技术路线2的示意图；
图3为本发明实验例所述材料对应的全血黏附照片。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
因此，以下对提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下结合实施例对本发明的特征和性能作进一步的详细描述。本发明采用了两种技术路线：（1）氧自由基活化剂活化心包膜表面后三氯硅烷氟化物化学修饰（图1）。（2）氧自由基活化剂活化心包膜表面后双键氟化物化学修饰心包膜（图2）。
本发明对基底材料没有限制，由于心包膜是目前已经广泛验证使用效果较好的人工心脏瓣膜材料，以下实施例中主要以心包膜为例对本发明进行展示，本领域技术人员可以很容易地将其替换为瓣膜、肠膜、脑膜、肺膜、血管、皮肤或韧带等其他生物组织。
实施例1
具体步骤如下：
a.将0.625vt%戊二醛交联的10cm*10cm猪心包膜浸入50mM过硫酸铵/50mM亚硫酸氢钠溶液中，进行基团活化2h；
b.通过液相沉积将三氯(1H,1H,2H,2H-全氟辛基)硅烷与心包膜进行化学结合,即将100毫升10vt%三氯(1H,1H,2H,2H-全氟辛基)硅烷溶液于60摄氏度浸泡10cm*10cm心包膜24h；
c.加入10毫升全氟萘烷润滑剂24h；
d.漂洗后采用25vt%异丙醇水溶液保存。
实施例2
具体步骤如下：
a.将0.625vt%戊二醛交联的10cm*10cm猪心包膜浸入50mM过硫酸铵/50mM亚硫酸氢钠溶液中，进行基团活化2h；
b.将100毫升10vt%烯丙基五氟苯溶液于室温浸泡10cm*10cm心包膜24h；
c.加入10毫升全氟萘烷润滑剂24h；
d.漂洗后采用25vt%异丙醇水溶液保存。
实施例3
具体步骤如下：
a.将0.625vt%戊二醛交联的10cm*10cm猪心包膜浸入50mM硝酸铈铵溶液中，进行基团活化2h；
b.将100毫升10vt%1H,1H,2H-全氟-1-辛烯溶液于室温浸泡10cm*10cm心包膜24h；
c.加入10毫升全氟萘烷润滑剂24h；
d.漂洗后采用25vt%异丙醇水溶液保存。
实施例4
具体步骤如下：
a.将0.625vt%戊二醛交联的10cm*10cm猪心包膜浸入50mM硝酸铈铵溶液中，进行基团活化2h；
b.将100毫升10vt%1H,1H,2H-全氟-1-十二烯溶液于室温浸泡10cm*10cm心包膜24h；
c.加入10毫升五氟苯酚二乙基三甲基烯酯润滑剂24h；
d.漂洗后采用25vt%异丙醇水溶液保存。
实验例
设置对照组与实施例制得的材料分别进行血小板及全血黏附测试。
对照组：将猪心包膜浸泡于0.625vt%的戊二醛溶液中交联1天。漂洗后采用25vt%异丙醇水溶液保存。
（1）全血黏附测试
使用枸橼酸钠采血管采集兔血，将样品（直径为12 mm）浸泡在1 mL全血中，37℃孵化2小时，之后吸出全血并采用1mL的PBS溶液清洗三次，每次5分钟，之后对全血黏附情况进行拍照分析。
结果如图3所示，实施例1-4组的全血黏附相比戊二醛对照组明显减少。
（2）血小板黏附测试
新鲜枸橼酸钠抗凝的兔血以1500rpm离心15分钟以获得富含血小板的上清液（PRP）。实验样品裁剪为12mm圆片并用PBS溶液清洗三次后，光滑面向上放置于24孔板中，并在每孔中加入1 mL新鲜富血小板血浆后37℃孵育1个小时（纯PRP被设置为阳性对照组）。孵育完成后，样品用PBS清洗三次以除去体系中未黏附的血小板，并使用LDH细胞毒性试剂盒测量细胞释放的LDH间接定量黏附在材料表面的血小板数量。反应完成后用酶标仪测量490nm处的吸光度，设置PBS为阴性对照。血小板的相对黏附量=（测试样品490nm吸光度 - 阴性对照490nm吸光度）/（阳性对照490nm吸光度 - 阴性对照490nm吸光度）。
结果如表1所示，实施例1-4组的血小板黏附相比戊二醛对照组明显减少。
表1 血小板黏附相对含量。
血小板黏附相对含量
戊二醛对照组 0.31 ± 0.05
实施例1 0.05 ± 0.01
实施例2 0.04 ± 0.01
实施例3 0.05 ± 0.01
实施例4 0.05 ± 0.01
上述实施例仅为本发明的优选实施方式之一，不应当用于限制本发明的保护范围，但凡在本发明的主体设计思想和精神上作出的毫无实质意义的改动或润色，其所解决的技术问题仍然与本发明一致的，均应当包含在本发明的保护范围之内。

Claims (19)

  1. 一种抗凝血的人工心脏瓣膜材料，其特征在于，包括生物心脏瓣膜材料、与所述生物心脏瓣膜材料化学结合的氟化物超润滑抗凝涂层、保持在所述氟化物超润滑抗凝涂层的润滑剂；所述氟化物超润滑抗凝涂层是通过化学气相沉积或液相沉积将三氯硅烷氟化物与生物心脏瓣膜材料进行化学结合而成；或者是通过将双键氟化物与生物心脏瓣膜材料进行化学结合而成。
  2. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述氟化物超润滑抗凝涂层的厚度为0.01-1μm。
  3. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述生物心脏瓣膜材料为猪或牛心包膜。
  4. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述三氯硅烷氟化物为三氯(1H,1H,2H,2H-全氟辛基)硅烷；所述双键氟化物为烯丙基五氟苯、1H,1H,2H-全氟-1-辛烯、1H,1H,2H-全氟-1-十二烯、2-(全氟辛基)乙基甲基丙烯酸酯中一种或几种。
  5. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述润滑剂为全氟萘烷或五氟苯酚二乙基三甲基烯酯。
  6. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述生物心脏瓣膜材料预先经过戊二醛交联处理。
  7. 一种如权利要求1所述的人工心脏瓣膜材料的制备方法，其特征在于，包括以下步骤：
    a．将戊二醛交联的生物心脏瓣膜材料，浸入氧自由基活化剂溶液中，进行基团活化；
    b．将生物心脏瓣膜材料与三氯硅烷氟化物或双键氟化物接触，使氟化物通过步骤a中的被活化的基团与生物心脏瓣膜材料进行化学结合，在生物心脏瓣膜材料表面形成氟化物超润滑抗凝涂层；
    c．向步骤b体系中加入润滑剂浸泡生物心脏瓣膜材料或取出生物心脏瓣膜材料并将其完全浸没于润滑剂中，使润滑剂保持在所述氟化物超润滑抗凝涂层。
  8. 根据权利要求7所述的制备方法，其特征在于，在步骤a中，将戊二醛交联的生物心脏瓣膜材料浸泡在5-500mM氧自由基活化剂溶液中1-24h。
  9. 根据权利要求8所述的制备方法，其特征在于，所述氧自由剂活化剂为过硫酸铵、亚硫酸氢钠或硝酸铈铵。
  10. 根据权利要求7所述的制备方法，其特征在于，在步骤b中，通过气相沉积或液相沉积将三氯硅烷氟化物与生物心脏瓣膜材料进行化学结合，或加入双键氟化物与生物心脏瓣膜材料进行化学结合。
  11. 根据权利要求10所述的制备方法，其特征在于，所述液相沉积包括向步骤a的体系中加入三氯硅烷氟化物溶液或双键氟化物溶液，继续浸泡生物心脏瓣膜材料1-24h；或将经步骤a处理后的生物心脏瓣膜材料取出，再完全浸泡于三氯硅烷氟化物溶液或双键氟化物溶液，保持1-24h。
  12. 根据权利要求11所述的制备方法，其特征在于，所述三氯硅烷氟化物溶液和双键氟化物溶液的浓度均为5-20vt%。
  13. 根据权利要求7所述的制备方法，其特征在于，步骤c中，浸泡或浸没的时间为1-24h。
  14. 根据权利要求7所述的制备方法，其特征在于，还包括步骤c处理后的生物心脏瓣膜材料经漂洗后，采用抑菌溶剂保存或采用醇溶液脱水、干燥后保存。
  15. 根据权利要求14所述的制备方法，其特征在于，漂洗时采用的漂洗液为去离子水、生理盐水和磷酸缓冲液一种或几种。
  16. 根据权利要求14所述的制备方法，其特征在于，所述抑菌溶剂保存具体为：将瓣膜材料浸泡于20-100vt%的异丙醇水溶液或70-100vt%的乙醇水溶液中保存。
  17. 根据权利要求14所述的制备方法，其特征在于，所述醇溶液脱水、干燥后保存具体为：
    将生物瓣膜材料浸泡于10-30vt%甘油与70-90vt%乙醇等体积混合的混合溶液或10-30vt%甘油、35-45vt%乙醇与35-45vt%异丙醇等体积混合的混合溶液中脱水4-24h，再进行干燥。
  18. 根据权利要求14所述的制备方法，其特征在于，所述干燥为自然风干。
  19. 一种如权利要求1所述的人工心脏瓣膜材料的应用，其特征在于，将其用于制作人工主动脉瓣膜、肺动脉瓣膜、静脉瓣膜、二尖瓣膜或三尖瓣膜。
PCT/CN2021/125823 2020-10-23 2021-10-22 一种抗凝血的人工心脏瓣膜材料及其制备方法与应用 WO2022083758A1 (zh)

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