WO2022083759A1 - 一种抗凝血抗钙化的人工心脏瓣膜材料及其制备方法与应用 - Google Patents

一种抗凝血抗钙化的人工心脏瓣膜材料及其制备方法与应用 Download PDF

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WO2022083759A1
WO2022083759A1 PCT/CN2021/125826 CN2021125826W WO2022083759A1 WO 2022083759 A1 WO2022083759 A1 WO 2022083759A1 CN 2021125826 W CN2021125826 W CN 2021125826W WO 2022083759 A1 WO2022083759 A1 WO 2022083759A1
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heart valve
valve material
fluoride
biological
preparation
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PCT/CN2021/125826
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English (en)
French (fr)
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雷洋
王云兵
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 WO2022083759A1 publication Critical patent/WO2022083759A1/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
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
    • A61F2/2475—Venous 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
    • A61L27/36—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
    • A61L27/36—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
    • A61L27/3641—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 site of application in 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
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification 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
    • 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
    • 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
    • A61L33/0005—Use of materials characterised by their function or physical properties
    • A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
    • A61L33/0041—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate characterised by the choice of an antithrombatic agent other than heparin
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/02—Treatment of implants to prevent calcification or mineralisation in vivo
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • 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
    • A61L2420/02—Methods for coating medical devices
    • 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 anticoagulation and anticalcification 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 and anticalcification artificial heart valve material.
  • a fluoride superlubricant anticoagulation coating on the surface of the biological heart valve material, the adhesion of platelets and whole blood can be significantly reduced. Improve the anticoagulant and anticalcification properties of the material.
  • An anticoagulant and anticalcification artificial heart valve material comprising a biological heart valve material, a fluoride superlubricant anticoagulant coating formed by modifying the biological heart valve material, amino fluoride or epoxy fluoride, and maintaining the fluoride Lubricant for superlubricating anticoagulant coatings.
  • the lubricant may remain on the surface of the fluoride superlubricating anticoagulation coating and/or within the coating structure.
  • the fluoride super-lubricating anticoagulant coating is coated on the surface of the biological heart valve material, as an effective barrier to protect the biological heart valve material, which is beneficial to reduce platelet adhesion and whole blood adhesion;
  • the amino fluoride or epoxy fluoride can improve the stability of the material, reduce the calcium ion binding site of the material, and achieve anti-calcification by consuming the active groups of the biological valve material, including aldehyde group, carboxyl group, hydroxyl group, and amino group.
  • 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.
  • the amino fluoride is: trifluoroacetamide (CAS number: 354-38-1), 2,2,2-trifluoroethylamine hydrochloride (CAS No.: 373-88-6), 2,3,4,5,6-Pentafluoroaniline (CAS No.: 771-60-8), Pentafluorophenylhydrazine (CAS No.: 828-73-9), 2, One or more of 2,3,3,4,4,4-heptafluorobutylamine (CAS number: 374-99-2);
  • the epoxy fluoride is: 3-(perfluoro-n-octyl) )-1,2-propylene oxide (CAS number: 38565-53-6), 1,4-bis(2',3'-epoxypropyl)perfluorobutane (CAS number: 791-22-0 ) one or more of them.
  • the amino fluoride can be chemically combined with the group on the surface of the biological valve material through the active amino group, and the
  • the lubricant is perfluorodecalin or pentafluorophenol diethyl trimethenate.
  • the lubricant has good biosafety and biocompatibility.
  • the biological heart valve material is pre-treated with glutaraldehyde cross-linking.
  • the present invention also provides a preparation method of the above-mentioned artificial heart valve material, characterized in that it comprises the following steps:
  • a fluoride superlubricant anticoagulant coating on the surface of the glutaraldehyde-crosslinked biological heart valve material, and the method of forming the fluoride superlubricant anticoagulant coating is to immerse the biological heart valve material in a compound containing amino fluoride or epoxy fluoride solution or vapor deposition of compounds;
  • the glutaraldehyde cross-linking in step a is specifically immersing the biological heart valve material in a 0.2-2.5 vt% glutaraldehyde solution for 1-7 days.
  • step a the concentrations of amino fluoride and epoxy fluoride in the solution are both 5-20vt%; the immersion time of the biological heart valve material is 1-24h.
  • the solution is an ethanol solution or a hydrochloric acid solution.
  • step b the amount of lubricant added to ensure complete immersion of the biological heart valve material is sufficient.
  • the preparation method further includes: after rinsing the biological heart valve material processed in step b, using a bacteriostatic solvent for preservation or using an alcohol solution for dehydration and drying before preservation.
  • a common rinsing solution such as deionized water can be used for rinsing, and more preferably, physiological saline or phosphate buffered saline (PBS) can be used for rinsing; wherein the pH of the phosphate buffer is 7-7.4.
  • physiological saline or phosphate buffered saline PBS
  • Physiological saline and phosphate buffer are water-soluble solvents typically used in biomaterial processing, which can ensure that the artificial heart valve material maintains an osmotic pressure compatible with the human body and avoids osmotic pressure imbalance after implantation 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: immersing the valve material in a 20-100 vt% isopropanol aqueous solution or a 70-100 vt% 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 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 selection of the solvent in all the above solutions mainly considers the solubility of each fluoride, and also needs to consider its potential side effects of damaging 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 anticoagulation and anticalcification artificial heart valve material of the present invention can significantly reduce platelet adhesion and whole blood adhesion by constructing a fluoride superlubricant anticoagulant coating on the surface of the biological heart valve material, and greatly improve the material's performance. Anticoagulant properties, while improving the anticalcification properties of the material.
  • 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, only simple solution immersion or vapor deposition is required, 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 features and performances of the present invention will be further described in detail below in conjunction with the embodiments.
  • the present invention adopts two technical routes: (1) Amino fluoride chemically modifies the pericardium (Fig. 1). (2) Epoxy fluoride chemically modified pericardium ( Figure 2).
  • 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. After that, the whole blood adhesion situation was photographed and analyzed.
  • 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.
  • test group samples and control group samples that have been cut into 1cm ⁇ 1cm were cleaned, and 0.1 mL of 3% sodium pentobarbital was injected into the abdominal cavity of juvenile SD rats about 20 days old for anesthesia, and the fur on the muscles on both sides of the spine was shaved. Routine disinfection with iodine and alcohol.
  • One sample of the test group was subcutaneously implanted on the right back, and one sample of the control group was subcutaneously implanted on the left back, and the skin incision was sutured. After 60 days, the animals were euthanized by cervical dislocation and the grafts were removed. The host tissue on the graft surface was carefully removed and rinsed with saline.
  • Example 1 0.32 ⁇ 0.24
  • Example 2 0.35 ⁇ 0.21
  • Example 3 0.43 ⁇ 0.12
  • Example 4 0.42 ⁇ 0.11

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Abstract

本发明公开了一种抗凝血抗钙化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。所述抗凝血抗钙化的人工心脏瓣膜材料包括生物心脏瓣膜材料、氨基氟化物或环氧氟化物修饰所述生物心脏瓣膜材料形成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。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，同时提高材料的抗钙化性能。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

一种抗凝血抗钙化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抗钙化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同时具有良好的抗钙化效果的人工心脏瓣膜材料问世。
 技术问题
本发明针对上述问题，开发了一种抗凝血抗钙化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及抗钙化性能。
 技术解决方案
本发明包含以下技术方案：
一种抗凝血抗钙化的人工心脏瓣膜材料，包括生物心脏瓣膜材料、氨基氟化物或环氧氟化物修饰所述生物心脏瓣膜材料形成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。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。
一方面，所述氟化物超润滑抗凝涂层包覆于生物心脏瓣膜材料的表面，作为保护生物心脏瓣膜材料的有效屏障，有利于减少血小板粘附以及全血粘附；另一方面，所述氨基氟化物或环氧氟化物能够通过消耗生物瓣膜材料的活性基团，包括醛基、羧基、羟基、氨基，可以提高材料的稳定性，减少材料的钙离子结合位点，实现抗钙化。
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。
作为可选方式，在上述人工心脏瓣膜材料中，所述氨基氟化物为：三氟乙酰胺（CAS号：354-38-1），2,2,2-三氟乙胺盐酸盐（CAS号：373-88-6），2,3,4,5,6-五氟苯胺（CAS号：771-60-8），五氟苯肼（CAS号：828-73-9），2,2,3,3,4,4,4-七氟丁胺（CAS号：374-99-2）中的一种或几种；所述环氧氟化物为：3-(全氟正辛基)-1,2-环氧丙烷（CAS号：38565-53-6），1,4-二(2’,3’-环氧丙基)全氟丁烷（CAS号：791-22-0）中的一种或几种。所述氨基氟化物可以通过活性氨基与生物瓣膜材料表面的基团进行化学结合，所环氧氟化物可以通过环氧基团与生物瓣膜材料表面的活性基团进行化学结合。
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。
作为可选方式，所述生物心脏瓣膜材料预先经过戊二醛交联处理。
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.在戊二醛交联的生物心脏瓣膜材料表面形成氟化物超润滑抗凝涂层，形成氟化物超润滑抗凝涂层的方式为将生物心脏瓣膜材料浸入含有氨基氟化物或环氧氟化物的溶液中或气相沉积法；
b. 加入润滑剂使其保持在所述氟化物超润滑抗凝涂层，或将步骤a处理后的生物心脏瓣膜材料完全浸没于润滑剂中保持1-24h。
作为可选方式，在上述制备方法中，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，步骤a中，所述溶液中氨基氟化物和环氧氟化物的浓度均为5-20vt%；生物心脏瓣膜材料的浸入时间为1-24h。
作为可选方式，步骤a中，所述溶液为乙醇溶液或盐酸溶液。
作为可选方式，步骤b中，加入润滑剂的量确保完全浸没生物心脏瓣膜材料即可。
作为可选方式，所述制备方法还包括：步骤b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，漂洗时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，可以确保人工心脏瓣膜材料保持与人体兼容的渗透压，避免将其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%的异丙醇水溶液或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。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。
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）本发明所述抗凝血抗钙化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，同时提高材料的抗钙化性能。
（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，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）氨基氟化物化学修饰心包膜（图1）。（2）环氧氟化物化学修饰心包膜（图2）。
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100毫升10vt%三氟乙酰胺溶液中24h；
b.加入10毫升全氟萘烷润滑剂24h；
c.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100毫升10vt%2,2,2-三氟乙胺盐酸盐溶液中24h；
b.加入10毫升五氟苯酚二乙基三甲基烯酯润滑剂24h；
c.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100毫升10vt% 的3-(全氟正辛基)-1,2-环氧丙烷溶液中24h；
b.加入10毫升全氟萘烷润滑剂24h；
c.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100毫升10vt% 的1,4-二(2’,3’-环氧丙基)全氟丁烷溶液中24h；
b.加入10毫升五氟苯酚二乙基三甲基烯酯润滑剂24h；
c.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。
血小板黏附相对黏附量
戊二醛对照组 0.32 ± 0.06
实施例1 0.07 ± 0.01
实施例2 0.06 ± 0.01
实施例3 0.07 ± 0.02
实施例4 0.07 ± 0.01
（3）钙化性能测试
已裁剪成1cm×1cm的试验组样品和对照组样品清洗好，向20天左右幼年SD大鼠的腹腔注射3%戊巴比妥钠0.1 mL进行麻醉，剃除脊柱两旁肌肉上的皮毛，用碘酒和酒精进行常规消毒。右侧背部皮下植入试验组样品1个，左侧背部皮下植入对照组样品1个，缝合皮肤切口。60天后，采用颈椎脱臼法对动物进行安乐死，取出移植物。小心除去移植物表面的宿主组织，生理盐水冲洗干净。冷冻干燥后称量干重，之后采用6N浓盐酸在95摄氏度水浴锅中消解直到无可见固体颗粒，之后采用电感耦合等离子体发射光谱仪进行钙元素的定量分析。对实施例和对照组制得的材料分别进行抗钙化性能测试。结果如下表2所示。由表2可知，实施例1-4组的挂钙量相比于戊二醛对照组减少。
表2 挂钙量。
挂钙量 µg/mg
戊二醛交联组 25.26 ± 3.56
实施例1 0.32 ± 0.24
实施例2 0.35 ± 0.21
实施例3 0.43 ± 0.12
实施例4 0.42 ± 0.11
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 (18)

  1. 一种抗凝血抗钙化的人工心脏瓣膜材料，其特征在于，包括生物心脏瓣膜材料、氨基氟化物或环氧氟化物修饰所述生物心脏瓣膜材料形成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。
  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选自心包膜、瓣膜、肠膜、脑膜、肺膜、血管、皮肤和韧带中的一种。
  4. 根据权利要求3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。
  5. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述氨基氟化物为三氟乙酰胺、2,2,2-三氟乙胺盐酸盐、2,3,4,5,6-五氟苯胺、五氟苯肼、2,2,3,3,4,4,4-七氟丁胺中的一种或几种；所述环氧氟化物为3-(全氟正辛基)-1,2-环氧丙烷和1,4-二(2’,3’-环氧丙基)全氟丁烷中的一种或几种。
  6. 根据权利要求1所述的人工心脏瓣膜材料，其特征在于，所述润滑剂为全氟萘烷或五氟苯酚二乙基三甲基烯酯。
  7. 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。
  8. 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. 在戊二醛交联的生物心脏瓣膜材料表面形成氟化物超润滑抗凝涂层，形成氟化物超润滑抗凝涂层的方式为将生物心脏瓣膜材料浸入含有氨基氟化物或环氧氟化物的溶液中或气相沉积法；
    b.加入润滑剂使其保持在所述氟化物超润滑抗凝涂层，或将步骤a处理后的生物心脏瓣膜材料完全浸没于润滑剂中保持1-24h。
  9. 根据权利要求8所述的制备方法，其特征在于，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。
  10. 根据权利要求8所述的制备方法，其特征在于，步骤a中，所述溶液中氨基氟化物和环氧氟化物的浓度均为5-20vt%；生物心脏瓣膜材料的浸入时间为1-24h。
  11. 根据权利要求8所述的制备方法，其特征在于，步骤a中，所述溶液为乙醇溶液或盐酸溶液。
  12. 根据权利要求8所述的制备方法，其特征在于，步骤b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。
  13. 根据权利要求12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、生理盐水和磷酸盐缓冲液中一种。
  14. 根据权利要求13所述的制备方法，其特征在于，所述磷酸盐缓冲液的pH为7-7.4。
  15. 根据权利要求12所述的制备方法，其特征在于，所述抑菌溶剂保存具体为：
    将步骤b处理后的生物瓣膜材料浸泡于20-100vt%的异丙醇水溶液或70-100vt%的乙醇水溶液中保存。
  16. 根据权利要求12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。
  17. 根据权利要求12所述的制备方法，其特征在于，所述干燥为自然风干。
  18. 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。
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