CN103721300B - Anticoagulation coating material and preparation method thereof - Google Patents
Anticoagulation coating material and preparation method thereof Download PDFInfo
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- CN103721300B CN103721300B CN201310700680.3A CN201310700680A CN103721300B CN 103721300 B CN103721300 B CN 103721300B CN 201310700680 A CN201310700680 A CN 201310700680A CN 103721300 B CN103721300 B CN 103721300B
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- 239000011248 coating agent Substances 0.000 title abstract description 107
- 230000010100 anticoagulation Effects 0.000 title description 4
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Abstract
本发明公开了一种抗凝血涂层材料及制备方法。所述抗凝血涂层材料，包括透明质酸钠和水蛭素，透明质酸钠形成凝胶网络，水蛭素通过酰胺键与透明质酸钠共价结合。所述抗凝血涂层材料制备方法，包括以下步骤：（1）配置MES缓冲液；（2）配制透明质酸钠溶液，其浓度为1mg/ml至20mg/ml；（3）配制水蛭素溶液，其浓度为1mg/ml至10mg/ml；（4）将上述两种溶液均匀混合，使得水蛭素与透明质酸钠的质量比例为1:1至1:10；（5）向步骤（4）中得到的混合溶液中加入交联剂，均匀混合，使得水蛭素与透明质酸钠共价交联。本发明提供的抗凝血涂层材料，抗凝血活性较高，效果稳定，持续时间长，生物相容性好，且制备方法简单，条件温和，适合工业大规模生产。
The invention discloses an anticoagulant coating material and a preparation method. The anticoagulant coating material includes sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently combined with the sodium hyaluronate through an amide bond. The preparation method of the anticoagulant coating material comprises the following steps: (1) preparing MES buffer solution; (2) preparing sodium hyaluronate solution with a concentration of 1 mg/ml to 20 mg/ml; (3) preparing hirudin solution, the concentration of which is 1 mg/ml to 10 mg/ml; (4) uniformly mix the above two solutions so that the mass ratio of hirudin to sodium hyaluronate is 1:1 to 1:10; (5) to step ( 4) Add a cross-linking agent to the mixed solution obtained in 4), and mix evenly, so that hirudin and sodium hyaluronate are covalently cross-linked. The anticoagulant coating material provided by the invention has high anticoagulant activity, stable effect, long duration, good biocompatibility, simple preparation method and mild conditions, and is suitable for large-scale industrial production.
Description
技术领域technical field
本发明属于生物医用材料领域，更具体地，涉及一种抗凝血涂层材料及其制备方法。The invention belongs to the field of biomedical materials, and more specifically relates to an anticoagulant coating material and a preparation method thereof.
背景技术Background technique
抗凝血生物医用材料是一类重要的生物材料，被广泛应用于与人类血液和组织相接触的医用材料上，如血液透析系统、体外循环系统、人工心脏瓣膜、心脏起搏器、人工血管、血管支架、外科手术线和导管等。进入21世纪，心血管疾病发病率和死亡率在不断上升，心血管疾病的防治已成为国内外医学界关注的重点。抗凝血生物医用材料有着重要的应用价值。Anticoagulant biomedical materials are an important class of biological materials, which are widely used in medical materials in contact with human blood and tissues, such as hemodialysis systems, extracorporeal circulation systems, artificial heart valves, cardiac pacemakers, artificial blood vessels , vascular stents, surgical lines and catheters, etc. In the 21st century, the morbidity and mortality of cardiovascular diseases are on the rise, and the prevention and treatment of cardiovascular diseases has become the focus of medical circles at home and abroad. Anticoagulant biomedical materials have important application value.
目前已有抗凝血涂层，主要是采用肝素作为抗凝血因子，将肝素通过物理吸附或者化学结合的方法固定在材料表面。采用物理吸附的方法，将材料肝素化，肝素容易流失，抗凝血效果不长；采用化学结合的方法，将材料肝素化，由于肝素为大生物分子，共价结合在金属支架表面后，其构象受到限制，可能造成抗凝血活性降低。另外用于固定肝素的涂层聚合物还存在生物相容性问题，不能促进内皮愈合，对减少再狭窄率也没有明显贡献。At present, there are anticoagulant coatings, mainly using heparin as an anticoagulant factor, and heparin is fixed on the surface of the material by physical adsorption or chemical combination. The material is heparinized by physical adsorption, and the heparin is easy to lose, and the anticoagulant effect is not long; the material is heparinized by chemical combination, because heparin is a large biomolecule, after being covalently bonded to the surface of the metal stent, its Conformational constraints may result in reduced anticoagulant activity. In addition, the coating polymer used to immobilize heparin still has biocompatibility problems, cannot promote endothelial healing, and does not significantly contribute to reducing the rate of restenosis.
目前还没有一种具有理想抗凝血作用且不影响其生物相容性的抗凝血涂层材料。At present, there is no anticoagulant coating material that has ideal anticoagulant effect and does not affect its biocompatibility.
发明内容Contents of the invention
针对现有技术的以上缺陷或改进需求，本发明提供了一种抗凝血涂层材料，其目的在于通过将分子量较小、抗凝血效果稳定的抗凝血因子水蛭素与具有良好生物相容性的透明质酸钠凝胶网络共价结合，由此解决目前抗凝血涂层材料抗凝血效果不稳定或者不长久、生物相容性差的技术问题。In view of the above defects or improvement needs of the prior art, the present invention provides an anticoagulant coating material, the purpose of which is to combine the anticoagulant factor hirudin, which has a small molecular weight and stable anticoagulant effect, with a good biological phase. The capacitive sodium hyaluronate gel network is covalently bonded, thereby solving the technical problems of unstable or short-term anticoagulant effect and poor biocompatibility of current anticoagulant coating materials.
为实现上述目的，按照本发明的一个方面，提供了一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。In order to achieve the above object, according to one aspect of the present invention, an anticoagulant coating material is provided, including sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin forms a gel network through an amide bond. Covalently bound to the sodium hyaluronate.
优选地，所述的抗凝血涂层材料，其水蛭素与所述透明质酸钠的质量比例为1:1至1:10。Preferably, in the anticoagulant coating material, the mass ratio of hirudin to sodium hyaluronate is 1:1 to 1:10.
优选地，所述的抗凝血涂层材料，其水蛭素与所述透明质酸钠的质量比例为1:3至1:5。Preferably, the anticoagulant coating material has a mass ratio of hirudin to sodium hyaluronate of 1:3 to 1:5.
优选地，所述的抗凝血涂层材料，还包括交联剂。Preferably, the anticoagulant coating material further includes a crosslinking agent.
优选地，所述的抗凝血涂层材料，其交联剂为1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺和N-羟基琥珀酰亚胺。Preferably, the crosslinking agent of the anticoagulant coating material is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide.
按照本发明的另一方面，提供了一种抗凝血涂层材料的制备方法，包括以下步骤：According to another aspect of the present invention, a kind of preparation method of anticoagulant coating material is provided, comprising the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液（0.1mol/L，pH=6.4）。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution (0.1mol/L, pH=6.4).
（2）使用步骤（1）配制的MES缓冲溶液，加入透明质酸钠，浓度为1mg/ml至20mg/ml，充分溶解制得透明质酸钠溶液；(2) Using the MES buffer solution prepared in step (1), add sodium hyaluronate at a concentration of 1 mg/ml to 20 mg/ml, fully dissolve to obtain a sodium hyaluronate solution;
（3）使用步骤（1）配制的MES缓冲溶液，加入水蛭素，浓度为1mg/ml至10mg/ml，充分溶解制得水蛭素溶液；(3) Using the MES buffer solution prepared in step (1), add hirudin at a concentration of 1 mg/ml to 10 mg/ml, and fully dissolve the hirudin solution;
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合，使得水蛭素与透明质酸钠的质量比例为1:1至1:10；(4) Evenly mix the sodium hyaluronate solution prepared in step (2) with the hirudin solution prepared in step (3), so that the mass ratio of hirudin to sodium hyaluronate is 1:1 to 1:10;
（5）向步骤（4）中得到的混合溶液中加入交联剂，均匀混合，使得水蛭素与透明质酸钠共价交联，即制得抗凝血涂层材料。(5) Add a cross-linking agent to the mixed solution obtained in step (4), and mix evenly, so that hirudin and sodium hyaluronate are covalently cross-linked, and an anticoagulant coating material is obtained.
优选地，所述抗凝血涂层材料制备方法，其步骤（3）中的水蛭素溶液含有质量浓度为0.1%至1.0%的吐温80。Preferably, in the preparation method of the anticoagulant coating material, the hirudin solution in step (3) contains Tween 80 with a mass concentration of 0.1% to 1.0%.
优选地，所述抗凝血涂层材料制备方法，其步骤（5）中使用的交联剂为新鲜配制的1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺溶液和N-羟基琥珀酰亚胺溶液。Preferably, in the preparation method of the anticoagulant coating material, the cross-linking agent used in step (5) is freshly prepared 1-ethyl-3-(3-dimethylaminopropyl)-carbide Amine solution and N-hydroxysuccinimide solution.
优选地，所述抗凝血涂层材料制备方法，其水蛭素、1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺和N-羟基琥珀酰亚胺的质量比例为1:5:1。Preferably, the preparation method of the anticoagulant coating material, the quality of its hirudin, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide The ratio is 1:5:1.
优选地，所述抗凝血涂层材料制备方法，还包括：Preferably, the preparation method of the anticoagulant coating material also includes:
（6）将步骤（5）制得的抗凝血涂层材料进行透析，去除交联剂。(6) Dialyzing the anticoagulant coating material prepared in step (5) to remove the cross-linking agent.
总体而言，通过本发明所构思的以上技术方案与现有技术相比，由于采用分子量较小水蛭素与生物相容性较好的透明质酸钠交联物作为抗凝血涂层材料，能够取得下列有益效果：Generally speaking, compared with the prior art, the above technical scheme conceived by the present invention adopts the cross-linked sodium hyaluronate with smaller molecular weight hirudin and better biocompatibility as the anticoagulant coating material, The following beneficial effects can be obtained:
（1）所述抗凝血涂层材料中包含有能直接抑制凝血酶的水蛭素作为抗凝血材料。水蛭素通过阻塞凝血酶的底物结合基团致使凝血酶失去活性，其自由羟基与凝血酶自由氨基通过静电引力而结合，产生的络合物在凝血酶保持活性的整个pH范围内不会解离，因此水蛭素具有抗凝血活性高、结构稳定、不易失活的特点。因此所述抗凝血涂层材料抗凝血活性较高，不易因构象变化导致抗凝血因子失活，抗凝血效果稳定。透明质酸钠单独作为涂层，具有一定的抗凝血作用，如附图2所示，血小板黏附数量在1200个/mm2至2000个/mm2之间，比正常水平低，本发明提供的经水蛭素修饰后的透明质酸钠涂层，能发挥显著的抗凝血效果，血小板黏附数量下降到500个/mm2以下，如图1所示。由此可见本发明提供的抗凝血涂层材料能发挥显著的抗凝血效果。(1) The anticoagulant coating material contains hirudin, which can directly inhibit thrombin, as an anticoagulant material. Hirudin deactivates thrombin by blocking the substrate-binding group of thrombin, and its free hydroxyl group binds to the free amino group of thrombin through electrostatic attraction, and the resulting complex will not decompose within the entire pH range where thrombin remains active Therefore, hirudin has the characteristics of high anticoagulant activity, stable structure, and not easy to inactivate. Therefore, the anticoagulant coating material has high anticoagulant activity, is not easy to inactivate anticoagulant factors due to conformational changes, and has a stable anticoagulant effect. Sodium hyaluronate is used alone as a coating, which has a certain anticoagulant effect. As shown in Figure 2, the number of platelets adhered is between 1200/ mm2 and 2000/ mm2 , which is lower than the normal level. The present invention provides The hirudin-modified sodium hyaluronate coating can exert a significant anticoagulant effect, and the number of platelets adhered to less than 500/mm 2 , as shown in Figure 1. It can be seen that the anticoagulant coating material provided by the present invention can exert a significant anticoagulant effect.
（2）由于水蛭素是小分子肽，因此单独作为抗凝血涂层材料，流失严重导致抗凝血时间短。本发明采用粘性较强的交联呈网状的透明质酸钠固定水蛭素，水蛭素与透明质酸钠通过酰胺键共价结合，形成稳定的凝胶网络，可持续长久的发挥抗凝血功效。(2) Since hirudin is a small molecular peptide, it is used as an anticoagulant coating material alone, and the loss is serious, resulting in a short anticoagulant time. The present invention adopts highly viscous cross-linked sodium hyaluronate to immobilize hirudin, and hirudin and sodium hyaluronate are covalently bonded through amide bonds to form a stable gel network, which can last for a long time to play an anticoagulant role. effect.
（3）透明质酸钠能提供高亲水性表层，适合长期与血液接触的表面，同时具有一定的抗凝血效果和良好的生物相容性，能与水蛭素协同作用，加强其抗凝血效果，使用本发明提供的抗凝血涂层材料，对植入物进行改造，使得植入物本身具有长期稳定的抗凝血效果，能帮助减少抗凝血药的使用，从而尽量避免或减轻抗凝血药的毒副作用。(3) Sodium hyaluronate can provide a highly hydrophilic surface layer, which is suitable for surfaces that are in contact with blood for a long time. At the same time, it has a certain anticoagulant effect and good biocompatibility, and can work synergistically with hirudin to strengthen its anticoagulation blood effect, use the anticoagulant coating material provided by the present invention to modify the implant, so that the implant itself has a long-term stable anticoagulant effect, which can help reduce the use of anticoagulant drugs, thereby avoiding or Reduce the side effects of anticoagulants.
（4）本发明提供的抗凝血涂层材料制备方法，针对水蛭素谷氨酸和天门冬氨酸含量较高的特点，采用酰胺键使水蛭素与透明质酸钠共价结合，能使水蛭素与透明质酸钠结合稳定，从而使抗凝血涂层材料发挥长久的抗凝血效果。目前尚无文献报道能使水蛭素与透明质酸钠之间形成稳定结合的方法。(4) The preparation method of the anticoagulant coating material provided by the present invention aims at the high content of hirudin glutamic acid and aspartic acid, adopts amide bonds to covalently combine hirudin and sodium hyaluronate, and can make The combination of hirudin and sodium hyaluronate is stable, so that the anticoagulant coating material can exert a long-term anticoagulant effect. At present, there is no documented method for forming a stable combination between hirudin and sodium hyaluronate.
（5）由于本发明提供的抗凝血涂层材料制备方法，采用交联剂帮助形成酰胺键，能大幅提高反应效率，且所选择的交联剂可通过透析去除，残留少，几乎没有毒副作用。(5) Due to the preparation method of the anticoagulant coating material provided by the present invention, the cross-linking agent is used to help form amide bonds, which can greatly improve the reaction efficiency, and the selected cross-linking agent can be removed by dialysis, with little residue and almost no toxicity side effect.
（6）本发明提供的抗凝血涂层材料制备方法，工艺较为简单，反应条件温和，适合大批量生产。(6) The preparation method of the anticoagulant coating material provided by the present invention has a relatively simple process and mild reaction conditions, and is suitable for mass production.
优选方案，通过对水蛭素和透明质酸钠的质量比例范围、交联剂的种类和交联剂添加比例做优化，使得抗凝血涂层材料在抗凝血效果、制备难度、生产时间和生产成本之间取得平衡，获得成本较为低廉，生产相对简单，抗凝血效果好、抗凝血时间长的抗凝血涂层材料。The preferred solution is to optimize the mass ratio range of hirudin and sodium hyaluronate, the type of crosslinking agent and the addition ratio of the crosslinking agent, so that the anticoagulant coating material has the advantages of anticoagulant effect, preparation difficulty, production time and A balance is achieved between production costs, and an anticoagulant coating material with relatively low cost, relatively simple production, good anticoagulant effect and long anticoagulant time is achieved.
附图说明Description of drawings
图1是本发明提供的抗凝血涂层材料进行血小板黏附测试的扫描电子显微镜照片；Fig. 1 is the scanning electron microscope photograph that anticoagulant coating material provided by the present invention carries out platelet adhesion test;
图2是透明质酸钠涂层进行血小板粘附测试后的扫描电子显微镜照片。Figure 2 is a scanning electron micrograph of sodium hyaluronate coating after platelet adhesion test.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，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仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.
透明质酸钠是一种酸性粘多糖透明质酸的钠盐。透明质酸在人体各种软组织中分布广泛，可以形成凝胶，透明质酸具有高度粘弹性、渗透性和独特的流变学特性以及良好的生物相容性，并且透明质酸形成的凝胶还是一种可吸收生物材料。Sodium hyaluronate is the sodium salt of the acidic mucopolysaccharide hyaluronic acid. Hyaluronic acid is widely distributed in various soft tissues of the human body and can form gels. Hyaluronic acid has high viscoelasticity, permeability, unique rheological properties and good biocompatibility, and the gel formed by hyaluronic acid It is also an absorbable biomaterial.
水蛭素是一种由65个氨基酸组成的低分子量多肽，其中谷氨酸和天门冬酰胺的含量较高，分子量为7000左右，含有三个二硫键。水蛭素通过其自由羟基与凝血酶的自由氨基结合，致使凝血酶失去活性，在这个结合作用在凝血酶的活性pH范围内不会解离，因此水蛭素具有抗凝血活性高、结构稳定、不易失活等特点，此外，水蛭素后期纯化成本低，这些优点使得水蛭素成为一种抗凝、溶栓的理想原料。Hirudin is a low-molecular-weight polypeptide composed of 65 amino acids, of which the content of glutamic acid and asparagine is relatively high, the molecular weight is about 7000, and it contains three disulfide bonds. Hirudin binds to the free amino group of thrombin through its free hydroxyl group, causing thrombin to lose its activity. In this combination, it will not dissociate within the active pH range of thrombin. Therefore, hirudin has high anticoagulant activity, stable structure, Not easy to inactivate and other characteristics, in addition, the cost of later purification of hirudin is low, these advantages make hirudin an ideal raw material for anticoagulation and thrombolysis.
本发明提供的一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。所述水蛭素与所述透明质酸钠的质量比例为1:1至1:10，优选范围为1:3至1:5。为了缩短交联反应时间，促进酰胺键形成，所述抗凝血涂层材料还包括交联剂，如1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）。An anticoagulant coating material provided by the present invention comprises sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently bonded to the sodium hyaluronate through an amide bond . The mass ratio of the hirudin to the sodium hyaluronate is 1:1 to 1:10, preferably 1:3 to 1:5. In order to shorten the crosslinking reaction time and promote the formation of amide bonds, the anticoagulant coating material also includes a crosslinking agent, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ( EDC) and N-hydroxysuccinimide (NHS).
本发明提供的一种抗凝血涂层材料的制备方法，包括以下步骤：A kind of preparation method of anticoagulation coating material provided by the invention comprises the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液，浓度为0.1mol/L，调节pH值至6.4。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution with a concentration of 0.1mol/L and adjust the pH value to 6.4.
（2）配制透明质酸钠溶液：(2) Preparation of sodium hyaluronate solution:
取透明质酸钠（HA，分子量约200万）于2-(N-吗啡啉)乙磺酸缓冲溶液（MES，0.1M，pH=6.4）中，使其充分溶解得到透明质酸钠溶液，透明质酸钠溶液浓度为1mg/ml至20mg/ml。可采用磁力搅拌加速透明质酸钠溶解，搅拌12小时。制得的透明质酸钠溶液可密封冷冻保存备用。Take sodium hyaluronate (HA, molecular weight about 2 million) in 2-(N-morpholine) ethanesulfonic acid buffer solution (MES, 0.1M, pH=6.4), and dissolve it fully to obtain sodium hyaluronate solution. The sodium hyaluronate solution has a concentration of 1 mg/ml to 20 mg/ml. Magnetic stirring can be used to accelerate the dissolution of sodium hyaluronate, stirring for 12 hours. The prepared sodium hyaluronate solution can be sealed and frozen for future use.
（3）配制水蛭素溶液：(3) Preparation of hirudin solution:
取水蛭素于MES缓冲液中，使水蛭素均匀分散于MES缓冲液中。为增加水蛭素的溶解度，可添加表面活性剂，例如质量比例为0.1%至1.0%的吐温80。制得的水蛭素溶液可密封冷冻保存备用。Take the hirudin in the MES buffer, so that the hirudin is uniformly dispersed in the MES buffer. In order to increase the solubility of hirudin, a surfactant can be added, such as Tween 80 with a mass ratio of 0.1% to 1.0%. The prepared hirudin solution can be sealed and frozen for future use.
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合：(4) Evenly mix the sodium hyaluronate solution prepared in step (2) and the hirudin solution prepared in step (3):
在MES缓冲液中加入步骤（2）中制得的透明质酸钠溶液，然后加入水蛭素溶液，使得水蛭素与透明质酸钠的质量比例为1:1至1:10，混合均匀。Add the sodium hyaluronate solution prepared in step (2) to the MES buffer, and then add the hirudin solution so that the mass ratio of hirudin to sodium hyaluronate is 1:1 to 1:10, and mix well.
（5）向步骤（4）中得到的混合溶液中加入交联剂：(5) Add a crosslinking agent to the mixed solution obtained in step (4):
选择1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）溶液和N-羟基琥珀酰亚胺（NHS）溶液作为交联剂，即EDC/NHS交联剂。取新鲜配制的50mg/ml的EDC溶液，加入步骤（4）中得到的混合溶液中，加入比例为水蛭素与EDC质量比例1:5，混合均匀后静置15分钟。然后取新鲜配制的10mg/ml的NHS溶液，加入所述混合溶液，加入比例为水蛭素与NHS质量比例1:1。在避光条件下均匀混合，12小时候得到透明质酸钠与水蛭素交联混合物，即制得所述抗凝血涂层材料。Choose 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution as the crosslinking agent, that is, EDC/NHS crosslinking agent. Take the freshly prepared 50 mg/ml EDC solution, add it to the mixed solution obtained in step (4), and add the mass ratio of hirudin to EDC at a ratio of 1:5, mix well and let stand for 15 minutes. Then take the freshly prepared 10 mg/ml NHS solution and add it to the mixed solution at a mass ratio of hirudin to NHS of 1:1. Mix evenly under the condition of avoiding light, and obtain the cross-linked mixture of sodium hyaluronate and hirudin after 12 hours, that is, the anticoagulant coating material is prepared.
（6）将步骤（5）制得的抗凝血涂层材料进行透析，去除交联剂，从而纯化抗凝血涂层材料。(6) Dialyzing the anticoagulant coating material prepared in step (5) to remove the cross-linking agent, thereby purifying the anticoagulant coating material.
本发明提供的抗凝血涂层材料，以浸涂法修饰并常温干燥，即可修饰上具有抗凝血性能的涂层。对本发明提供的抗凝血涂层材料进行血小板黏附测试，测试后用扫面电子显微镜成像，照片如图1所示。仅采用透明质酸钠涂层材料进行血小板黏附测试，其测试结果如图2所示。对测试结果进行对比，可观察到，本发明提供的抗凝血涂层材料发挥了显著的抗凝血效果。The anticoagulant coating material provided by the present invention can be modified with a coating having anticoagulant properties by dip coating and drying at room temperature. The platelet adhesion test was carried out on the anticoagulant coating material provided by the present invention, and after the test, it was imaged with a scanning electron microscope, as shown in Figure 1 . Only the sodium hyaluronate coating material was used for the platelet adhesion test, and the test results are shown in Figure 2. Comparing the test results, it can be observed that the anticoagulant coating material provided by the present invention exerts a significant anticoagulant effect.
以下为实施例：The following are examples:
实施例1Example 1
一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。所述水蛭素与所述透明质酸钠的质量比例为1:1。所述抗凝血涂层材料还包括交联剂，1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）。An anticoagulant coating material comprises sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently bonded to the sodium hyaluronate through an amide bond. The mass ratio of the hirudin to the sodium hyaluronate is 1:1. The anticoagulant coating material also includes a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) .
所述抗凝血涂层材料的制备方法，包括以下步骤：The preparation method of described anticoagulant coating material, comprises the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液，浓度为0.1mol/L，调节pH值至6.4。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution with a concentration of 0.1mol/L and adjust the pH value to 6.4.
（2）配制透明质酸钠溶液。(2) Prepare sodium hyaluronate solution.
取透明质酸钠（HA，分子量约200万）于2-(N-吗啡啉)乙磺酸缓冲溶液（MES，0.1M，pH=6.4）中，使其充分溶解得到透明质酸钠溶液，透明质酸钠溶液浓度为1mg/ml。可采用磁力搅拌加速透明质酸钠溶解，搅拌12小时。制得的透明质酸钠溶液可密封冷冻保存备用。Take sodium hyaluronate (HA, molecular weight about 2 million) in 2-(N-morpholine) ethanesulfonic acid buffer solution (MES, 0.1M, pH=6.4), and dissolve it fully to obtain sodium hyaluronate solution. The concentration of sodium hyaluronate solution is 1mg/ml. Magnetic stirring can be used to accelerate the dissolution of sodium hyaluronate, stirring for 12 hours. The prepared sodium hyaluronate solution can be sealed and frozen for future use.
（3）配制水蛭素溶液。(3) Prepare hirudin solution.
取水蛭素于MES缓冲液中，水蛭素浓度为1mg/ml,通过磁力搅拌，使水蛭素均匀分散于MES缓冲液中，加入质量浓度为0.1%的吐温80。制得的水蛭素溶液可密封冷冻保存备用。Take hirudin in MES buffer solution, the concentration of hirudin is 1 mg/ml, and the hirudin is uniformly dispersed in MES buffer solution by magnetic stirring, and Tween 80 with a mass concentration of 0.1% is added. The prepared hirudin solution can be sealed and frozen for future use.
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合。(4) Evenly mix the sodium hyaluronate solution prepared in step (2) and the hirudin solution prepared in step (3).
在6mL MES缓冲液中加入步骤（2）中制得的透明质酸钠溶液20mL，然后加入步骤（3）制得的水蛭素溶液20mL，透明质酸钠与水蛭素的质量比例为1:1，搅拌混合均匀。Add 20 mL of the sodium hyaluronate solution prepared in step (2) to 6 mL of MES buffer, then add 20 mL of the hirudin solution prepared in step (3), the mass ratio of sodium hyaluronate to hirudin is 1:1 , stir to mix well.
（5）向步骤（4）中得到的混合溶液中加入交联剂。(5) Adding a crosslinking agent to the mixed solution obtained in step (4).
选择1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）溶液和N-羟基琥珀酰亚胺（NHS）溶液作为交联剂，即EDC/NHS交联剂。取新鲜配制的50mg/ml的EDC溶液2mL，加入步骤（4）中得到的混合溶液中，水蛭素与EDC质量比例1:5，混合均匀后静置15分钟。然后取新鲜配制的10mg/ml的NHS溶液2mL，加入所述混合溶液，水蛭素与NHS质量比例1:1。在避光条件下均匀混合，12小时候得到透明质酸钠与水蛭素交联混合物，即制得所述抗凝血涂层材料。Choose 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution as the crosslinking agent, that is, EDC/NHS crosslinking agent. Take 2 mL of freshly prepared 50 mg/ml EDC solution, add it to the mixed solution obtained in step (4), the mass ratio of hirudin and EDC is 1:5, mix well and let stand for 15 minutes. Then take 2 mL of freshly prepared 10 mg/ml NHS solution and add to the mixed solution, the mass ratio of hirudin and NHS is 1:1. Mix evenly under the condition of avoiding light, and obtain the cross-linked mixture of sodium hyaluronate and hirudin after 12 hours, that is, the anticoagulant coating material is obtained.
（6）将步骤（5）制得的抗凝血涂层材料进行透析，去除交联剂，从而纯化抗凝血涂层材料。(6) Dialyzing the anticoagulant coating material prepared in step (5) to remove the cross-linking agent, thereby purifying the anticoagulant coating material.
将所述抗凝血涂层材料采用浸凃法涂覆在测试材料聚乙烯（PE）片上，对聚乙烯片做红外光谱检测，观察到1650-1750cm-1（C=O）和1200-1320cm-1（C-N）的酰胺（R1-CO-NH-R2）峰增强，说明透明质酸钠与水蛭素发生酰胺键交联。The anticoagulant coating material was coated on the test material polyethylene (PE) sheet by dip coating method, and the polyethylene sheet was detected by infrared spectroscopy, and it was observed that 1650-1750cm -1 (C=O) and 1200-1320cm The amide (R1-CO-NH-R2) peak of -1 (CN) was enhanced, indicating that sodium hyaluronate and hirudin had amide bond cross-linking.
对所述抗凝血涂层材料进行抗凝血测试：Carry out anticoagulant test to described anticoagulant coating material:
将所述抗凝血涂层材料，采用浸涂法涂覆在聚乙烯片上，将聚乙烯片剪成2cm*2cm的正方形，将新鲜血液离心收集富血小板血浆，接种于涂覆了抗凝血涂层的聚乙烯片上，孵育固定脱水后，通过扫描电子显微镜观察血小板粘附情况，将未修饰水蛭素的透明质酸钠涂层作为对照组，结果表明，修饰水蛭素的透明质酸钠涂层的单位面积血小板粘附数量为未添加水蛭素的透明质酸钠涂层的样品的45%，共价结合水蛭素后提高了涂层的抗凝血能力。The anticoagulant coating material is coated on a polyethylene sheet by dip coating, the polyethylene sheet is cut into a square of 2cm*2cm, the fresh blood is centrifuged to collect platelet-rich plasma, and inoculated on the anticoagulant coated On the coated polyethylene sheet, after incubation, fixation and dehydration, the platelet adhesion was observed by scanning electron microscope, and the unmodified hirudin sodium hyaluronate coating was used as the control group. The results showed that the modified hirudin sodium hyaluronate coating The number of platelet adhesion per unit area of the layer is 45% of that of the sodium hyaluronate coating sample without adding hirudin, and the anticoagulant ability of the coating is improved after covalently binding hirudin.
实施例2Example 2
一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。所述水蛭素与所述透明质酸钠的质量比例为1:3。所述抗凝血涂层材料还包括交联剂，1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）。An anticoagulant coating material comprises sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently bonded to the sodium hyaluronate through an amide bond. The mass ratio of the hirudin to the sodium hyaluronate is 1:3. The anticoagulant coating material also includes a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) .
所述抗凝血涂层材料的制备方法，包括以下步骤：The preparation method of described anticoagulant coating material, comprises the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液，浓度为0.1mol/L，调节pH值至6.4。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution with a concentration of 0.1mol/L and adjust the pH value to 6.4.
（2）配制透明质酸钠溶液。(2) Prepare sodium hyaluronate solution.
取透明质酸钠（HA，分子量约200万）于2-(N-吗啡啉)乙磺酸缓冲溶液（MES，0.1M，pH=6.4）中，使其充分溶解得到透明质酸钠溶液，透明质酸钠溶液浓度为3mg/ml。可采用磁力搅拌加速透明质酸钠溶解，搅拌12小时。制得的透明质酸钠溶液可密封冷冻保存备用。Take sodium hyaluronate (HA, molecular weight about 2 million) in 2-(N-morpholine) ethanesulfonic acid buffer solution (MES, 0.1M, pH=6.4), and dissolve it fully to obtain sodium hyaluronate solution. The concentration of sodium hyaluronate solution is 3mg/ml. Magnetic stirring can be used to accelerate the dissolution of sodium hyaluronate, stirring for 12 hours. The prepared sodium hyaluronate solution can be sealed and frozen for future use.
（3）配制水蛭素溶液。(3) Prepare hirudin solution.
取水蛭素于MES缓冲溶液中，水蛭素浓度为1mg/ml,通过磁力搅拌，使水蛭素均匀分散于MES缓冲液中，加入质量浓度为0.1%的吐温80。制得的水蛭素溶液可密封冷冻保存备用。Take hirudin in MES buffer solution, the concentration of hirudin is 1 mg/ml, and the hirudin is uniformly dispersed in MES buffer solution by magnetic stirring, and Tween 80 with a mass concentration of 0.1% is added. The prepared hirudin solution can be sealed and frozen for future use.
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合。(4) Evenly mix the sodium hyaluronate solution prepared in step (2) and the hirudin solution prepared in step (3).
在6mL MES缓冲液中加入步骤（2）中制得的透明质酸钠溶液20mL，然后加入步骤（3）制得的水蛭素溶液20mL，水蛭素与透明质酸钠的质量比例为1:3，搅拌混合均匀。Add 20 mL of the sodium hyaluronate solution prepared in step (2) to 6 mL of MES buffer, then add 20 mL of the hirudin solution prepared in step (3), the mass ratio of hirudin to sodium hyaluronate is 1:3 , stir to mix well.
（5）向步骤（4）中得到的混合溶液中加入交联剂。(5) Adding a crosslinking agent to the mixed solution obtained in step (4).
选择1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）溶液和N-羟基琥珀酰亚胺（NHS）溶液作为交联剂，即EDC/NHS交联剂。取新鲜配制的50mg/ml的EDC溶液2mL，加入步骤（4）中得到的混合溶液中，水蛭素与EDC质量比例1:5，混合均匀后静置15分钟。然后取新鲜配制的10mg/ml的NHS溶液2mL，加入所述混合溶液，水蛭素与NHS质量比例1:1。在避光条件下均匀混合，12小时候得到透明质酸钠与水蛭素交联混合物，即制得所述抗凝血涂层材料。Choose 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution as the crosslinking agent, that is, EDC/NHS crosslinking agent. Take 2 mL of freshly prepared 50 mg/ml EDC solution, add it to the mixed solution obtained in step (4), the mass ratio of hirudin and EDC is 1:5, mix well and let stand for 15 minutes. Then take 2 mL of freshly prepared 10 mg/ml NHS solution and add to the mixed solution, the mass ratio of hirudin and NHS is 1:1. Mix evenly under the condition of avoiding light, and obtain the cross-linked mixture of sodium hyaluronate and hirudin after 12 hours, that is, the anticoagulant coating material is prepared.
将所述抗凝血涂层材料采用浸凃法涂覆在测试材料聚乙烯（PE）片上，对聚乙烯片做红外光谱检测，观察到1650-1750cm-1（C=O）和1200-1320cm-1（C-N）的酰胺（R1-CO-NH-R2）峰增强，说明透明质酸钠与水蛭素发生酰胺键交联。The anticoagulant coating material was coated on the test material polyethylene (PE) sheet by dip coating method, and the polyethylene sheet was detected by infrared spectroscopy, and it was observed that 1650-1750cm -1 (C=O) and 1200-1320cm The amide (R1-CO-NH-R2) peak of -1 (CN) was enhanced, indicating that sodium hyaluronate and hirudin had amide bond cross-linking.
对所述抗凝血涂层材料进行抗凝血测试：Carry out anticoagulant test to described anticoagulant coating material:
将所述抗凝血涂层材料，采用浸涂法涂覆在聚乙烯片上，将聚乙烯片剪成2cm*2cm的正方形，将新鲜血液离心收集富血小板血浆，接种于涂覆了抗凝血涂层的聚乙烯片上，孵育固定脱水后，通过扫描电子显微镜观察血小板粘附情况，将未修饰水蛭素的透明质酸钠涂层作为对照组，结果表明，修饰水蛭素的透明质酸钠涂层的单位面积血小板粘附数量为未添加水蛭素的透明质酸钠涂层的样品的23%，共价结合水蛭素后提高了涂层的抗凝血能力。The anticoagulant coating material is coated on a polyethylene sheet by dip coating, the polyethylene sheet is cut into a square of 2cm*2cm, the fresh blood is centrifuged to collect platelet-rich plasma, and inoculated on the anticoagulant coated On the coated polyethylene sheet, after incubation, fixation and dehydration, the platelet adhesion was observed by scanning electron microscope, and the unmodified hirudin sodium hyaluronate coating was used as the control group. The results showed that the modified hirudin sodium hyaluronate coating The number of platelet adhesion per unit area of the layer was 23% of that of the sodium hyaluronate coating sample without adding hirudin, and the anticoagulant ability of the coating was improved after covalently binding hirudin.
实施例3Example 3
一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。所述水蛭素与所述透明质酸钠的质量比例为1:4。所述抗凝血涂层材料还包括交联剂，1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）。An anticoagulant coating material comprises sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently bonded to the sodium hyaluronate through an amide bond. The mass ratio of the hirudin to the sodium hyaluronate is 1:4. The anticoagulant coating material also includes a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) .
所述抗凝血涂层材料的制备方法，包括以下步骤：The preparation method of described anticoagulant coating material, comprises the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液，浓度为0.1mol/L，调节pH值至6.4。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution with a concentration of 0.1mol/L and adjust the pH value to 6.4.
（2）配制透明质酸钠溶液。(2) Prepare sodium hyaluronate solution.
取透明质酸钠（HA，分子量约200万）于2-(N-吗啡啉)乙磺酸缓冲溶液（MES，0.1M，pH=6.4）中，使其充分溶解得到透明质酸钠溶液，透明质酸钠溶液浓度为5mg/ml。可采用磁力搅拌加速透明质酸钠溶解，搅拌12小时。制得的透明质酸钠溶液可密封冷冻保存备用。Take sodium hyaluronate (HA, molecular weight about 2 million) in 2-(N-morpholine) ethanesulfonic acid buffer solution (MES, 0.1M, pH=6.4), and dissolve it fully to obtain sodium hyaluronate solution. The concentration of sodium hyaluronate solution is 5mg/ml. Magnetic stirring can be used to accelerate the dissolution of sodium hyaluronate, stirring for 12 hours. The prepared sodium hyaluronate solution can be sealed and frozen for future use.
（3）配制水蛭素溶液。(3) Prepare hirudin solution.
取水蛭素于MES缓冲溶液中，水蛭素浓度为2.5mg/ml,通过磁力搅拌，使水蛭素均匀分散于MES缓冲液中，加入质量浓度为0.5%的吐温80。制得的水蛭素溶液可密封冷冻保存备用。Take hirudin in the MES buffer solution, the concentration of hirudin is 2.5 mg/ml, and the hirudin is uniformly dispersed in the MES buffer solution by magnetic stirring, and Tween 80 with a mass concentration of 0.5% is added. The prepared hirudin solution can be sealed and frozen for future use.
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合。(4) Evenly mix the sodium hyaluronate solution prepared in step (2) and the hirudin solution prepared in step (3).
在6mL MES缓冲液中加入步骤（2）中制得的透明质酸钠溶液8mL，然后加入步骤（3）制得的水蛭素溶液4mL，水蛭素与透明质酸钠的质量比例为1:4，搅拌混合均匀。Add 8 mL of sodium hyaluronate solution prepared in step (2) to 6 mL of MES buffer, then add 4 mL of hirudin solution prepared in step (3), the mass ratio of hirudin to sodium hyaluronate is 1:4 , stir to mix well.
（5）向步骤（4）中得到的混合溶液中加入交联剂。(5) Adding a crosslinking agent to the mixed solution obtained in step (4).
选择1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）溶液和N-羟基琥珀酰亚胺（NHS）溶液作为交联剂，即EDC/NHS交联剂。取新鲜配制的50mg/ml的EDC溶液1mL，加入步骤（4）中得到的混合溶液中，水蛭素与EDC质量比例1:5，混合均匀后静置15分钟。然后取新鲜配制的10mg/ml的NHS溶液1mL，加入所述混合溶液，水蛭素与NHS质量比例1:1。在避光条件下均匀混合，12小时候得到透明质酸钠与水蛭素交联混合物，即制得所述抗凝血涂层材料。Choose 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution as the crosslinking agent, that is, EDC/NHS crosslinking agent. Take 1 mL of freshly prepared 50 mg/ml EDC solution, add it to the mixed solution obtained in step (4), the mass ratio of hirudin and EDC is 1:5, mix well and let stand for 15 minutes. Then take 1 mL of freshly prepared 10 mg/ml NHS solution and add to the mixed solution, the mass ratio of hirudin and NHS is 1:1. Mix evenly under the condition of avoiding light, and obtain the cross-linked mixture of sodium hyaluronate and hirudin after 12 hours, that is, the anticoagulant coating material is obtained.
（6）将步骤（5）制得的抗凝血涂层材料进行透析，去除交联剂，从而纯化抗凝血涂层材料。(6) Dialyzing the anticoagulant coating material prepared in step (5) to remove the cross-linking agent, thereby purifying the anticoagulant coating material.
将所述抗凝血涂层材料采用浸凃法涂覆在测试材料聚乙烯（PE）片上，对聚乙烯片做红外光谱检测，观察到1650-1750cm-1（C=O）和1200-1320cm-1（C-N）的酰胺（R1-CO-NH-R2）峰增强，说明透明质酸钠与水蛭素发生酰胺键交联。The anticoagulant coating material was coated on the test material polyethylene (PE) sheet by dip coating method, and the polyethylene sheet was detected by infrared spectroscopy, and it was observed that 1650-1750cm -1 (C=O) and 1200-1320cm The amide (R1-CO-NH-R2) peak of -1 (CN) was enhanced, indicating that sodium hyaluronate and hirudin had amide bond cross-linking.
对所述抗凝血涂层材料进行抗凝血测试：Carry out anticoagulant test to described anticoagulant coating material:
将所述抗凝血涂层材料，采用浸涂法涂覆在聚乙烯片上，将聚乙烯片剪成2cm*2cm的正方形，将新鲜血液离心收集富血小板血浆，接种于涂覆了抗凝血涂层的聚乙烯片上，孵育固定脱水后，通过扫描电子显微镜观察血小板粘附情况，将未修饰水蛭素的透明质酸钠涂层作为对照组，结果表明，修饰水蛭素的透明质酸钠涂层的单位面积血小板粘附数量为未添加水蛭素的透明质酸钠涂层的样品的25%，共价结合水蛭素后提高了涂层的抗凝血能力。The anticoagulant coating material is coated on a polyethylene sheet by dip coating, the polyethylene sheet is cut into a square of 2cm*2cm, the fresh blood is centrifuged to collect platelet-rich plasma, and inoculated on the anticoagulant coated On the coated polyethylene sheet, after incubation, fixation and dehydration, the platelet adhesion was observed by scanning electron microscope, and the unmodified hirudin sodium hyaluronate coating was used as the control group. The results showed that the modified hirudin sodium hyaluronate coating The number of platelet adhesion per unit area of the layer is 25% of that of the sodium hyaluronate coating sample without adding hirudin, and the anticoagulant ability of the coating is improved after covalently binding hirudin.
实施例4Example 4
一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。所述水蛭素与所述透明质酸钠的质量比例为1:5。所述抗凝血涂层材料还包括交联剂，1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）。An anticoagulant coating material comprises sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently bonded to the sodium hyaluronate through an amide bond. The mass ratio of the hirudin to the sodium hyaluronate is 1:5. The anticoagulant coating material also includes a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) .
所述抗凝血涂层材料的制备方法，包括以下步骤：The preparation method of described anticoagulant coating material, comprises the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液，浓度为0.1mol/L，调节pH值至6.4。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution with a concentration of 0.1mol/L and adjust the pH value to 6.4.
（2）配制透明质酸钠溶液：(2) Preparation of sodium hyaluronate solution:
取透明质酸钠（HA，分子量约200万）于2-(N-吗啡啉)乙磺酸缓冲溶液（MES，0.1M，pH=6.4）中，使其充分溶解得到透明质酸钠溶液，透明质酸钠溶液浓度为10mg/ml。可采用磁力搅拌加速透明质酸钠溶解，搅拌12小时。制得的透明质酸钠溶液可密封冷冻保存备用。Take sodium hyaluronate (HA, molecular weight about 2 million) in 2-(N-morpholine) ethanesulfonic acid buffer solution (MES, 0.1M, pH=6.4), and dissolve it fully to obtain sodium hyaluronate solution. The concentration of sodium hyaluronate solution is 10mg/ml. Magnetic stirring can be used to accelerate the dissolution of sodium hyaluronate, stirring for 12 hours. The prepared sodium hyaluronate solution can be sealed and frozen for future use.
（3）配制水蛭素溶液。(3) Prepare hirudin solution.
取水蛭素于MES缓冲溶液中，水蛭素浓度为5mg/ml,通过磁力搅拌，使水蛭素均匀分散于MES缓冲液中，加入质量浓度为0.5%的吐温80。制得的水蛭素溶液可密封冷冻保存备用。Take hirudin in the MES buffer solution, the concentration of hirudin is 5 mg/ml, and the hirudin is uniformly dispersed in the MES buffer solution by magnetic stirring, and Tween 80 with a mass concentration of 0.5% is added. The prepared hirudin solution can be sealed and frozen for future use.
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合。(4) Evenly mix the sodium hyaluronate solution prepared in step (2) and the hirudin solution prepared in step (3).
在2mL MES缓冲液中加入步骤（2）中制得的透明质酸钠溶液10mL，然后加入步骤（3）制得的水蛭素溶液4mL，水蛭素与透明质酸钠的质量比例为1:5，搅拌混合均匀。Add 10 mL of the sodium hyaluronate solution prepared in step (2) to 2 mL of MES buffer, then add 4 mL of the hirudin solution prepared in step (3), the mass ratio of hirudin to sodium hyaluronate is 1:5 , stir to mix well.
（5）向步骤（4）中得到的混合溶液中加入交联剂。(5) Adding a crosslinking agent to the mixed solution obtained in step (4).
选择1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）溶液和N-羟基琥珀酰亚胺（NHS）溶液作为交联剂，即EDC/NHS交联剂。取新鲜配制的50mg/ml的EDC溶液2mL，加入步骤（4）中得到的混合溶液中，水蛭素与EDC质量比例1:5，混合均匀后静置15分钟。然后取新鲜配制的10mg/ml的NHS溶液2mL，加入所述混合溶液，水蛭素与NHS质量比例1:1。在避光条件下均匀混合，12小时候得到透明质酸钠与水蛭素交联混合物，即制得所述抗凝血涂层材料。Choose 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution as the crosslinking agent, that is, EDC/NHS crosslinking agent. Take 2 mL of freshly prepared 50 mg/ml EDC solution, add it to the mixed solution obtained in step (4), the mass ratio of hirudin and EDC is 1:5, mix well and let stand for 15 minutes. Then take 2 mL of freshly prepared 10 mg/ml NHS solution and add to the mixed solution, the mass ratio of hirudin and NHS is 1:1. Mix evenly under the condition of avoiding light, and obtain the cross-linked mixture of sodium hyaluronate and hirudin after 12 hours, that is, the anticoagulant coating material is prepared.
将所述抗凝血涂层材料采用浸凃法涂覆在测试材料聚乙烯（PE）片上，对聚乙烯片做红外光谱检测，观察到1650-1750cm-1（C=O）和1200-1320cm-1（C-N）的酰胺（R1-CO-NH-R2）峰增强，说明透明质酸钠与水蛭素发生酰胺键交联。The anticoagulant coating material was coated on the test material polyethylene (PE) sheet by dip coating method, and the polyethylene sheet was detected by infrared spectroscopy, and it was observed that 1650-1750cm -1 (C=O) and 1200-1320cm The amide (R1-CO-NH-R2) peak of -1 (CN) was enhanced, indicating that sodium hyaluronate and hirudin had amide bond cross-linking.
对所述抗凝血涂层材料进行抗凝血测试：Carry out anticoagulant test to described anticoagulant coating material:
将所述抗凝血涂层材料，采用浸涂法涂覆在聚乙烯片上，将聚乙烯片剪成2cm*2cm的正方形，将新鲜血液离心收集富血小板血浆，接种于涂覆了抗凝血涂层的聚乙烯片上，孵育固定脱水后，通过扫描电子显微镜观察血小板粘附情况，将未修饰水蛭素的透明质酸钠涂层作为对照组，结果表明，修饰水蛭素的透明质酸钠涂层的单位面积血小板粘附数量为未添加水蛭素的透明质酸钠涂层的样品的30%，共价结合水蛭素后提高了涂层的抗凝血能力。The anticoagulant coating material is coated on a polyethylene sheet by dip coating, the polyethylene sheet is cut into a square of 2cm*2cm, the fresh blood is centrifuged to collect platelet-rich plasma, and inoculated on the anticoagulant coated On the coated polyethylene sheet, after incubation, fixation and dehydration, the platelet adhesion was observed by scanning electron microscope, and the unmodified hirudin sodium hyaluronate coating was used as the control group. The results showed that the modified hirudin sodium hyaluronate coating The number of platelet adhesion per unit area of the layer is 30% of that of the sodium hyaluronate coating sample without adding hirudin, and the anticoagulant ability of the coating is improved after covalently binding hirudin.
实施例5Example 5
一种抗凝血涂层材料，包括透明质酸钠和水蛭素，所述透明质酸钠形成凝胶网络，所述水蛭素通过酰胺键与所述透明质酸钠共价结合。所述水蛭素与所述透明质酸钠的质量比例为1:10。所述抗凝血涂层材料还包括交联剂，1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）。An anticoagulant coating material comprises sodium hyaluronate and hirudin, the sodium hyaluronate forms a gel network, and the hirudin is covalently bonded to the sodium hyaluronate through an amide bond. The mass ratio of the hirudin to the sodium hyaluronate is 1:10. The anticoagulant coating material also includes a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) .
所述抗凝血涂层材料的制备方法，包括以下步骤：The preparation method of described anticoagulant coating material, comprises the following steps:
（1）配制2-(N-吗啡啉)乙磺酸（MES）缓冲溶液，浓度为0.1mol/L，调节pH值至6.4。(1) Prepare 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution with a concentration of 0.1mol/L and adjust the pH value to 6.4.
（2）配制透明质酸钠溶液。(2) Prepare sodium hyaluronate solution.
取透明质酸钠（HA，分子量约200万）于2-(N-吗啡啉)乙磺酸缓冲溶液（MES，0.1M，pH=6.4）中，使其充分溶解得到透明质酸钠溶液，透明质酸钠溶液浓度为20mg/ml。可采用磁力搅拌加速透明质酸钠溶解，搅拌12小时。制得的透明质酸钠溶液可密封冷冻保存备用。Take sodium hyaluronate (HA, molecular weight about 2 million) in 2-(N-morpholine) ethanesulfonic acid buffer solution (MES, 0.1M, pH=6.4), and dissolve it fully to obtain sodium hyaluronate solution. The concentration of sodium hyaluronate solution is 20mg/ml. Magnetic stirring can be used to accelerate the dissolution of sodium hyaluronate, stirring for 12 hours. The prepared sodium hyaluronate solution can be sealed and frozen for future use.
（3）配制水蛭素溶液。(3) Prepare hirudin solution.
取水蛭素于MES缓冲溶液中，水蛭素浓度为10mg/ml,通过磁力搅拌，使水蛭素均匀分散于MES缓冲液中，加入质量浓度为1.0%的吐温80。制得的水蛭素溶液可密封冷冻保存备用。Take hirudin in MES buffer solution, the concentration of hirudin is 10 mg/ml, and the hirudin is uniformly dispersed in MES buffer solution by magnetic stirring, and Tween 80 with a mass concentration of 1.0% is added. The prepared hirudin solution can be sealed and frozen for future use.
（4）将步骤（2）中配制的透明质酸钠溶液和步骤（3）中配制的水蛭素溶液均匀混合。(4) Evenly mix the sodium hyaluronate solution prepared in step (2) and the hirudin solution prepared in step (3).
在4mL MES缓冲液中加入步骤（2）中制得的透明质酸钠溶液10mL，然后加入步骤（3）制得的水蛭素溶液2mL，水蛭素与透明质酸钠的质量比例为1:10，搅拌混合均匀。Add 10 mL of the sodium hyaluronate solution prepared in step (2) to 4 mL of MES buffer, then add 2 mL of the hirudin solution prepared in step (3), the mass ratio of hirudin to sodium hyaluronate is 1:10 , stir to mix well.
（5）向步骤（4）中得到的混合溶液中加入交联剂。(5) Adding a crosslinking agent to the mixed solution obtained in step (4).
选择1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺（EDC）溶液和N-羟基琥珀酰亚胺（NHS）溶液作为交联剂，即EDC/NHS交联剂。取新鲜配制的50mg/ml的EDC溶液2mL，加入步骤（4）中得到的混合溶液中，水蛭素与EDC质量比例1:5，混合均匀后静置15分钟。然后取新鲜配制的10mg/ml的NHS溶液2mL，加入所述混合溶液，水蛭素与NHS质量比例1:1。在避光条件下均匀混合，12小时候得到透明质酸钠与水蛭素交联混合物，即制得所述抗凝血涂层材料。Choose 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution as the crosslinking agent, that is, EDC/NHS crosslinking agent. Take 2 mL of freshly prepared 50 mg/ml EDC solution, add it to the mixed solution obtained in step (4), the mass ratio of hirudin and EDC is 1:5, mix well and let stand for 15 minutes. Then take 2 mL of freshly prepared 10 mg/ml NHS solution and add to the mixed solution, the mass ratio of hirudin and NHS is 1:1. Mix evenly under the condition of avoiding light, and obtain the cross-linked mixture of sodium hyaluronate and hirudin after 12 hours, that is, the anticoagulant coating material is obtained.
将所述抗凝血涂层材料采用浸凃法涂覆在测试材料聚乙烯（PE）片上，对聚乙烯片做红外光谱检测，观察到1650-1750cm-1（C=O）和1200-1320cm-1（C-N）的酰胺（R1-CO-NH-R2）峰增强，说明透明质酸钠与水蛭素发生酰胺键交联。The anticoagulant coating material was coated on the test material polyethylene (PE) sheet by dip coating method, and the polyethylene sheet was detected by infrared spectroscopy, and it was observed that 1650-1750cm -1 (C=O) and 1200-1320cm The amide (R1-CO-NH-R2) peak of -1 (CN) was enhanced, indicating that sodium hyaluronate and hirudin had amide bond cross-linking.
对所述抗凝血涂层材料进行抗凝血测试：Carry out anticoagulant test to described anticoagulant coating material:
将所述抗凝血涂层材料，采用浸涂法涂覆在聚乙烯片上，将聚乙烯片剪成2cm*2cm的正方形，将新鲜血液离心收集富血小板血浆，接种于涂覆了抗凝血涂层的聚乙烯片上，孵育固定脱水后，通过扫描电子显微镜观察血小板粘附情况，将未修饰水蛭素的透明质酸钠涂层作为对照组，结果表明，修饰水蛭素的透明质酸钠涂层的单位面积血小板粘附数量为未添加水蛭素的透明质酸钠涂层的样品的50%，共价结合水蛭素后提高了涂层的抗凝血能力。The anticoagulant coating material is coated on a polyethylene sheet by dip coating, the polyethylene sheet is cut into a square of 2cm*2cm, the fresh blood is centrifuged to collect platelet-rich plasma, and inoculated on the anticoagulant coated On the coated polyethylene sheet, after incubation, fixation and dehydration, the platelet adhesion was observed by scanning electron microscope, and the unmodified hirudin sodium hyaluronate coating was used as the control group. The results showed that the modified hirudin sodium hyaluronate coating The number of platelet adhesion per unit area of the layer is 50% of that of the sodium hyaluronate coating sample without adding hirudin, and the anticoagulant ability of the coating is improved after covalently binding hirudin.
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.
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