CN114632194A - Coating material with long-acting NO catalytic release function, preparation method and application - Google Patents

Abstract

Translated fromChinese

本发明公开了具有长效NO催化释放的涂层材料及制备方法和应用，涉及生物材料技术领域。先利用含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子反应，将一氧化氮催化释放分子通过螯合作用引入，然后再与胺基化合物反应得到改性胺基化合物；利用改性胺基化合物与含羧基的酚类化合物反应得到富酚‑胺缩合化合物，然后将基材置于富酚‑胺缩合化合物溶液中碱性条件下进行沉积得到涂层。该涂层可实现具有可控一氧化氮催化释放的功能，是一种具有高化学稳定性涂层；能够改善现有的酚胺涂层化学稳定性差，提高涂层进一步功能化修饰的效率，并与材料表面具有优异的粘附性能，在极端条件下使用不易变质损坏，具有优异的使用安全性。

The invention discloses a coating material with long-acting NO catalytic release, a preparation method and application, and relates to the technical field of biological materials. First, the tetraaza heterocyclic compound containing carboxyl group or NHS modified is reacted with nitric oxide catalyzed release molecule, the nitric oxide catalyzed release molecule is introduced through chelation, and then reacted with amine compound to obtain modified amine compound; The modified amine compound is reacted with a phenolic compound containing a carboxyl group to obtain a phenol-amine-rich condensation compound, and then the substrate is placed in a solution of the phenol-amine-rich condensation compound for deposition under alkaline conditions to obtain a coating. The coating can realize the function of controllable catalytic release of nitric oxide, and is a coating with high chemical stability; it can improve the poor chemical stability of the existing phenolamine coating, and improve the efficiency of further functional modification of the coating. And it has excellent adhesion performance with the surface of the material, it is not easy to deteriorate and damage under extreme conditions, and has excellent safety in use.

Description

Translated fromChinese

具有长效NO催化释放的涂层材料及制备方法和应用Coating material with long-term NO catalytic release and preparation method and application

技术领域technical field

本发明涉及生物材料技术领域，具体而言，涉及具有长效NO催化释放的涂层材料及制备方法和应用。The present invention relates to the technical field of biological materials, in particular, to a coating material with long-term NO catalytic release, a preparation method and application thereof.

背景技术Background technique

在自然界中，海洋贻贝通过分泌含各种蛋白质的足丝蛋白，能与各种有机和无机材料表面强烈结合。足丝蛋白与表面最初接触时，其中的邻苯二酚基团与界面形成以共价、非共价的相互作用为主，如电荷-电荷相互作用、π-π相互作用、阳离子-π作用、配位作用、氢键作用和疏水效应等。这种依赖于时间尺度的粘附，受到环境中pH值和其他离子等因素的调节。由于这种复杂的酚胺化学反应，使酚的氧化速度和产物得不到很好的调节，多种氧化产物参与涂层的聚合，生成大量的酚类低聚物。尽管能与各种有机和无机表面形成强附着力的黏附涂层，但是其聚合过程中非共价键形成的低聚体使得涂层在偏酸碱条件下的化学稳定性能较差，无法满足其在一些极端条件下的应用。同时，涂层表面保留具有二次反应活性的伯胺基主要由酚类化合物的贡献，远远不能满足涂层表面的再次功能化修饰。In nature, marine mussels can bind strongly to the surface of various organic and inorganic materials by secreting byssin proteins containing various proteins. When the byssin first contacts with the surface, the catechol groups in the interface are mainly formed by covalent and non-covalent interactions, such as charge-charge interaction, π-π interaction, and cation-π interaction. , coordination, hydrogen bonding and hydrophobic effects. This timescale-dependent adhesion is regulated by factors such as pH and other ions in the environment. Due to this complex phenol-amine chemical reaction, the oxidation rate and products of phenol cannot be well regulated, and various oxidation products participate in the polymerization of the coating, resulting in a large number of phenolic oligomers. Although it can form adhesive coatings with strong adhesion with various organic and inorganic surfaces, the oligomers formed by non-covalent bonds during the polymerization process make the coatings have poor chemical stability under acidic and alkaline conditions, which cannot meet the its application under some extreme conditions. At the same time, the primary amine groups with secondary reactivity retained on the surface of the coating are mainly contributed by phenolic compounds, which are far from satisfying the re-functionalization of the coating surface.

体内天然血管中NO由内皮细胞分泌，是维持心血管体内平衡和调节血管舒张的重要因子。NO在天然血管中发挥着促进内皮细胞的黏附增殖、抑制血小板激活和平滑肌增殖的重要作用。近些年来，“金属-酚-胺”表面化学改性策略得到快速的发展，选用具有NO催化活性的Cu²⁺或有机硒类化合物可以实现涂层表面的长期催化释放。NO in natural blood vessels in the body is secreted by endothelial cells and is an important factor in maintaining cardiovascular homeostasis and regulating vasodilation. NO plays an important role in promoting endothelial cell adhesion and proliferation, inhibiting platelet activation and smooth muscle proliferation in native blood vessels. In recent years, the "metal-phenol-amine" surface chemical modification strategy has been rapidly developed, and the long-term catalytic release of the coating surface can be achieved by selecting Cu²⁺ or organoselenium compounds with NO catalytic activity.

但是，目前的NO-催化型材料的实现主要依赖于在涂层表面共价接枝或者通过表面基团螯合具有催化释放NO的活性分子，其释放量受限于表面活性分子接枝量及催化效率，难以满足应用于不同生理环境中的大剂量NO和稳定剂量需求的生理环境。However, the realization of the current NO-catalytic materials mainly relies on covalent grafting on the coating surface or chelating active molecules with catalytic NO release through surface groups, and the release amount is limited by the amount of surface active molecule grafting and Catalytic efficiency, it is difficult to meet the physiological environment that requires large doses of NO and stable doses in different physiological environments.

鉴于此，特提出本发明。In view of this, the present invention is proposed.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供具有长效NO催化释放的涂层材料及制备方法和应用，旨在制备具有一氧化氮催化释放的高化学稳定性涂层。The purpose of the present invention is to provide a coating material with long-term NO catalytic release, a preparation method and application thereof, and to prepare a high chemical stability coating with catalytic release of nitric oxide.

本发明是这样实现的：The present invention is realized in this way:

第一方面，本发明提供一种具有长效NO催化释放的涂层材料的制备方法，包括：利用含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子反应得到螯合有一氧化氮催化释放分子的四氮杂环化合物，将螯合有一氧化氮催化释放分子的四氮杂环化合物与胺基化合物反应得到改性胺基化合物；In a first aspect, the present invention provides a method for preparing a coating material with long-term catalytic release of NO, comprising: reacting a tetraaza heterocyclic compound containing a carboxyl group or an NHS modification with a catalytic release molecule of nitric oxide to obtain chelated monoxide. The tetraaza heterocyclic compound of nitrogen catalyzed release molecule is reacted with the tetraaza heterocyclic compound of chelated nitrogen monoxide catalyzed release molecule and the amine compound to obtain the modified amine compound;

利用含羧基的酚类化合物与改性胺基化合物反应得到富酚-胺缩合化合物；Utilize carboxyl-containing phenolic compounds to react with modified amine-based compounds to obtain rich phenol-amine condensation compounds;

将基材置于富酚-胺缩合化合物的溶液中，在碱性条件下进行涂层沉积。The substrate is placed in a solution rich in a phenol-amine condensation compound, and the coating is deposited under alkaline conditions.

在可选的实施方式中，含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子在0-240℃的条件下超声反应0.1h-3h，离心纯化之后再与胺基化合物在0-240℃的条件下反应0.001h-240h；In an optional embodiment, the carboxyl-containing or NHS-modified tetraaza heterocyclic compound and nitric oxide catalyzed release molecules are subjected to ultrasonic reaction at 0-240°C for 0.1h-3h, and after centrifugal purification 0.001h-240h under the condition of 0-240℃;

优选地，含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子的反应温度为0-30℃，反应时间为0.1h-0.5h；与胺基化合物的反应温度为0-30℃，反应时间为0.01h-24h。Preferably, the reaction temperature of the carboxyl-containing or NHS-modified tetraaza heterocyclic compound and the nitric oxide catalytically released molecule is 0-30°C, and the reaction time is 0.1h-0.5h; the reaction temperature with the amine compound is 0-30°C ℃, the reaction time is 0.01h-24h.

在可选的实施方式中，一氧化氮催化释放分子选自氯化铜、氯化亚铜、溴化铜、溴化亚铜、碘化铜、碘化亚铜、硫酸铜、硒代胱氨酸、胱氨酸、L-硒代胱胺基乙酸和半胱氨酸及其衍生物中的至少一种；In an alternative embodiment, the nitric oxide catalytically releasing molecule is selected from the group consisting of cupric chloride, cuprous chloride, cupric bromide, cuprous bromide, cupric iodide, cuprous iodide, copper sulfate, selenocystamine at least one of acid, cystine, L-selenocystaminoacetic acid and cysteine and derivatives thereof;

优选地，一氧化氮催化释放分子为含有二价铜离子或一价铜离子的化合物，一氧化氮催化释放分子的用量是控制铜离子与四氮杂环的摩尔比为0.01-1：5；优选为0.01-1：2。Preferably, the nitric oxide catalytically released molecules are compounds containing divalent copper ions or monovalent copper ions, and the amount of the nitric oxide catalytically released molecules is controlled to control the molar ratio of copper ions to tetraaza heterocycles to be 0.01-1:5; It is preferably 0.01-1:2.

在可选的实施方式中，胺基化合物选自脂肪类长链胺基化合物和芳香聚胺类化合物中的至少一种；In an optional embodiment, the amine compound is selected from at least one of aliphatic long-chain amine compounds and aromatic polyamine compounds;

优选地，胺基化合物选自聚烯丙胺、聚乙烯胺、多聚赖氨酸、聚乙二醇双(胺)、聚醚胺、聚酰胺、三聚氰胺、聚N-异丙基丙烯酰胺和壳聚糖及其衍生物中至少一种；更优选为聚烯丙胺；Preferably, the amine-based compound is selected from the group consisting of polyallylamine, polyvinylamine, polylysine, polyethylene glycol bis(amine), polyetheramine, polyamide, melamine, polyN-isopropylacrylamide and shell At least one of polysaccharides and derivatives thereof; more preferably polyallylamine;

优选地，胺基化合物的用量是控制羧基与胺基摩尔比为0.001-2:1；优选为0.005-0.02:1。Preferably, the amount of the amine-based compound is to control the molar ratio of carboxyl group to amine group to be 0.001-2:1; preferably 0.005-0.02:1.

在可选的实施方式中，含羧基或NHS修饰的四氮杂环化合物选自1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸、NHS修饰的1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸、三叔丁基1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸和1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸三叔丁酯及其衍生物中的至少一种；优选为1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸；In an optional embodiment, the carboxyl-containing or NHS-modified tetraaza heterocyclic compound is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, NHS-modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1, At least one of 4,7,10-tetraacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester and derivatives thereof; preferably is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

优选地，利用含羧基四氮杂环化合物溶液与pH值为4-10的酸性缓冲溶液混合，与缩合剂在0-240℃的条件下反应0.001h-240h之后再与一氧化氮催化释放分子反应；Preferably, the carboxyl-containing tetraaza heterocyclic compound solution is mixed with an acidic buffer solution with a pH value of 4-10, reacted with a condensing agent at a temperature of 0-240 ° C for 0.001h-240h, and then catalyzed with nitric oxide to release molecules reaction;

优选地，含羧基四氮杂环化合物溶液的浓度为0.01ng/mL-5g/mL，更优选为0.1-1mg/mL。Preferably, the concentration of the carboxyl group-containing tetraazaheterocyclic compound solution is 0.01 ng/mL-5 g/mL, more preferably 0.1-1 mg/mL.

在可选的实施方式中，将含羧基的酚类化合物先与pH值为4-10的缓冲溶液混合，与缩合剂在0-240℃的条件下反应0.001h-240h之后再与改性胺基化合物反应，控制反应温度为0-240℃，反应时间为0.001h-240h；In an optional embodiment, the carboxyl-containing phenolic compound is first mixed with a buffer solution with a pH value of 4-10, reacted with a condensing agent at 0-240°C for 0.001h-240h, and then reacted with the modified amine base compound reaction, control the reaction temperature to be 0-240°C, and the reaction time to be 0.001h-240h;

优选地，与缩合剂的反应温度为0-30℃，反应时间为0.5-48h；与改性胺基化合物的反应温度为0-30℃，反应时间为0.5-48h；Preferably, the reaction temperature with the condensing agent is 0-30°C, and the reaction time is 0.5-48h; the reaction temperature with the modified amine compound is 0-30°C, and the reaction time is 0.5-48h;

优选地，含羧基的酚类化合物的用量是控制羧基与改性胺基化合物中的胺基的摩尔比为0.001-2:1，优选为0.005-0.02:1；Preferably, the dosage of the carboxyl-containing phenolic compound is to control the molar ratio of the carboxyl group to the amine group in the modified amine group compound to be 0.001-2:1, preferably 0.005-0.02:1;

优选地，含羧基的酚类化合物为单胺酚类化合物；更优选地，含羧基的酚类化合物选自咖啡酸、氢化咖啡酸、二羟基苯丙氨酸、没食子酸、单宁酸、阿魏酸、2,3-二羟基苯甲酸和3,4-二羟基苯甲酸及其衍生物中的至少一种。Preferably, the carboxyl-containing phenolic compound is a monoamine phenolic compound; more preferably, the carboxyl-containing phenolic compound is selected from caffeic acid, hydrogenated caffeic acid, dihydroxyphenylalanine, gallic acid, tannic acid, At least one of salicylic acid, 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid and derivatives thereof.

在可选的实施方式中，含羧基四氮杂环化合物和含羧基的酚类化合物与缩合剂反应过程中所采用的缩合剂均选自N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、4-二甲氨基吡啶、碳化二亚胺盐酸盐、1,3-二异丙基碳二亚胺和二环己基碳二亚胺中的至少一种；In an optional embodiment, the condensing agents used in the reaction of the carboxyl-containing tetraaza heterocyclic compound and the carboxyl-containing phenolic compound with the condensing agent are all selected from N-(3-dimethylaminopropyl)-N '-Ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, carbodiimide hydrochloride, 1,3-diisopropylcarbodiimide and bicyclic at least one of hexylcarbodiimide;

优选地，缩合剂的用量是控制缩合剂与羧基的摩尔比为0.1-10:1；Preferably, the consumption of the condensing agent is to control the molar ratio of the condensing agent to the carboxyl group to be 0.1-10:1;

优选地，含羧基四氮杂环化合物和含羧基的酚类化合物与缩合剂反应过程中所采用的缓冲溶液的pH值为5-8；Preferably, the pH value of the buffer solution used in the reaction process of the carboxyl-containing tetraaza heterocyclic compound and the carboxyl-containing phenolic compound and the condensing agent is 5-8;

优选地，缓冲溶液选自2-(N-吗啉代)乙磺酸缓冲液、柠檬酸-氢氧化钠-盐酸缓冲液和磷酸氢二钠-磷酸二氢钠缓冲液中的至少一种。Preferably, the buffer solution is selected from at least one of 2-(N-morpholino)ethanesulfonic acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

在可选的实施方式中，将基材置于富酚-胺缩合化合物的溶液中，加入碱性缓冲液调节pH值为7-14，在0-240℃的条件下反应0.001h-240h；In an optional embodiment, the substrate is placed in a solution rich in phenol-amine condensation compound, an alkaline buffer is added to adjust the pH to 7-14, and the reaction is performed at 0-240°C for 0.001h-240h;

优选地，基材与富酚-胺缩合化合物的反应温度为0-45℃，反应时间为0.1-48h，反应pH值为7-11，反应后进行清洗、干燥；Preferably, the reaction temperature of the substrate and the phenol-rich-amine condensation compound is 0-45° C., the reaction time is 0.1-48 h, the reaction pH is 7-11, and cleaning and drying are performed after the reaction;

优选地，在将基材与富酚-胺缩合化合物反应完成之后进行功能化修饰，功能化修饰是与带有羧基、巯基或NHS化的生物活性分子反应；Preferably, functional modification is performed after the reaction of the substrate with the phenol-rich amine condensation compound is completed, and the functional modification is to react with a bioactive molecule with a carboxyl group, a sulfhydryl group or an NHS compound;

优选地，碱性缓冲液为浓度0.00001g/L-40g/L的氢氧化钠溶液；Preferably, the alkaline buffer is a sodium hydroxide solution with a concentration of 0.00001g/L-40g/L;

优选地，基材选自金属材料、无机材料和高分子材料中的至少一种；Preferably, the base material is selected from at least one of metal materials, inorganic materials and polymer materials;

更优选地，基材选自不锈钢、铁及其合金、镁合金、锌锰合金、钴基合金、钛合金、四氧化三铁纳米粒子、陶瓷、二氧化硅、聚甲基丙烯酸甲酯、聚苯乙烯、硅橡胶和聚氨酯中的至少一种。More preferably, the substrate is selected from stainless steel, iron and its alloys, magnesium alloys, zinc-manganese alloys, cobalt-based alloys, titanium alloys, ferric oxide nanoparticles, ceramics, silica, polymethyl methacrylate, poly At least one of styrene, silicone rubber and polyurethane.

第二方面，本发明提供一种具有长效NO催化释放的涂层材料，通过前述实施方式中任一项的制备方法制备而得。In a second aspect, the present invention provides a coating material with long-term NO catalytic release, which is prepared by the preparation method of any one of the foregoing embodiments.

第三方面，本发明提供前述实施方式的具有长效NO催化释放的涂层材料在制备血液植入器件中的应用。In a third aspect, the present invention provides the application of the coating material with long-term NO catalytic release of the foregoing embodiments in the preparation of blood implantation devices.

本发明具有以下有益效果：先利用含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子反应，将一氧化氮催化释放分子通过螯合作用引入，然后再与胺基化合物反应得到改性胺基化合物；利用改性胺基化合物与含羧基的酚类化合物反应得到富酚-胺缩合化合物，然后将基材置于富酚-胺缩合化合物溶液中碱性条件下进行沉积得到涂层。该涂层可实现具有可控一氧化氮催化释放的功能，是一种具有高化学稳定性涂层；能够改善现有的酚胺涂层化学稳定性差，提高涂层进一步功能化修饰的效率，并与材料表面具有优异的粘附性能，在极端条件下使用不易变质损坏，具有优异的使用安全性。The invention has the following beneficial effects: firstly, the tetraaza heterocyclic compound containing carboxyl group or NHS modification is used to react with nitric oxide catalyzed release molecules, the nitric oxide catalyzed release molecules are introduced through chelation, and then reacted with amine compounds to obtain Modified amine-based compound; the modified amine-based compound is reacted with a carboxyl-containing phenolic compound to obtain a phenol-rich condensate compound, and then the substrate is placed in a solution of the phenol-amine-rich condensate compound for deposition under alkaline conditions to obtain a coating. Floor. The coating can realize the function of controllable catalytic release of nitric oxide, and is a coating with high chemical stability; it can improve the poor chemical stability of the existing phenolamine coating, and improve the efficiency of further functional modification of the coating. And it has excellent adhesion performance with the surface of the material, it is not easy to deteriorate and damage under extreme conditions, and has excellent safety in use.

附图说明Description of drawings

为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

图1为该方法制备的涂层化学稳定性检测结果；Fig. 1 is the coating chemical stability test result prepared by this method;

图2为对比例涂层中一氧化氮催化释放检测结果；Fig. 2 is the detection result of the catalytic release of nitric oxide in the coating of the comparative example;

图3为该方法制备的涂层催化释放一氧化氮检测结果。Fig. 3 is the detection result of catalytically releasing nitric oxide from the coating prepared by this method.

具体实施方式Detailed ways

为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

本发明实施例提供一种具有长效NO催化释放的涂层材料的制备方法，包括以下步骤：An embodiment of the present invention provides a method for preparing a coating material with long-term NO catalytic release, comprising the following steps:

S1、改性胺基化合物的制备S1. Preparation of modified amine compounds

利用含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子反应得到螯合有一氧化氮催化释放分子的四氮杂环化合物，将螯合有一氧化氮催化释放分子的四氮杂环化合物与胺基化合物反应得到改性胺基化合物。Using carboxyl or NHS-modified tetraaza heterocyclic compounds with nitric oxide catalyzed release molecules to obtain tetraaza heterocyclic compounds chelating nitric oxide catalyzing release molecules, and chelating nitric oxide catalyzing release molecules of tetraaza heterocyclic compounds The compound reacts with an amine-based compound to obtain a modified amine-based compound.

需要说明的是，四氮杂环类化合物的选用可以实现Cu²⁺等一氧化氮催化释放分子的高效固定螯合，不仅提升了NO长久稳定的释放效率，还能根据实际应用部位对NO需求而改变催化活性分子接枝量的实现可控的NO催化释放。It should be noted that the selection of tetraaza heterocyclic compounds can realize the efficient immobilization and chelation of nitric oxide catalytically released molecules such as Cu²⁺ , which not only improves the long-term and stable release efficiency of NO, but also can meet the demand for NO according to the actual application site. The controllable NO catalytic release can be achieved by changing the amount of grafted catalytically active molecules.

在一些实施例中，含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子在0-240℃的条件下超声反应0.1h-3h，离心纯化之后再与胺基化合物在0-240℃的条件下反应0.001h-240h；优选地，含羧基或NHS修饰的四氮杂环化合物与一氧化氮催化释放分子的反应温度为0-30℃，反应时间为0.1h-0.5h；与胺基化合物的反应温度为0-30℃，反应时间为0.01h-24h；胺基化合物的用量是控制羧基与胺基摩尔比为0.001-2:1；优选为0.005-0.02:1。将四氮杂环化合物与一氧化氮催化释放分子在超声条件下螯合，进而实现具有NO催化活性分子的固定。随后加入胺基化合物，反应结束后，采用透析方法纯化合成的分子备用。通过对两步反应的反应温度、反应时间和用量比进行优化，以使反应充分，增加铜离子和胺基化合物的引入量。In some embodiments, the carboxyl- or NHS-modified tetraaza heterocyclic compound and nitric oxide catalyzed release molecules are subjected to ultrasonic reaction at 0-240° C. for 0.1h-3h, and after centrifugal purification, they are then reacted with amine compounds at 0-240°C. The reaction is carried out under the condition of 240°C for 0.001h-240h; preferably, the reaction temperature of the carboxyl-containing or NHS-modified tetraaza heterocyclic compound and nitric oxide catalyzed and released molecules is 0-30°C, and the reaction time is 0.1h-0.5h; The reaction temperature with the amine compound is 0-30°C, and the reaction time is 0.01h-24h; the amount of the amine compound is controlled to the molar ratio of carboxyl group to amine group to be 0.001-2:1; preferably 0.005-0.02:1. The tetraaza heterocyclic compound is chelated with nitric oxide catalytically released molecules under ultrasonic conditions, thereby realizing the immobilization of NO catalytically active molecules. Subsequently, an amine-based compound was added, and after the reaction was completed, the synthesized molecule was purified by dialysis for use. By optimizing the reaction temperature, reaction time and dosage ratio of the two-step reaction, the reaction is sufficient and the introduction amount of copper ions and amine compounds is increased.

需要说明的是，在分子合成过程中，分子的浓度、反应温度和时间对于分子的合成效果具有显著影响，一般合成反应在37℃下就能完成，且合成时间不宜过短，反应短于0.5小时并不能得到理想的功能分子。It should be noted that in the process of molecular synthesis, the concentration of the molecule, reaction temperature and time have a significant impact on the synthesis effect of the molecule. Generally, the synthesis reaction can be completed at 37 °C, and the synthesis time should not be too short, and the reaction is shorter than 0.5 Hours do not yield ideal functional molecules.

在一些实施例中，含羧基或NHS修饰的四氮杂环化合物选自1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(DOTA)、NHS修饰的1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(NHS-DOTA)、三叔丁基1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸和1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸三叔丁酯及其衍生物中的至少一种；优选为1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸。上述几种固定NO催化活性的功能分子为较为常见和易得，本发明实施例所指的四氮杂环化合物并不仅限于上述几种。In some embodiments, the carboxyl-containing or NHS-modified tetraazacyclic compound is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), NHS Modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (NHS-DOTA), tri-tert-butyl 1,4,7,10-tetraazacyclo In dodecane-1,4,7,10-tetraacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester and its derivatives At least one of ; preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. The above-mentioned several functional molecules for fixing NO catalytic activity are relatively common and readily available, and the tetraaza heterocyclic compounds referred to in the embodiments of the present invention are not limited to the above-mentioned types.

在一些实施例中，一氧化氮催化释放分子选自氯化铜、氯化亚铜、溴化铜、溴化亚铜、碘化铜、碘化亚铜、硫酸铜、硒代胱氨酸、胱氨酸、L-硒代胱胺基乙酸和半胱氨酸及其衍生物中的至少一种，可以为一种或几种。上述几种NO催化活性物质为较为常见和易得，本发明实施例所指的一氧化氮催化释放分子并不仅限于上述几种。In some embodiments, the nitric oxide catalytically releasing molecule is selected from the group consisting of cupric chloride, cuprous chloride, cupric bromide, cuprous bromide, cupric iodide, cuprous iodide, copper sulfate, selenocystine, At least one of cystine, L-selenocystaminoacetic acid, cysteine and derivatives thereof may be one or more. The above-mentioned several kinds of NO catalytically active substances are relatively common and readily available, and the nitric oxide catalytically released molecules referred to in the embodiments of the present invention are not limited to the above-mentioned ones.

在优选的实施例中，一氧化氮催化释放分子为含有二价铜离子或一价铜离子的化合物，一氧化氮催化释放分子的用量是控制铜离子与四氮杂环的摩尔比为0.01-1：5；优选为0.01-1：2。通过对一氧化氮催化释放分子进行优选，有利于提升所制备得到涂层材料的NO释放的稳定性。In a preferred embodiment, the nitric oxide catalytically released molecules are compounds containing divalent copper ions or monovalent copper ions, and the amount of the nitric oxide catalytically released molecules is controlled to control the molar ratio of copper ions to tetraaza heterocycles to be 0.01- 1:5; preferably 0.01-1:2. By optimizing the catalytic release molecules of nitric oxide, it is beneficial to improve the stability of NO release of the prepared coating material.

在一些实施例中，胺基化合物选自脂肪类长链胺基化合物和芳香聚胺类化合物中的至少一种，可以为一种或几种。优选地，胺基化合物选自聚烯丙胺、聚乙烯胺、多聚赖氨酸、聚乙二醇双(胺)、聚醚胺、聚酰胺、三聚氰胺、聚N-异丙基丙烯酰胺和壳聚糖及其衍生物中至少一种；更优选为聚烯丙胺。通过对胺基化合物进行优选，有利于提升胺基的引入量，便于后续进一步引入其他功能分子。In some embodiments, the amine-based compound is selected from at least one of aliphatic long-chain amine-based compounds and aromatic polyamine-based compounds, and may be one or more of them. Preferably, the amine-based compound is selected from the group consisting of polyallylamine, polyvinylamine, polylysine, polyethylene glycol bis(amine), polyetheramine, polyamide, melamine, polyN-isopropylacrylamide and shell At least one of polysaccharides and derivatives thereof; more preferably polyallylamine. By optimizing the amine-based compound, it is beneficial to increase the amount of amine-based introduction and facilitate the subsequent introduction of other functional molecules.

若采用的四氮杂环化合物没有NHS的修饰仅含有羧基，则需要进行羧基活化之后再与一氧化氮催化释放分子反应。在实际操作过程中，利用含羧基四氮杂环化合物溶液与pH值为4-10的酸性缓冲溶液混合，与缩合剂在0-240℃的条件下反应0.001h-240h之后再与一氧化氮催化释放分子反应；含羧基四氮杂环化合物溶液的浓度为0.01ng/mL-5g/mL。If the tetraaza heterocyclic compound used has no NHS modification and only contains a carboxyl group, it is necessary to activate the carboxyl group and then react with nitric oxide to catalyze the release of the molecule. In the actual operation process, the carboxyl-containing tetraaza heterocyclic compound solution is mixed with an acidic buffer solution with a pH value of 4-10, reacted with a condensing agent at 0-240 ° C for 0.001h-240h, and then reacted with nitric oxide. Catalytic release molecular reaction; the concentration of carboxyl-containing tetraaza heterocyclic compound solution is 0.01ng/mL-5g/mL.

在优选的实施例中，含羧基四氮杂环化合物溶液的浓度为0.1-1mg/mL，反应过程中所采用的缓冲溶液的pH值为5-8，缩合剂的用量是控制缩合剂与羧基的摩尔比为0.1-10:1，以达到充分活化的效果，增加胺基的引入量。In a preferred embodiment, the concentration of the carboxyl-containing tetraaza heterocyclic compound solution is 0.1-1 mg/mL, the pH value of the buffer solution used in the reaction process is 5-8, and the consumption of the condensing agent is to control the condensing agent and the carboxyl group. The molar ratio of amine group is 0.1-10:1, in order to achieve the effect of full activation and increase the introduction of amine groups.

在一些实施例中，含羧基四氮杂环化合物和含羧基的酚类化合物与缩合剂反应过程中所采用的缩合剂均选自N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS)、4-二甲氨基吡啶(DMAP)、碳化二亚胺盐酸盐(EDCI)、1,3-二异丙基碳二亚胺(DIC)和二环己基碳二亚胺(DCC)中的至少一种，可以为一种或几种。优选为EDC和NHS。In some embodiments, the condensing agent used in the reaction of the carboxyl-containing tetraaza heterocyclic compound and the carboxyl-containing phenolic compound with the condensing agent is selected from N-(3-dimethylaminopropyl)-N'- Ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), carbodiimide hydrochloride (EDCI), 1,3-di At least one of isopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) may be one or more. Preferred are EDC and NHS.

在一些实施例中，缓冲溶液选自2-(N-吗啉代)乙磺酸(MES)缓冲液、柠檬酸-氢氧化钠-盐酸缓冲液和磷酸氢二钠-磷酸二氢钠缓冲液中的至少一种，优选为MES缓冲溶液。In some embodiments, the buffer solution is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer At least one of them is preferably MES buffer solution.

在优选的实施例中，改性胺基化合物可以为大环多胺螯合铜离子改性的聚烯丙胺(DOTA-Cu/pPAM)、NHS-修饰后的大环多胺螯合铜离子改性的聚烯丙胺(DOTA-Cu/pPAM)及其衍生物。In a preferred embodiment, the modified amine compound can be polyallylamine modified by macrocyclic polyamine chelated copper ion (DOTA-Cu/pPAM), NHS-modified macrocyclic polyamine chelated copper ion modified Polyallylamine (DOTA-Cu/pPAM) and its derivatives.

S2、富酚-胺缩合化合物的制备S2, the preparation of rich phenol-amine condensation compound

利用含羧基的酚类化合物与改性胺基化合物反应得到富酚-胺缩合化合物，通过羧基酚类与胺基化合物的缩合增加了酚羟基与基底界面的结合效率，含碳量较多的氨基化合物的引入，也增加了涂层的交联聚合，使其具有更加优异的化学耐受性，能适用极酸、极碱和强氧化性环境。The phenolic compounds containing carboxyl groups are reacted with modified amine compounds to obtain phenol-rich condensed compounds. The condensation of carboxyl phenols and amine compounds increases the binding efficiency between the phenolic hydroxyl and the substrate interface. The introduction of the compound also increases the cross-linking polymerization of the coating, making it more excellent in chemical resistance and suitable for extremely acidic, extremely alkaline and strong oxidizing environments.

在实际操作过程中，将含羧基的酚类化合物先与pH值为4-10的缓冲溶液混合，与缩合剂在0-240℃的条件下反应0.001h-240h之后再与改性胺基化合物反应，控制反应温度为0-240℃，反应时间为0.001h-240h。含羧基的酚类化合物的用量是控制羧基与改性胺基化合物中的胺基的摩尔比为0.001-2:1。控制范围温度、时间和原料用量在上述范围内均能够完成反应。In the actual operation, the carboxyl-containing phenolic compound is first mixed with a buffer solution with a pH value of 4-10, reacted with a condensing agent at 0-240°C for 0.001h-240h, and then reacted with the modified amine compound For the reaction, the reaction temperature is controlled to be 0-240°C, and the reaction time is 0.001h-240h. The dosage of the phenolic compound containing carboxyl group is to control the molar ratio of the carboxyl group to the amine group in the modified amine group compound to be 0.001-2:1. The reaction can be completed by controlling the temperature, time and raw material dosage within the above-mentioned ranges.

在优选的实施例中，与缩合剂的反应温度为0-30℃，反应时间为0.5-48h；与改性胺基化合物的反应温度为0-30℃，反应时间为0.5-48h，反应完成之后采用透析方法在无氧水中纯化合成的分子备用。含羧基的酚类化合物的用量是控制羧基与改性胺基化合物中的胺基的摩尔比为0.005-0.02:1。通过对反应温度、时间和原料的用量比进行优化，以使反应更充分进行。In a preferred embodiment, the reaction temperature with the condensing agent is 0-30°C, and the reaction time is 0.5-48h; the reaction temperature with the modified amine compound is 0-30°C, the reaction time is 0.5-48h, and the reaction is completed. The synthesized molecules were then purified in deoxygenated water by dialysis. The dosage of the carboxyl-containing phenolic compound is to control the molar ratio of the carboxyl group to the amine group in the modified amine group compound to be 0.005-0.02:1. By optimizing the reaction temperature, time and raw material dosage ratio, the reaction can be carried out more fully.

需要说明的是，在富酚-胺缩合反应过程中，分子的浓度、溶液中氧气含量、反应温度和时间对于分子合成效果具有显著影响，一般在25℃下就能合成，合成时间不宜过短，反应时间短于0.5小时并不能得到理想的分子。It should be noted that in the process of phenol-rich-amine condensation reaction, the concentration of the molecule, the oxygen content in the solution, the reaction temperature and the time have a significant impact on the molecular synthesis effect. Generally, it can be synthesized at 25 °C, and the synthesis time should not be too short. , the reaction time is shorter than 0.5 hours and the ideal molecule cannot be obtained.

在一些实施例中，含羧基的酚类化合物可以为单胺酚类化合物，具体种类不限。具体地，含羧基的酚类化合物选自咖啡酸、氢化咖啡酸、二羟基苯丙氨酸、没食子酸、单宁酸、阿魏酸、2,3-二羟基苯甲酸和3,4-二羟基苯甲酸及其衍生物中的至少一种，可以为一种或几种。上述几种含羧基酚类化合物为较为常见和易得，本发明实施例所指的含羧基酚类化合物并不仅限于上述几种。In some embodiments, the carboxyl group-containing phenolic compound may be a monoamine phenolic compound, and the specific type is not limited. Specifically, the carboxyl-containing phenolic compound is selected from caffeic acid, hydrogenated caffeic acid, dihydroxyphenylalanine, gallic acid, tannic acid, ferulic acid, 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid At least one of hydroxybenzoic acid and its derivatives may be one or more. The above-mentioned several carboxyl-containing phenolic compounds are relatively common and readily available, and the carboxyl-containing phenolic compounds referred to in the embodiments of the present invention are not limited to the above-mentioned types.

与步骤S1类似，含羧基的酚类化合物与缩合剂反应过程中所采用的缩合剂选自N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、4-二甲氨基吡啶、碳化二亚胺盐酸盐、1,3-二异丙基碳二亚胺和二环己基碳二亚胺中的至少一种；可以为一种或几种，优选为EDC和NHS。缩合剂的用量是控制缩合剂与羧基的摩尔比为0.1-10:1为宜。优选地，含羧基四氮杂环化合物和含羧基的酚类化合物与缩合剂反应过程中所采用的缓冲溶液的pH值为5-8；缓冲溶液选自2-(N-吗啉代)乙磺酸(MES)缓冲液、柠檬酸-氢氧化钠-盐酸缓冲液和磷酸氢二钠-磷酸二氢钠缓冲液中的至少一种。Similar to step S1, the condensing agent used in the reaction process of the carboxyl-containing phenolic compound and the condensing agent is selected from N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, At least one of N-hydroxysuccinimide, 4-dimethylaminopyridine, carbodiimide hydrochloride, 1,3-diisopropylcarbodiimide and dicyclohexylcarbodiimide; may One or more, preferably EDC and NHS. The dosage of the condensing agent is to control the molar ratio of the condensing agent to the carboxyl group to be 0.1-10:1. Preferably, the pH value of the buffer solution used during the reaction of the carboxyl-containing tetraaza heterocyclic compound and the carboxyl-containing phenolic compound with the condensing agent is 5-8; the buffer solution is selected from 2-(N-morpholino)ethyl At least one of a sulfonic acid (MES) buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, and a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

S3、涂层沉积S3, coating deposition

将基材置于富酚-胺缩合化合物的溶液中，在碱性条件下进行涂层沉积，获得一种持续催化稳定释放一氧化氮以及较高化学稳定性、粘附性能强的涂层。The substrate is placed in a solution rich in a phenol-amine condensation compound, and the coating is deposited under alkaline conditions to obtain a coating with continuous catalytic and stable release of nitric oxide, high chemical stability and strong adhesion.

在实际操作过程中，将基材置于富酚-胺缩合化合物的溶液中，加入碱性缓冲液调节pH值为7-14，在0-240℃的条件下反应0.001h-240h；优选地，基材与富酚-胺缩合化合物的反应温度为0-45℃，反应时间为0.1-48h，反应pH值为7-11，反应后进行清洗、干燥。通过对反应温度、时间和pH值进行优化以保证基材上的沉积量。In the actual operation process, the substrate is placed in a solution rich in phenol-amine condensation compound, an alkaline buffer is added to adjust the pH to 7-14, and the reaction is carried out at 0-240°C for 0.001h-240h; preferably , the reaction temperature of the substrate and the phenol-rich-amine condensation compound is 0-45° C., the reaction time is 0.1-48h, the reaction pH is 7-11, and the reaction is washed and dried after the reaction. Reaction temperature, time and pH are optimized to ensure deposition on the substrate.

基材的种类不限，可以根据具体的需求进行选择，如可以为金属材料、无机材料和高分子材料等。具体而言，基材可以选自不锈钢、铁及其合金、镁合金、锌锰合金、钴基合金、钛合金、四氧化三铁纳米粒子、陶瓷、二氧化硅、聚甲基丙烯酸甲酯、聚苯乙烯、硅橡胶和聚氨酯中的至少一种，可以为一种或几种。以上几种材料适合适用于邻酚类聚合改性层的制备，且广泛用于生物材料的制备。The types of substrates are not limited, and can be selected according to specific needs, such as metal materials, inorganic materials, and polymer materials. Specifically, the substrate can be selected from stainless steel, iron and its alloys, magnesium alloys, zinc-manganese alloys, cobalt-based alloys, titanium alloys, iron tetroxide nanoparticles, ceramics, silica, polymethyl methacrylate, At least one of polystyrene, silicone rubber and polyurethane may be one or more. The above materials are suitable for the preparation of ortho-phenolic polymer modified layers, and are widely used in the preparation of biological materials.

在一些实施例中，碱性缓冲液为浓度0.00001g/L-40g/L的氢氧化钠溶液，也可以为其他碱性溶液。In some embodiments, the alkaline buffer is a sodium hydroxide solution with a concentration of 0.00001 g/L-40 g/L, or other alkaline solutions.

由于涂层表面保留丰富的伯胺基使其具有广泛的二次反应接枝功能基团的反应特性，赋予材料具有多重的生物功能应用前景。在一些实施例中，在将基材与富酚-胺缩合化合物反应完成之后进行功能化修饰，功能化修饰是与带有羧基、巯基或NHS化的生物活性分子反应，如肝素、比伐芦定、透明质酸等。Due to the abundant primary amine groups retained on the surface of the coating, it has a wide range of reactive characteristics of secondary reaction grafting functional groups, which endows the material with multiple biological functional application prospects. In some embodiments, functional modification is performed after the reaction of the substrate with the phenol-rich amine-rich condensate compound, and the functional modification is to react with a bioactive molecule with a carboxyl group, a sulfhydryl group or an NHS compound, such as heparin, bivalirru Ding, hyaluronic acid, etc.

本发明实施例提供一种具有长效NO催化释放的涂层材料，通过上述制备方法制备而得，是一种持续催化稳定释放一氧化氮以及较高化学稳定性、粘附性能强的涂层，可以在制备血液植入器件中得到应用。The embodiment of the present invention provides a coating material with long-term NO catalytic release, which is prepared by the above preparation method, and is a coating with continuous catalytic and stable release of nitric oxide, high chemical stability and strong adhesion performance. , which can be used in the preparation of blood implanted devices.

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。The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

实施例1Example 1

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL含羧基的DOTA溶液与pH调至5.6的MES酸性缓冲溶液混合，加入EDC和NHS(EDC与羧基的摩尔比为5:1，NHS与羧基摩尔比为1:1，下同)，在37℃下反应0.5h后，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.01：1的聚烯丙胺(重均分子量8,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL carboxyl-containing DOTA solution with MES acid buffer solution with pH adjusted to 5.6, and add EDC and NHS (the molar ratio of EDC and carboxyl groups is 5:1, and the molar ratio of NHS and carboxyl groups is 1:1, the same below) , after reacting for 0.5h at 37°C, adding CuCl₂ with a molar ratio of DOTA 1:1, ultrasonically reacting for 0.2h, centrifuging for purification, and then adding polyallylamine (weight average) with a molar ratio of carboxyl and amine groups of 0.01:1 Molecular weight 8,000, 20wt.% aqueous solution), after reacting at 37°C for 24h, the synthesized molecule (DOTA-Cu/pPAM) was purified by dialysis method for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL氢化咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS(EDC与羧基的摩尔比为5:1，NHS与羧基摩尔比为1:1，下同)，在37℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.01：1的DOTA-Cu/pPAM，在10℃下反应24h后采用透析方法在无氧水中纯化合成的分子备用。Dissolve 1 mg/mL hydrogenated caffeic acid in a completely anaerobic MES buffer solution with pH adjusted to 5.6, add EDC and NHS (the molar ratio of EDC to carboxyl group is 5:1, the molar ratio of NHS to carboxyl group is 1:1, and the following The same), after reacting at 37 °C for 0.5 h, DOTA-Cu/pPAM with a molar ratio of carboxyl group to DOTA-Cu/pPAM amine group of 0.01:1 was added, and the reaction was performed at 10 °C for 24 h by dialysis in oxygen-free water. The synthesized molecule was purified for use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified was placed in the molecular solution obtained in step B, an alkaline anaerobic buffer was added to adjust the pH to 9, the reaction was carried out at 37° C. for 24 hours, and the coating was obtained after washing and drying.

实施例2Example 2

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL含4羧基的DOTA的溶液与pH调至5.6的MES酸性缓冲溶液混合，加入EDC和NHS，在37℃下反应0.5h后，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.1h，离心纯化，随后加入羧基与胺基摩尔比为0.01：1的聚烯丙胺(重均分子量8,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL solution of DOTA containing 4 carboxyl groups with MES acid buffer solution adjusted to pH 5.6, add EDC and NHS, react at 37 °C for 0.5 h, add CuCl₂ with a molar ratio of 1:1 to DOTA, Ultrasonic reaction for 0.1h, centrifugation for purification, then polyallylamine (weight average molecular weight 8,000, 20wt.% aqueous solution) with a molar ratio of carboxyl group to amine group of 0.01:1 was added. Molecules (DOTA-Cu/pPAM) are ready for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在37℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.01：1的DOTA-Cu/pPAM，在4℃下反应24h后采用透析方法在无氧水中纯化合成的分子备用。1 mg/mL caffeic acid was dissolved in a completely anaerobic MES buffer solution with pH adjusted to 5.6, EDC and NHS were added, and after reacting at 37 °C for 0.5 h, the molar ratio of the carboxyl group to the DOTA-Cu/pPAM amine group was: 0.01:1 DOTA-Cu/pPAM was reacted at 4 °C for 24 h and the synthesized molecules were purified by dialysis in deoxygenated water for later use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified was placed in the molecular solution obtained in step B, an alkaline anaerobic buffer was added to adjust the pH to 9, the reaction was carried out at 37° C. for 24 hours, and the coating was obtained after washing and drying.

实施例3Example 3

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL含羧基的DOTA溶液与pH调至5.6的MES酸性缓冲溶液混合，加入EDC和NHS，在37℃下反应0.5h后，加入与DOTA摩尔比为1：1的CuCl2，超声反应0.5h，离心纯化，随后加入羧基与胺基摩尔比为0.01：1的聚烯丙胺(重均分子量8,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL carboxyl-containing DOTA solution with MES acid buffer solution adjusted to pH 5.6, add EDC and NHS, react at 37 °C for 0.5 h, add CuCl2 with a molar ratio of 1:1 to DOTA, and ultrasonically react 0.5 h, centrifuged for purification, then polyallylamine (weight-average molecular weight 8,000, 20 wt.% aqueous solution) with a molar ratio of carboxyl group to amine group of 0.01:1 was added, and after reacting at 37 °C for 24 h, the synthesized molecule (DOTA) was purified by dialysis -Cu/pPAM) for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL 2,3-二羟基苯甲酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在25℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.01：1的DOTA-Cu/pPAM，在10℃下反应24h后采用透析方法在无氧水中纯化合成的分子备用。Dissolve 1 mg/mL 2,3-dihydroxybenzoic acid in a completely anaerobic MES buffer solution with pH adjusted to 5.6, add EDC and NHS, react at 25 °C for 0.5 h, add carboxyl and DOTA-Cu/pPAM The DOTA-Cu/pPAM with an amine molar ratio of 0.01:1 was reacted at 10 °C for 24 h and the synthesized molecules were purified by dialysis in deoxygenated water for later use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified was placed in the molecular solution obtained in step B, an alkaline anaerobic buffer was added to adjust the pH to 9, the reaction was carried out at 37° C. for 24 hours, and the coating was obtained after washing and drying.

实施例4Example 4

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将2mg/mL含羧基的DOTA溶液与pH调至5.6的MES酸性缓冲溶液混合，加入EDC和NHS，在37℃下反应0.5h后，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.01：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 2 mg/mL carboxyl-containing DOTA solution with MES acid buffer solution adjusted to pH 5.6, add EDC and NHS, react at 37 °C for 0.5 h, add CuCl₂ with a molar ratio of 1:1 to DOTA, and react by ultrasonic 0.2h, centrifuged for purification, then polyallylamine (weight-average molecular weight 17,000, 20wt.% aqueous solution) with a molar ratio of carboxyl groups to amine groups was added to 0.01:1, and after reacting at 37°C for 24h, the synthesized molecules were purified by dialysis ( DOTA-Cu/pPAM) for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL 2,3-二羟基苯甲酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在25℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.01：1的DOTA-Cu/pPAM，在25℃下反应24h后采用透析方法在无氧水中纯化合成的分子备用。Dissolve 1 mg/mL 2,3-dihydroxybenzoic acid in a completely anaerobic MES buffer solution with pH adjusted to 5.6, add EDC and NHS, react at 25 °C for 0.5 h, add carboxyl and DOTA-Cu/pPAM The DOTA-Cu/pPAM with an amine molar ratio of 0.01:1 was reacted at 25 °C for 24 h and the synthesized molecules were purified by dialysis in deoxygenated water for later use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified was placed in the molecular solution obtained in step B, an alkaline anaerobic buffer was added to adjust the pH to 9, the reaction was carried out at 37° C. for 24 hours, and the coating was obtained after washing and drying.

实施例5Example 5

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将2mg/mL含羧基的DOTA溶液与pH调至5.6的MES酸性缓冲溶液混合，加入EDC和NHS，在37℃下反应0.5h后，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.1h，离心纯化，随后加入羧基与胺基摩尔比为0.01：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 2 mg/mL carboxyl-containing DOTA solution with MES acid buffer solution adjusted to pH 5.6, add EDC and NHS, react at 37 °C for 0.5 h, add CuCl₂ with a molar ratio of 1:1 to DOTA, and react by ultrasonic 0.1h, centrifuged for purification, then polyallylamine (weight-average molecular weight 17,000, 20wt.% aqueous solution) with a molar ratio of carboxyl groups to amine groups was added, and after reacting at 37 °C for 24h, the synthesized molecules were purified by dialysis ( DOTA-Cu/pPAM) for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL氢化咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在25℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.01：1的DOTA-Cu/pPAM，在4℃下反应12h后采用透析方法在无氧水中纯化合成的分子备用。Dissolve 1 mg/mL hydrogenated caffeic acid in a completely anaerobic MES buffer solution with pH adjusted to 5.6, add EDC and NHS, and react at 25 °C for 0.5 h, then add the molar ratio of carboxyl group to DOTA-Cu/pPAM amine group DOTA-Cu/pPAM of 0.01:1, reacted at 4 °C for 12 h, and purified the synthesized molecule in deoxygenated water by dialysis method for use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified is placed in the molecular solution obtained in step B, an alkaline anaerobic buffer is added to adjust the pH to 9, the reaction is carried out at 37° C. for 24 hours, and the coating is obtained after washing and drying.

实施例6Example 6

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL含羧基的DOTA溶液与pH调至5.6的MES酸性缓冲溶液混合，加入EDC和NHS，在37℃下反应0.5h后，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.19：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL carboxyl-containing DOTA solution with MES acid buffer solution adjusted to pH 5.6, add EDC and NHS, react at 37 °C for 0.5 h, add CuCl₂ with a molar ratio of 1:1 to DOTA, and perform ultrasonic reaction 0.2h, centrifuged for purification, then polyallylamine (weight-average molecular weight 17,000, 20wt.% aqueous solution) with a molar ratio of carboxyl groups to amine groups of 0.19:1 was added, and after reacting at 37 °C for 24h, the synthesized molecules were purified by dialysis ( DOTA-Cu/pPAM) for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在37℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.01：1的DOTA-Cu/pPAM，在10℃下反应12h后采用透析方法在无氧水中纯化合成的分子备用。1 mg/mL caffeic acid was dissolved in a completely anaerobic MES buffer solution with pH adjusted to 5.6, EDC and NHS were added, and after reacting at 37 °C for 0.5 h, the molar ratio of the carboxyl group to the DOTA-Cu/pPAM amine group was: The DOTA-Cu/pPAM of 0.01:1 was reacted at 10 °C for 12 h and the synthesized molecules were purified by dialysis in deoxygenated water for use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在25℃下反应12h，清洗干燥后得到所述涂层。The sample to be modified is placed in the molecular solution obtained in step B, an alkaline anaerobic buffer is added to adjust the pH to 9, the reaction is carried out at 25° C. for 12 h, and the coating is obtained after washing and drying.

实施例7Example 7

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL NHS-DOTA溶液与pH调至8.5的PBS缓冲溶液混合中，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.2：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL NHS-DOTA solution with PBS buffer solution whose pH was adjusted to 8.5, add CuCl₂ with a molar ratio of 1:1 to DOTA, ultrasonically react for 0.2 h, purify by centrifugation, and then add a molar ratio of carboxyl group to amine group of 0.2:1 polyallylamine (weight-average molecular weight 17,000, 20 wt.% aqueous solution) was reacted at 37° C. for 24 h, and the synthesized molecule (DOTA-Cu/pPAM) was purified by dialysis method for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将2.3mg/mL氢化咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在37℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.19：1的DOTA-Cu/pPAM，在10℃下反应12h后采用透析方法在无氧水中纯化合成的分子备用。Dissolve 2.3 mg/mL hydrogenated caffeic acid in a completely anaerobic MES buffer solution with pH adjusted to 5.6, add EDC and NHS, react at 37 °C for 0.5 h, add carboxyl and DOTA-Cu/pPAM amino molar DOTA-Cu/pPAM with a ratio of 0.19:1 was reacted at 10 °C for 12 h and the synthesized molecules were purified by dialysis in deoxygenated water for use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应12h，清洗干燥后得到所述涂层。The sample to be modified is placed in the molecular solution obtained in step B, an alkaline anaerobic buffer is added to adjust the pH to 9, the reaction is carried out at 37° C. for 12 h, and the coating is obtained after washing and drying.

实施例8Example 8

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL NHS-DOTA溶液与pH调至8.5的PBS缓冲溶液混合，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.1：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL NHS-DOTA solution with PBS buffer solution with pH adjusted to 8.5, add CuCl₂ with a molar ratio of 1:1 to DOTA, ultrasonically react for 0.2 h, centrifuge for purification, and then add carboxyl to amine molar ratio of 0.1 : 1 of polyallylamine (weight average molecular weight 17,000, 20 wt.% aqueous solution), after reacting at 37° C. for 24 h, the synthesized molecule (DOTA-Cu/pPAM) was purified by dialysis method for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在37℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.19：1的DOTA-Cu/pPAM，在4℃下反应36h后采用透析方法在无氧水中纯化合成的分子备用。1 mg/mL caffeic acid was dissolved in a completely anaerobic MES buffer solution with pH adjusted to 5.6, EDC and NHS were added, and after reacting at 37 °C for 0.5 h, the molar ratio of the carboxyl group to the DOTA-Cu/pPAM amine group was: DOTA-Cu/pPAM of 0.19:1 was reacted at 4 °C for 36 h, and the synthesized molecules were purified by dialysis in deoxygenated water for use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在25℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified is placed in the molecular solution obtained in step B, an alkaline anaerobic buffer is added to adjust the pH to 9, the reaction is carried out at 25° C. for 24 hours, and the coating is obtained after washing and drying.

实施例9Example 9

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用316L不锈钢作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, and selects 316L stainless steel as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL NHS-DOTA溶液与pH调至8.5的PBS缓冲溶液混合，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.5：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL NHS-DOTA solution with PBS buffer solution with pH adjusted to 8.5, add CuCl₂ with a molar ratio of 1:1 to DOTA, ultrasonically react for 0.2 h, centrifuge for purification, and then add carboxyl to amine molar ratio of 0.5 : 1 of polyallylamine (weight average molecular weight 17,000, 20 wt.% aqueous solution), after reacting at 37° C. for 24 h, the synthesized molecule (DOTA-Cu/pPAM) was purified by dialysis method for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL 2,3-二羟基苯甲酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在25℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.3：1的DOTA-Cu/pPAM，在4℃下反应24h后采用透析方法在无氧水中纯化合成的分子备用。Dissolve 1 mg/mL 2,3-dihydroxybenzoic acid in a completely anaerobic MES buffer solution with pH adjusted to 5.6, add EDC and NHS, react at 25 °C for 0.5 h, add carboxyl and DOTA-Cu/pPAM The DOTA-Cu/pPAM with an amine molar ratio of 0.3:1 was reacted at 4 °C for 24 h and the synthesized molecules were purified by dialysis in deoxygenated water for later use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应12h，清洗干燥后得到所述涂层。The sample to be modified is placed in the molecular solution obtained in step B, an alkaline anaerobic buffer is added to adjust the pH to 9, the reaction is carried out at 37° C. for 12 h, and the coating is obtained after washing and drying.

实施例10Example 10

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用镍钛合金作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, selecting nickel-titanium alloy as the base material prepared by the coating, which includes the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL NHS-DOTA溶液与pH调至8.5的PBS缓冲溶液混合，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.01：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL NHS-DOTA solution with PBS buffer solution with pH adjusted to 8.5, add CuCl₂ with a molar ratio of 1:1 to DOTA, ultrasonically react for 0.2 h, purify by centrifugation, and then add carboxyl to amine molar ratio of 0.01 : 1 of polyallylamine (weight average molecular weight 17,000, 20 wt.% aqueous solution), after reacting at 37° C. for 24 h, the synthesized molecule (DOTA-Cu/pPAM) was purified by dialysis method for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将3mg/mL咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在37℃下反应0.2h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.1：1的DOTA-Cu/pPAM，在5℃下反应24h后采用透析方法在无氧水中纯化合成的分子备用。3 mg/mL caffeic acid was dissolved in a completely anaerobic MES buffer solution with pH adjusted to 5.6, EDC and NHS were added, and after reacting at 37 °C for 0.2 h, the molar ratio of the carboxyl group to the amine group of DOTA-Cu/pPAM was: 0.1:1 DOTA-Cu/pPAM was reacted at 5 °C for 24 h and the synthesized molecules were purified by dialysis in deoxygenated water for later use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified was placed in the molecular solution obtained in step B, an alkaline anaerobic buffer was added to adjust the pH to 9, the reaction was carried out at 37° C. for 24 hours, and the coating was obtained after washing and drying.

实施例11Example 11

本实施例提供一种具有长效NO催化释放的涂层材料的制备方法，选用铁及其合金作为涂层制备的基底材料，其包括以下步骤：The present embodiment provides a method for preparing a coating material with long-term NO catalytic release, selecting iron and its alloy as the base material prepared by the coating, and comprising the following steps:

A、一氧化氮催化活性分子改性胺基化合物A. Nitric oxide catalytically active molecularly modified amine compounds

将1mg/mL NHS-DOTA溶液与pH调至8.5的PBS缓冲溶液混合，加入与DOTA摩尔比为1：1的CuCl₂，超声反应0.2h，离心纯化，随后加入羧基与胺基摩尔比为0.5：1的聚烯丙胺(重均分子量17,000,20wt.％水溶液)，在37℃下反应24h后，采用透析方法纯化合成的分子(DOTA-Cu/pPAM)备用。Mix 1 mg/mL NHS-DOTA solution with PBS buffer solution with pH adjusted to 8.5, add CuCl₂ with a molar ratio of 1:1 to DOTA, ultrasonically react for 0.2 h, centrifuge for purification, and then add carboxyl to amine molar ratio of 0.5 : 1 of polyallylamine (weight average molecular weight 17,000, 20 wt.% aqueous solution), after reacting at 37° C. for 24 h, the synthesized molecule (DOTA-Cu/pPAM) was purified by dialysis method for use.

B、富酚-胺化合物缩合B. Condensation of rich phenol-amine compounds

将1mg/mL咖啡酸溶于完全无氧、pH调至5.6的MES缓冲溶液中，加入EDC和NHS，在37℃下反应0.5h后，加入羧基与DOTA-Cu/pPAM的胺基摩尔比为0.5：1的DOTA-Cu/pPAM，在25℃下反应12h后采用透析方法在无氧水中纯化合成的分子备用。1 mg/mL caffeic acid was dissolved in a completely anaerobic MES buffer solution with pH adjusted to 5.6, EDC and NHS were added, and after reacting at 37 °C for 0.5 h, the molar ratio of the carboxyl group to the DOTA-Cu/pPAM amine group was: DOTA-Cu/pPAM of 0.5:1 was reacted at 25 °C for 12 h and the synthesized molecules were purified by dialysis in deoxygenated water for later use.

C、涂层制备C, coating preparation

将待改性样品置于B步骤所得分子溶液中，加入碱性无氧缓冲液调节pH至9，在37℃下反应24h，清洗干燥后得到所述涂层。The sample to be modified was placed in the molecular solution obtained in step B, an alkaline anaerobic buffer was added to adjust the pH to 9, the reaction was carried out at 37° C. for 24 hours, and the coating was obtained after washing and drying.

对比例1Comparative Example 1

与实施例1的区别在于：使用传统儿茶酚胺类化合物通过氧化自聚合形成涂层，且未引入胺基化合物。The difference from Example 1 is that the traditional catecholamine compound is used to form the coating by oxidative self-polymerization, and no amine compound is introduced.

对比例2Comparative Example 2

与实施例1的区别在于：改变铜离子的引入方式，对比例2通过涂层表面胺基共价固定大环多胺，随后螯合铜；而本发明方法是将螯合铜离子的大环多胺接枝于胺基化合物长链引入涂层，进而实现涂层中铜离子的固定。The difference from Example 1 is that the introduction mode of copper ions is changed, and in Comparative Example 2, macrocyclic polyamines are covalently fixed by amine groups on the coating surface, and then copper is chelated; and the method of the present invention is to chelate the macrocyclic copper ions. The polyamine is grafted onto the long chain of the amine compound and introduced into the coating, thereby realizing the fixation of copper ions in the coating.

试验例1Test Example 1

检测实施例1和对比例1涂层的化学稳定性，结果见图1所示。The chemical stability of the coatings of Example 1 and Comparative Example 1 was tested, and the results are shown in FIG. 1 .

检测方法：将实施例1中获得的涂层浸没于强酸(pH＝0)、强碱(pH＝14)和强氧化性(H₂O₂，质量分数30％)24h后，对涂层表面的形貌与中性条件下(pH＝8.5)的聚合涂层进行对比并拍照记录。Detection method: After immersing the coating obtained in Example 1 in strong acid (pH=0), strong alkali (pH=14) and strong oxidizing property (H₂ O₂ ,mass fraction 30%) for 24 hours, the surface of the coating was tested. The morphology was compared with that of the polymerized coating under neutral conditions (pH=8.5) and photographed.

从图1可知，通过本制备方法得到的NO催化释放涂层具有较强的化学稳定性能，在极端强酸、强碱和强氧化条件下依旧能保持完整的涂层。证明该方法制备的涂层相较于传统儿茶酚涂层具有更为优异的化学稳定性，即使在极端环境下也不会破坏涂层本身的完整性，具有更为宽广的应用领域。It can be seen from Figure 1 that the NO catalytic release coating obtained by this preparation method has strong chemical stability, and can still maintain a complete coating under extreme strong acid, strong alkali and strong oxidation conditions. It is proved that the coating prepared by this method has better chemical stability than the traditional catechol coating, and will not destroy the integrity of the coating itself even in extreme environments, and has a wider application field.

试验例2Test Example 2

检测对比例2涂层的NO催化释放检测效果图，结果见图2所示。The detection effect diagram of NO catalytic release of the coating of Comparative Example 2 is shown in Figure 2.

测试方法：使用化学发光NO分析仪(NOA)(Seivers 280i，Boulder，CO)在37℃的PBS中测量NO催化释放速率效果。Test method: The NO catalytic release rate effect was measured using a chemiluminescence NO analyzer (NOA) (Seives 280i, Boulder, CO) in PBS at 37°C.

图2可知，对比例2方法制备得到的涂层在早期NO催化释放时出现较为明显的突释，随着时间的推移其NO催化释放能力逐渐降低；将涂层拉出后再次插入反应液中，其催化释放NO能力相比较于第一次显著性地降低，其结果也暗示出采用分步接枝功能分子螯合铜离子的方式对于稳定催化释放NO具有一定的局限性。较低的铜离子利用率使其NO催化释放稳定性下降，致使其催化速率以及稳定性均明显下降。Figure 2 shows that the coating prepared by the method of Comparative Example 2 showed a relatively obvious burst release in the early NO catalytic release, and its NO catalytic release capacity gradually decreased with the passage of time; after the coating was pulled out, it was inserted into the reaction solution again. , its ability to catalyze the release of NO is significantly lower than that of the first time, and the results also suggest that the step-by-step grafting of functional molecules to chelate copper ions has certain limitations for the stable catalytic release of NO. The lower copper ion utilization rate reduces the stability of NO catalytic release, resulting in a significant decrease in its catalytic rate and stability.

试验例3Test Example 3

检测实施例1涂层的NO催化释放检测效果图，结果见图3所示。The detection effect diagram of NO catalytic release of the coating of Example 1 is shown, and the results are shown in FIG. 3 .

测试方法：同检测例2一致。Test method: the same as Test Example 2.

从图3可知，通过本制备方法得到的涂层在早期NO催化释放时的突释相比于对比例2涂层得到显著性的降低，NO突释降低了5倍左右。其NO催化释放能力随着时间的推移并未出现对比例2涂层催化释放NO过程中的下降趋势，且一直保持稳定的催化释放能力以及随着时间的增加还保持最初的NO催化释放能力。该检测结果直接证明该方法制备的涂层具有长效稳定催化释放NO释放的性能。It can be seen from Fig. 3 that the burst release of the coating obtained by this preparation method in the early catalytic release of NO is significantly reduced compared with the coating of Comparative Example 2, and the burst release of NO is reduced by about 5 times. Its NO catalytic release ability did not show a downward trend in the process of catalytic NO release of Comparative Example 2 over time, and it has always maintained a stable catalytic release ability and also maintained the initial NO catalytic release ability with the increase of time. The test results directly prove that the coating prepared by this method has the performance of long-term and stable catalytic release of NO.

以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A method for preparing a coating material with long-acting NO catalytic release is characterized by comprising the following steps: reacting carboxyl-or NHS-modified tetraazacyclo compound with nitric oxide catalytic release molecules to obtain a tetraazacyclo compound chelated with nitric oxide catalytic release molecules, and reacting the tetraazacyclo compound chelated with nitric oxide catalytic release molecules with an amino compound to obtain a modified amino compound;

reacting a carboxyl-containing phenolic compound with the modified amino compound to obtain a phenol-amine-rich condensation compound;

the substrate is placed in the solution of the phenol-amine rich condensation compound and the coating deposition is carried out under alkaline conditions.

2. The preparation method of claim 1, wherein the carboxyl-or NHS-modified tetraazacyclic compound is subjected to an ultrasonic reaction with the nitric oxide-catalyzed releasing molecule at 0-240 ℃ for 0.1h-3h, and after centrifugal purification is subjected to a reaction with the amine-based compound at 0-240 ℃ for 0.001h-240 h;

preferably, the reaction temperature of the carboxyl-or NHS-modified tetraazacyclic compound and the nitric oxide catalytic release molecule is 0-30 ℃, and the reaction time is 0.1-0.5 h; the reaction temperature of the amino compound is 0-30 ℃, and the reaction time is 0.01-24 h.

3. The method of claim 2, wherein the nitric oxide catalytically releasing molecule is selected from at least one of copper chloride, cuprous chloride, cupric bromide, cuprous bromide, cupric iodide, cuprous iodide, cupric sulfate, selenocysteine, cystine, L-selenocysteineacetic acid, and cysteine, and derivatives thereof;

preferably, the nitric oxide catalytic release molecule is a compound containing cupric ions or monovalent copper ions, and the dosage of the nitric oxide catalytic release molecule is controlled by controlling the molar ratio of the cupric ions to the tetraazacyclo-nitrogen to be 0.01-1: 5; preferably 0.01 to 1: 2.

4. the production method according to claim 2, wherein the amine-based compound is at least one selected from the group consisting of an aliphatic long-chain amine-based compound and an aromatic polyamine-based compound;

preferably, the amine-based compound is selected from at least one of polyallylamine, polyvinylamine, polylysine, polyethylene glycol bis (amine), polyetheramine, polyamide, melamine, poly-N-isopropylacrylamide and chitosan and derivatives thereof; more preferably polyallylamine;

preferably, the dosage of the amine compound is to control the molar ratio of carboxyl to amine to be 0.001-2: 1; preferably 0.005-0.02: 1.

5. The method according to claim 2, wherein the carboxyl-or NHS-modified tetraazacyclo compound is at least one selected from the group consisting of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, NHS-modified 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, tri-tert-butyl-1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, and derivatives thereof; preferably 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid;

preferably, carboxyl-containing tetraazacyclic compound solution is mixed with acidic buffer solution with the pH value of 4-10, and the mixture reacts with the condensing agent for 0.001-240 h at the temperature of 0-240 ℃ and then reacts with the nitric oxide catalytic release molecule;

preferably, the concentration of the carboxyl-containing tetraazacyclic compound solution is 0.01ng/mL to 5g/mL, more preferably 0.1 to 1 mg/mL.

6. The preparation method of claim 5, wherein the phenolic compound containing carboxyl is mixed with the buffer solution with the pH value of 4-10, and reacts with the condensing agent for 0.001h-240h at 0-240 ℃, and then reacts with the modified amino compound, wherein the reaction temperature is controlled to be 0-240 ℃ and the reaction time is controlled to be 0.001h-240 h;

preferably, the reaction temperature of the condensation agent is 0-30 ℃, and the reaction time is 0.5-48 h; the reaction temperature of the modified amino compound is 0-30 ℃, and the reaction time is 0.5-48 h;

preferably, the amount of the phenolic compound containing carboxyl is controlled to be 0.001-2:1, preferably 0.005-0.02:1 in molar ratio of carboxyl to amine in the modified amine compound;

preferably, the carboxyl group-containing phenolic compound is a monoamine phenolic compound; more preferably, the phenolic compound having a carboxyl group is at least one selected from the group consisting of caffeic acid, hydrogenated caffeic acid, dihydroxyphenylalanine, gallic acid, tannic acid, ferulic acid, 2, 3-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid, and derivatives thereof.

7. The method according to claim 6, wherein the condensing agent used in the reaction of the carboxyl group-containing tetraazacyclic compound and the carboxyl group-containing phenolic compound with the condensing agent is selected from at least one of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 4-dimethylaminopyridine, carbodiimide hydrochloride, 1, 3-diisopropylcarbodiimide, and dicyclohexylcarbodiimide;

preferably, the amount of the condensing agent is controlled to be 0.1 to 10:1 in terms of the molar ratio of the condensing agent to the carboxyl group;

preferably, the pH value of the buffer solution adopted in the reaction process of the carboxyl-containing tetraazacyclic compound and the carboxyl-containing phenolic compound and the condensing agent is 5-8;

preferably, the buffer solution is selected from at least one of 2- (N-morpholino) ethanesulfonic acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

8. The method according to claim 1, wherein the substrate is placed in the solution of the phenol-amine rich condensed compound, an alkaline buffer is added to adjust the pH value to 7-14, and the reaction is carried out at 0-240 ℃ for 0.001-240 h;

preferably, the reaction temperature of the base material and the rich phenol-amine condensation compound is 0-45 ℃, the reaction time is 0.1-48h, the reaction pH value is 7-11, and the base material and the rich phenol-amine condensation compound are cleaned and dried after reaction;

preferably, the functionalized modification is carried out after the reaction of the substrate with the phenol-amine rich condensation compound is completed, the functionalized modification being a reaction with a biologically active molecule bearing a carboxyl group, a thiol group or a NHS-functionalization;

preferably, the alkaline buffer solution is a sodium hydroxide solution with the concentration of 0.00001g/L-40 g/L;

preferably, the substrate is selected from at least one of a metal material, an inorganic material, and a polymer material;

more preferably, the substrate is selected from at least one of stainless steel, iron and its alloys, magnesium alloys, zinc-manganese alloys, cobalt-based alloys, titanium alloys, ferroferric oxide nanoparticles, ceramics, silica, polymethyl methacrylate, polystyrene, silicone rubber, and polyurethane.

9. Coating material with long-lasting NO catalytic release, characterized in that it is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a coating material according to claim 9 with long-acting NO catalytic release for the preparation of a blood implant device.

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