mPEG修饰对316L不锈钢表面生物相容性的影响研究

发布时间：2018-06-10 08:14

  本文选题：316L不锈钢 + 聚乙二醇　； 参考：《吉林大学》2015年博士论文

【摘要】：研究背景： 冠心病（Coronary Heart Disease，CHD）是当前引起人类死亡的首要原因，冠状动脉内支架植入术已成为严重冠心病患者的首选治疗方法。2013年全球冠状动脉介入治疗达到430万例，我国冠状动脉介入治疗例数超过40万例，然而在临床上广泛应用的血管内支架仍然存在着诸多不足。第一代支架属于金属裸支架（Bare Metal Stent，BMS），血管内皮细胞过度增生容易导致管腔再狭窄。第二代药物洗脱支架（Drug Eluting Stent，DES）引进了载体（Polymer）和药物（Drug）系统，减少了管腔内再狭窄，但聚合物的高致敏性可引发血管的慢性及持续性炎症、内皮化不完全等问题，最终导致（极）晚期血栓形成和支架内再狭窄。第三代药物洗脱支架主要分为生物可吸收支架，主要存在刚性和支撑性方面差等严重不足。因此，开发具有良好的刚性及生物相容性的高分子涂层支架是一项紧迫的任务。 316L不锈钢具有适宜的机械特性和优良的抗腐蚀性，是制造裸支架及药物涂层支架的重要材料，在临床上具有广泛的应用。同时在金属材料表面接枝聚乙二醇(Polyethylene Glycol，PEG)等亲水性涂层材料可以有效改善材料的血液相容性，同时减少316L不锈钢释放的镍离子所引起的过敏、致癌等不良反应。PEG是由乙二醇单体聚合而成的以羟基结尾的线性或分支状聚醚高分子化合物，具有亲水性高、排斥体积大、无毒、无致敏性等特点，是在材料表面接枝改性中应用最为广泛的亲水性高分子材料。当PEG分子和其它分子或材料相结合时,它的优良性能也会随之转移到结合物中。将聚乙二醇作为材料的一部分，将赋予材料新的特性和功能，如在水性溶液中，PEG分子可以产生较大的流体力学体积，具有空间屏障作用，被认为是阻止蛋白质的非特异性吸附和细胞粘附的最有效亲水性高分子。 材料表面接枝改性可分为表面物理吸附和共价连接，化学连接较物理连接更为稳定，这对置入血管中的医疗材料至关重要。多种表面接枝改性技术已经应用在316L不锈钢表面修饰上，包括在材料表面修饰硅烷化环氧树脂前体后连接PEG链，射频等离子体旋转涂膜交联技术以及通过聚乙烯亚胺薄膜改性的醛基PEG链等方法，其中，应用共价键改性聚乙二醇及其衍生物具有操作简单、稳定性强等优点。因此，通过共价键将甲氧基聚乙二醇（Methoxy PolyethyleneGlycol,mPEG）接枝改性316L不锈钢在改善血液及生物相容性方面具有重要的作用。 本论文的研究目的是在316L不锈钢表面修饰聚乙二醇涂层，通过研究mPEG涂层对316L不锈钢表面生物相容性的影响，为新型药物洗脱支架的制备奠定实验及理论基础。材料与方法： ①硅烷化mPEG单体制备：称量mPEG2000（0.01mol）放入500ml干燥三口烧瓶，在110℃下减压干燥2h，用200ml无水四氢呋喃（Tetrahydrofuran, THF）充分溶解。在氮气保护下依次向该溶液内加3-异氰丙基三乙氧基硅烷（0.025mol）及二月硅酸二丁基锡（0.001mol，催化剂），在氮气保护下持续反应48h后，生成硅烷化聚乙二醇。用正己烷沉降两次后，将目标产物放在真空干燥箱内减压干燥过夜，通过傅里叶变换红外光谱仪（Fourier Transform Infrared，FTIR）及400M核磁共振氢谱（Proton nuclear magnetic resonance,1HNMR）检测产物化学结构。 ②不锈钢表面mPEG接枝改性：316L不锈钢片（10×10×2mm3）经打磨抛光及在15%HCl中浸泡过夜，依次用丙酮、乙醇及去离子水进行超声清洗，以氮气吹干后，将不锈钢片于水虎鱼溶液中浸泡20min，以MilliQ water充分润洗，氮气吹干备用。配制50mg/ml硅烷化PEG的乙醇-水（95∶5v/v%）混合溶液，用醋酸将溶液的pH值调至4.5,磁力搅拌器搅拌24h后，，将备用316L不锈钢片在室温下浸泡3h，再依次用去离子水及无水乙醇润洗，再于110℃烘箱内干燥1h，以乙醇-水溶液超声处理5min进一步去除不锈钢表面物理粘附的mPEG。通过对mPEG接枝改性前后的不锈钢表面分别进行接触角、X射线光电子能谱（X-ray photoelectron spectroscopy,XPS）及原子力显微镜（Atomic Force Microscope,AFM）检测，表征材料表面的特性。 ③生物相容性检测：依次通过纤维蛋白原粘附实验、血小板激活及粘附实验、人脐静脉内皮细胞（Human Umbilical Vein EndothelialCells,HUVEC）粘附实验及细胞毒性实验进行生物相容性检测，探讨mPEG涂层对对316L不锈钢表面的生物相容性的影响。 结果： ①mPEG与3-异氰丙基三乙氧基硅烷在二月硅酸二丁基锡催化下反应生成硅烷化聚乙二醇，并通过硅氧共价键接枝到316L不锈钢表面。 ②经mPEG改性后，XPS测得316L不锈钢表面碳、硅元素含量明显增加，铁、铬元素含量较前明显下降，具有显著统计学差异；经过对碳元素、氧元素的核心图谱分析，证实了316L不锈钢表面碳氢化合物含量增加，而金属氧化物等含量下降；利用接触角测量仪显示改性后水接触角明显降低，材料亲水性显著增强；原子力显微镜显示改性后材料表面粗糙程度增加，表面形态及粗糙度均发生显著变化。 ③纤维蛋白原粘附实验、血小板激活及粘附实验、 HUVEC粘附及细胞毒性实验证明，mPEG涂层可显著抑制316L不锈钢表面纤维蛋白原的粘附，并降低血小板激活程度及粘附数量；修饰后的材料能支持HUVEC在表面的粘附和增殖，无明显细胞毒性。 结论： 1、mPEG可以通过硅氧共价键对316L不锈钢表面进行接枝改性。 2、mPEG改性后不锈钢表面微粗糙度及亲水性发生显著变化，有利于增强材料表面生物相容性。 3、mPEG改性后材料能明显减少血小板和纤维蛋白原的激活和粘附，支持HUVEC的粘附和增殖，具有良好的生物相容性。
[Abstract]:Research background:
Coronary Heart Disease (CHD) is the primary cause of human death. Coronary stent implantation has become the first choice for patients with severe coronary heart disease (CAD). 4 million 300 thousand cases of coronary artery interventional therapy are achieved in.2013 years. More than 400 thousand cases of coronary artery interventional therapy in China are more than 400 thousand cases, but they are widely used in clinical practice. There are still many shortcomings in the endovascular stent. The first generation of stent is Bare Metal Stent (BMS), and the hyperproliferation of vascular endothelial cells is easy to lead to the restenosis of the lumen. The second generation drug eluting stent (Drug Eluting Stent, DES) introduced the carrier (Polymer) and drug (Drug) system to reduce the restenosis in the lumen, but the polymer High sensitivity can lead to chronic and persistent inflammation of blood vessels, incomplete endothelialization, and eventually lead to late thrombosis and stent restenosis. The third generation of drug eluting stents are mainly divided into bioabsorbable scaffolds, which are mainly rigid and supportive. Therefore, the development has a good rigidity and life. Material compatibility of polymer coated stent is an urgent task.
316L stainless steel has suitable mechanical properties and excellent corrosion resistance. It is an important material for the manufacture of bare scaffolds and drug coated stents. It is widely used clinically. At the same time, the hydrophilic coating material such as Polyethylene Glycol (PEG) graft on the surface of metal material can improve the blood compatibility of the material. The adverse reaction, such as allergy and carcinogenesis, caused by reducing the nickel ions released by 316L stainless steel, is a linear or branched polyether polymer composed of ethylene glycol monomers. It has the characteristics of high hydrophilicity, large rejection volume, nontoxic, and no sensitization, and is the most widely used in the grafting modification of material surface. Water based polymer materials. When PEG molecules are combined with other molecules or materials, their excellent properties will also be transferred to the conjugates. Polyethylene glycol is used as part of the material to give new properties and functions. For example, in aqueous solution, PEG molecules can produce a larger volume of fluid mechanics and have a space barrier effect. It is considered to be the most effective hydrophilic polymer to prevent protein non-specific adsorption and cell adhesion.
The surface grafting modification of materials can be divided into surface physical adsorption and covalent connection, and chemical connections are more stable than physical connections. This is very important for the medical materials placed in the blood vessels. A variety of surface grafting modification techniques have been applied to the surface modification of 316L stainless steel, including the connection of the PEG chain after the surface modification of the silane epoxy resin precursor. The technology of radio frequency plasma spin coating crosslinking and the modified aldehyde group PEG chain through polyethylenimide film, in which the modified polyethylene glycol and its derivatives with covalent bond have the advantages of simple operation and strong stability. Therefore, the graft copolymerization of methoxy polyglycol (Methoxy PolyethyleneGlycol, mPEG) by covalent bond is used to modify 316L Stainless steel plays an important role in improving blood and biocompatibility.
The purpose of this study is to modify the polyethylene glycol coating on the surface of 316L stainless steel. By studying the effect of mPEG coating on the biocompatibility of 316L stainless steel surface, the experimental and theoretical basis for the preparation of new drug eluting stents is established.
(1) silanated mPEG single system: weighing mPEG2000 (0.01mol) into 500ml dry three mouthed flasks, decompressing and drying 2H at 110 C, fully dissolving with 200ml anhydrofuran (Tetrahydrofuran, THF), and adding 3- isocyanate triethoxyl silane (0.025mol) to the solution and two butyltin in February (0.001mol, catalysis) under nitrogen protection. After a continuous reaction of 48h under the protection of nitrogen, a silane polyethylene glycol was generated. After two times of n-hexane settlement, the target products were placed in a vacuum drying box and dried for the night. The Fourier transform infrared spectrometer (Fourier Transform Infrared, FTIR) and 400M nuclear magnetic resonance hydrogen spectrum (Proton nuclear magnetic resonance, 1HNMR) were detected. The chemical structure of the product.
(2) mPEG graft modification of stainless steel surface: 316L stainless steel sheet (10 x 10 x 2mm3) was polished and soaked overnight in 15%HCl, and then used acetone, ethanol and deionized water for ultrasonic cleaning. After blowing dry nitrogen, the stainless steel slices were soaked in the solution of the tiger fish with 20min, and MilliQ water was fully washed, nitrogen was blown to dry and prepared. 50mg/ml silane was prepared. The mixed solution of ethanol water (95: 5v/v%) of PEG was converted to 4.5 of the pH value of the solution with acetic acid. After stirring for 24h by magnetic stirrer, the standby 316L stainless steel slices were soaked at room temperature for 3h, and then washed with deionized water and anhydrous ethanol, and then dry 1H in the oven at 110 C, and the stainless steel surface was further removed by ultrasonic treatment of ethanol water solution to remove the surface of stainless steel. The adhesive mPEG. was detected by the contact angle of the stainless steel surface before and after the graft modification of mPEG, the X ray photoelectron spectroscopy (X-ray photoelectron spectroscopy, XPS) and the atomic force microscope (Atomic Force Microscope, AFM), to characterize the surface properties of the material.
(3) biocompatibility test: in turn, the biocompatibility of Human Umbilical Vein EndothelialCells (HUVEC) adhesion and cytotoxicity test was carried out by fibrinogen adhesion experiment, platelet activation and adhesion experiment, and the effect of mPEG coating on the biocompatibility of 316L stainless steel surface.
Result锛

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