本文選題：316L不銹鋼 + 聚乙二醇　； 參考：《吉林大學》2015年博士論文

【摘要】：研究背景： 冠心�。–oronary Heart Disease，CHD）是當前引起人類死亡的首要原因，冠狀動脈內支架植入術已成為嚴重冠心病患者的首選治療方法。2013年全球冠狀動脈介入治療達到430萬例，我國冠狀動脈介入治療例數超過40萬例，然而在臨床上廣泛應用的血管內支架仍然存在著諸多不足。第一代支架屬于金屬裸支架（Bare Metal Stent，BMS），血管內皮細胞過度增生容易導致管腔再狹窄。第二代藥物洗脫支架（Drug Eluting Stent，DES）引進了載體（Polymer）和藥物（Drug）系統(tǒng)，減少了管腔內再狹窄，但聚合物的高致敏性可引發(fā)血管的慢性及持續(xù)性炎癥、內皮化不完全等問題，最終導致（極）晚期血栓形成和支架內再狹窄。第三代藥物洗脫支架主要分為生物可吸收支架，主要存在剛性和支撐性方面差等嚴重不足。因此，開發(fā)具有良好的剛性及生物相容性的高分子涂層支架是一項緊迫的任務。 316L不銹鋼具有適宜的機械特性和優(yōu)良的抗腐蝕性，是制造裸支架及藥物涂層支架的重要材料，在臨床上具有廣泛的應用。同時在金屬材料表面接枝聚乙二醇(Polyethylene Glycol，PEG)等親水性涂層材料可以有效改善材料的血液相容性，同時減少316L不銹鋼釋放的鎳離子所引起的過敏、致癌等不良反應。PEG是由乙二醇單體聚合而成的以羥基結尾的線性或分支狀聚醚高分子化合物，具有親水性高、排斥體積大、無毒、無致敏性等特點，是在材料表面接枝改性中應用最為廣泛的親水性高分子材料。當PEG分子和其它分子或材料相結合時,它的優(yōu)良性能也會隨之轉移到結合物中。將聚乙二醇作為材料的一部分，將賦予材料新的特性和功能，如在水性溶液中，PEG分子可以產生較大的流體力學體積，具有空間屏障作用，被認為是阻止蛋白質的非特異性吸附和細胞粘附的最有效親水性高分子。 材料表面接枝改性可分為表面物理吸附和共價連接，化學連接較物理連接更為穩(wěn)定，這對置入血管中的醫(yī)療材料至關重要。多種表面接枝改性技術已經應用在316L不銹鋼表面修飾上，包括在材料表面修飾硅烷化環(huán)氧樹脂前體后連接PEG鏈，射頻等離子體旋轉涂膜交聯技術以及通過聚乙烯亞胺薄膜改性的醛基PEG鏈等方法，其中，應用共價鍵改性聚乙二醇及其衍生物具有操作簡單、穩(wěn)定性強等優(yōu)點。因此，通過共價鍵將甲氧基聚乙二醇（Methoxy PolyethyleneGlycol,mPEG）接枝改性316L不銹鋼在改善血液及生物相容性方面具有重要的作用。 本論文的研究目的是在316L不銹鋼表面修飾聚乙二醇涂層，通過研究mPEG涂層對316L不銹鋼表面生物相容性的影響，為新型藥物洗脫支架的制備奠定實驗及理論基礎。材料與方法： ①硅烷化mPEG單體制備：稱量mPEG2000（0.01mol）放入500ml干燥三口燒瓶，在110℃下減壓干燥2h，用200ml無水四氫呋喃（Tetrahydrofuran, THF）充分溶解。在氮氣保護下依次向該溶液內加3-異氰丙基三乙氧基硅烷（0.025mol）及二月硅酸二丁基錫（0.001mol，催化劑），在氮氣保護下持續(xù)反應48h后，生成硅烷化聚乙二醇。用正己烷沉降兩次后，將目標產物放在真空干燥箱內減壓干燥過夜，通過傅里葉變換紅外光譜儀（Fourier Transform Infrared，FTIR）及400M核磁共振氫譜（Proton nuclear magnetic resonance,1HNMR）檢測產物化學結構。 ②不銹鋼表面mPEG接枝改性：316L不銹鋼片（10×10×2mm3）經打磨拋光及在15%HCl中浸泡過夜，依次用丙酮、乙醇及去離子水進行超聲清洗，以氮氣吹干后，將不銹鋼片于水虎魚溶液中浸泡20min，以MilliQ water充分潤洗，氮氣吹干備用。配制50mg/ml硅烷化PEG的乙醇-水（95∶5v/v%）混合溶液，用醋酸將溶液的pH值調至4.5,磁力攪拌器攪拌24h后，，將備用316L不銹鋼片在室溫下浸泡3h，再依次用去離子水及無水乙醇潤洗，再于110℃烘箱內干燥1h，以乙醇-水溶液超聲處理5min進一步去除不銹鋼表面物理粘附的mPEG。通過對mPEG接枝改性前后的不銹鋼表面分別進行接觸角、X射線光電子能譜（X-ray photoelectron spectroscopy,XPS）及原子力顯微鏡（Atomic Force Microscope,AFM）檢測，表征材料表面的特性。 ③生物相容性檢測：依次通過纖維蛋白原粘附實驗、血小板激活及粘附實驗、人臍靜脈內皮細胞（Human Umbilical Vein EndothelialCells,HUVEC）粘附實驗及細胞毒性實驗進行生物相容性檢測，探討mPEG涂層對對316L不銹鋼表面的生物相容性的影響。 結果： ①mPEG與3-異氰丙基三乙氧基硅烷在二月硅酸二丁基錫催化下反應生成硅烷化聚乙二醇，并通過硅氧共價鍵接枝到316L不銹鋼表面。 ②經mPEG改性后，XPS測得316L不銹鋼表面碳、硅元素含量明顯增加，鐵、鉻元素含量較前明顯下降，具有顯著統(tǒng)計學差異；經過對碳元素、氧元素的核心圖譜分析，證實了316L不銹鋼表面碳氫化合物含量增加，而金屬氧化物等含量下降；利用接觸角測量儀顯示改性后水接觸角明顯降低，材料親水性顯著增強；原子力顯微鏡顯示改性后材料表面粗糙程度增加，表面形態(tài)及粗糙度均發(fā)生顯著變化。 ③纖維蛋白原粘附實驗、血小板激活及粘附實驗、 HUVEC粘附及細胞毒性實驗證明，mPEG涂層可顯著抑制316L不銹鋼表面纖維蛋白原的粘附，并降低血小板激活程度及粘附數量；修飾后的材料能支持HUVEC在表面的粘附和增殖，無明顯細胞毒性。 結論： 1、mPEG可以通過硅氧共價鍵對316L不銹鋼表面進行接枝改性。 2、mPEG改性后不銹鋼表面微粗糙度及親水性發(fā)生顯著變化，有利于增強材料表面生物相容性。 3、mPEG改性后材料能明顯減少血小板和纖維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錛

本文編號：2002554