鈦表面固定肝素—多聚賴氨酸納米顆粒以改善血液相容性的研究
發(fā)布時間：2018-10-29 20:55
【摘要】：心血管系統(tǒng)疾病是危害人類健康的重要疾病之一。目前,人工血管、人工心臟瓣膜、血管支架等各種人工器官或醫(yī)用裝置已廣泛應(yīng)用于心血管疾病的治療,但材料的血液相容性依然存在著嚴重的問題。為了改善生物材料的血液相容性,本論文通過在鈦材料表面構(gòu)建聚多巴胺涂層,并進一步在涂層上固定多聚賴氨酸(PLL)-肝素(Hep)納米顆粒,從而提高材料表面的血液相容性。 多巴胺(DM)在堿性條件下能發(fā)生自聚合,并可在鈦表面實現(xiàn)牢固粘接,形成具有多種官能團(酚羥基、醌基、氨基)的DM薄膜。多聚賴氨酸(PLL)可與肝素(Hep)通過自發(fā)靜電組裝作用形成具有三維結(jié)構(gòu)的納米顆粒。利用DM與伯氨基可發(fā)生麥克爾加成和西弗堿反應(yīng)的特性,可將含有氨基的Hep/PLL納米顆粒共價固定至樣品表面,構(gòu)建抗凝功能化表面。利用Zeta電位儀對不同工藝制備的納米顆粒進行尺寸及Zeta電位值的測定,并進行納米顆粒制備工藝的優(yōu)化。通過甲苯胺藍法對不同工藝納米顆粒中的肝素進行定量分析；傅里葉變換紅外光譜(FTIR)和X射線光電子能譜(XPS)顯示納米顆粒成功地固定到樣品表面；通過原子力顯微鏡(AFM)及水接觸角測試進行納米顆粒固定前后樣品表面形貌及親疏水性變化的表征；利用酸性橙定量氨基、甲苯胺藍法定量肝素及超微量電子天平對納米顆粒固定后樣品表面氨基含量、肝素含量及顆粒固定總量進行定量表征。 血小板粘附與激活實驗表明,鈦表面固定的納米顆�？娠@著抑制血小板粘附。纖維蛋白原變性結(jié)果表明,固定納米顆粒后樣品表面纖維蛋白原變性程度顯著降低,從而進一步降低血小板的聚集和激活。凝血時間檢測結(jié)果表明納米顆粒固定后,APTT及TT均有顯著延長,但PT對肝素不敏感,檢測結(jié)果無明顯變化。肝素動態(tài)釋放實驗及血小板粘附實驗表明,肝素初期釋放較快,有助于防止急性凝血發(fā)生,后期肝素釋放穩(wěn)定,有利于長期穩(wěn)定的抗凝。 進一步對材料表面修飾與生物相容性結(jié)果的關(guān)系研究表明,納米顆粒的形成受到溶液體系的離子濃度、pH環(huán)境以及參與納米顆粒形成的生物分子的分子量等因素的影響,在模擬正常人體血漿環(huán)境的弱堿性PBS中,納米顆粒能夠保持良好的穩(wěn)定性、均勻性,并發(fā)揮較強的抗凝作用。
[Abstract]:Cardiovascular disease is one of the most important diseases that harm human health. At present, artificial blood vessels, artificial heart valves, vascular stents and other artificial organs or medical devices have been widely used in the treatment of cardiovascular diseases, but there are still serious problems in the blood compatibility of materials. In order to improve the blood compatibility of biomaterials, a poly (dopamine) coating was constructed on the surface of titanium, and the poly-lysine (PLL) heparin (Hep) nanoparticles were further immobilized on the coating. So as to improve the surface of the material blood compatibility. Dopamine (DM) can be self-polymerized under alkaline conditions, and can be bonded firmly on titanium surface to form DM films with various functional groups (phenolic hydroxyl, quinone, amino). Poly-lysine (PLL) can interact with heparin (Hep) to form three-dimensional nanoparticles by spontaneous electrostatic assembly. Based on the characteristic that DM can react with primary amino group by Michael addition and sieverine, the Hep/PLL nanoparticles containing amino groups can be covalently immobilized on the surface of the sample to construct an anticoagulation-functionalized surface. The size and Zeta potential of the nanoparticles prepared by different processes were measured by Zeta potentiometer, and the preparation process of nanoparticles was optimized. Quantitative analysis of heparin in nanoparticles with different processes was carried out by toluidine blue method. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) showed that the nanoparticles were successfully immobilized on the surface of the samples. The surface morphology and hydrophobicity of the samples were characterized by atomic force microscope (AFM) (AFM) and water contact angle measurements before and after the nanoparticles were immobilized. The surface amino content, heparin content and total amount of particle fixation were quantitatively characterized by acid orange quantitative amino group, toluidine blue quantitative heparin and ultramicro electron balance. Platelet adhesion and activation test showed that titanium nanoparticles could significantly inhibit platelet adhesion. The results of fibrinogen denaturation showed that the degree of fibrinogen denaturation on the surface of the samples decreased significantly after the nanoparticles were fixed, which further reduced the aggregation and activation of platelet. The results of coagulation time test showed that both APTT and TT were significantly prolonged after immobilization of nanoparticles, but PT was not sensitive to heparin, and the results showed no obvious change. The dynamic release of heparin and platelet adhesion test showed that the initial release of heparin was faster, which was helpful to prevent the occurrence of acute coagulation, and to stabilize the release of heparin in the later stage, which was beneficial to long-term stable anticoagulation. Further studies on the relationship between surface modification and biocompatibility showed that the formation of nanoparticles was influenced by ionic concentration of solution system, pH environment and molecular weight of biomolecules involved in the formation of nanoparticles. In the weakly alkaline PBS which simulates the normal human plasma environment, the nanoparticles can maintain good stability, uniformity and play a strong anticoagulant effect.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：TB383.1;TB324
本文編號：2298798
[Abstract]:Cardiovascular disease is one of the most important diseases that harm human health. At present, artificial blood vessels, artificial heart valves, vascular stents and other artificial organs or medical devices have been widely used in the treatment of cardiovascular diseases, but there are still serious problems in the blood compatibility of materials. In order to improve the blood compatibility of biomaterials, a poly (dopamine) coating was constructed on the surface of titanium, and the poly-lysine (PLL) heparin (Hep) nanoparticles were further immobilized on the coating. So as to improve the surface of the material blood compatibility. Dopamine (DM) can be self-polymerized under alkaline conditions, and can be bonded firmly on titanium surface to form DM films with various functional groups (phenolic hydroxyl, quinone, amino). Poly-lysine (PLL) can interact with heparin (Hep) to form three-dimensional nanoparticles by spontaneous electrostatic assembly. Based on the characteristic that DM can react with primary amino group by Michael addition and sieverine, the Hep/PLL nanoparticles containing amino groups can be covalently immobilized on the surface of the sample to construct an anticoagulation-functionalized surface. The size and Zeta potential of the nanoparticles prepared by different processes were measured by Zeta potentiometer, and the preparation process of nanoparticles was optimized. Quantitative analysis of heparin in nanoparticles with different processes was carried out by toluidine blue method. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) showed that the nanoparticles were successfully immobilized on the surface of the samples. The surface morphology and hydrophobicity of the samples were characterized by atomic force microscope (AFM) (AFM) and water contact angle measurements before and after the nanoparticles were immobilized. The surface amino content, heparin content and total amount of particle fixation were quantitatively characterized by acid orange quantitative amino group, toluidine blue quantitative heparin and ultramicro electron balance. Platelet adhesion and activation test showed that titanium nanoparticles could significantly inhibit platelet adhesion. The results of fibrinogen denaturation showed that the degree of fibrinogen denaturation on the surface of the samples decreased significantly after the nanoparticles were fixed, which further reduced the aggregation and activation of platelet. The results of coagulation time test showed that both APTT and TT were significantly prolonged after immobilization of nanoparticles, but PT was not sensitive to heparin, and the results showed no obvious change. The dynamic release of heparin and platelet adhesion test showed that the initial release of heparin was faster, which was helpful to prevent the occurrence of acute coagulation, and to stabilize the release of heparin in the later stage, which was beneficial to long-term stable anticoagulation. Further studies on the relationship between surface modification and biocompatibility showed that the formation of nanoparticles was influenced by ionic concentration of solution system, pH environment and molecular weight of biomolecules involved in the formation of nanoparticles. In the weakly alkaline PBS which simulates the normal human plasma environment, the nanoparticles can maintain good stability, uniformity and play a strong anticoagulant effect.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：TB383.1;TB324
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