等离子体聚丙烯酸薄膜表面纤维连接蛋白的固定及其内皮细胞粘附行为
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摘要
本文采用低温等离子体聚合技术制备了含有羧基官能团的等离子体聚丙烯酸薄膜,并通过后续真空热处理以提高等离子体聚合薄膜的稳定性。在此基础上,利用聚丙烯酸薄膜表面的羧基为反应点,共价固定纤维连接蛋白(Fn)。本文采用漫反射傅立叶变换红外光谱仪(FTIR)、X射线光电子能谱(XPS)、甲苯胺蓝(TBO)法、原子力显微镜(AFM)、扫描电子显微镜(SEM)和接触角测量仪等测试手段和方法对各工艺薄膜的组成结构、表面物理化学性质进行了表征分析,并评价了等离子体聚丙烯酸薄膜在双蒸水中恒温动态浸泡的稳定性。通过人脐静脉内皮细胞(HUVEC)体外原代培养实验评价固定Fn前后等离子体聚丙烯酸薄膜表面的内皮细胞粘附和增殖行为。
FTIR图谱显示等离子体聚丙烯酸薄膜上保留了丙烯酸单体中的部分羧基官能团。TBO法检测的薄膜表面羧基浓度结果表明：随着有效功率的增大,等离子体聚丙烯酸薄膜表面的羧基浓度减少。接触角测试结果表明表面亲水性随有效功率的增大而减小。同时,等离子体聚丙烯酸薄膜在双蒸水中动态浸泡不同时间后,SEM和AFM观察结果显示随着有效功率的增大,等离子体聚丙烯酸薄膜的稳定性增大。这是由于功率提高,反应能量增大而导致薄膜交联度提高,从而使薄膜更加致密,减少溶胀现象。
为了进一步提高等离子体聚合薄膜的稳定性,本文对功率系列最大羧基浓度的等离子体聚丙烯酸薄膜在150℃和250℃下进行真空热处理,研究了不同真空热处理温度对等离子体聚丙烯酸薄膜性能的影响。结果显示150℃真空热处理对于等离子体聚丙烯酸薄膜稳定性的提高效果与200W下沉积的薄膜基本相同,而前者处理后的薄膜能够保留更多的羧基浓度。
通过FTIR和XPS结果显示纤连蛋白成功地固定在等离子体聚丙烯酸薄膜表面,纤连蛋白修饰的表面能够促进内皮细胞的粘附和增殖,但采用不同纤连蛋白浓度的溶液所制备的纤连蛋白修饰的等离子体聚丙烯酸薄膜表面的内皮细胞增殖效果无显著差异。ELISA检测结果表明固定了纤连蛋白的等离子体聚丙烯酸薄膜表面RGD肽段的暴露情况基本相同,因此Fn蛋白的促内皮化作用主要取决于RGD肽段的量。
综上所述,采用等离子体聚合技术,在医用不锈钢表面制备的等离子体聚丙烯酸薄膜能保留较大浓度的羧基基团。通过与增大射频功率来提高等离子体聚合薄膜的稳定性的途径相比,真空热处理能够同时提高薄膜交联度并有效地保留较高羧基浓度。固定纤连蛋白后的等离子体聚丙烯酸薄膜能够促进内皮细胞的粘附、铺展和增殖。内皮细胞的粘附与生长与样品表面RGD肽段的暴露情况密切相关。
Low temperature plasma polymerization technology was used to deposit plasma polymerized acrylic acid (PPAA) films which contained carboxyl group, and subsequently vacuum thermal treatment was used to improve the stability of PPAA films. Furthermore, reacted with-COOH of the PPAA film the the molecules of fibronectin (Fn) was covalently immobilized on the surface of PPAA. Fourier Transform Infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), toluidine blue-O(TBO) method and contact-angle measurement were used to investigate the component, structure and physiochemical property of PPAA films and Fn-immobilized PPAA films. The dynamical stability of PPAA films in distilled water was detected. Human umbilical vein endothelial cells (HUVECs) adhesion and proliferation testing in vitro were performed to evaluate the biological properties of Fn-immobilized PPAA films.
FTIR spectra indicated the existence of -COOH and C=O, which suggested that effective retention of the carboxylic functional groups. The results of TBO demonstrated that the concentration of -COOH on PPAA decreased as the effective power increased. Meanwhile, the stability of PPAA increased as the effective power increased. This was because of the enhancement of cross linking degree.
Different temperature of vacuum thermal treatment was used to improve the stability of PPAA films deposited at 50W. The results revealed that the stability of PPAA films after vacuum thermal treatment of 150℃was the same as the PPAA films deposited at 200W, but there was more -COOH concentration on the surface of PPAA films after vacuum thermal treatment of 150℃.
The FTIR and XPS results proved that the molecules of Fn were successfully immobilized on the PPAA's surface. The Fn-immobilized PPAA could enhance the attachment and coverage of HUVECs. The proliferation of HUVECs on the different Fn-immobilized film prepared by three kinds of Fn's concentration were not different from each other in statistics. The results of ELISA showed that exposure of RGD peptide on different Fn-immobilized films were almost the same. Therefore, the exposure of RGD peptide was essential for adhesion and proliferation of the HUVECs on Fn-immobilized PPAA films.
All these results illustrated that plasma polymerization is an effective way to deposit the functional films on stainless steel. Compared to the increase of effective power, vacuum thermal treatment either can enhance stability of the film or keep more functional groups on the film. The immobilization of Fn on the PPAA films can significantly improve adhesion and proliferation of the HUVECs. The exposure of RGD peptide may be related to the changes of configuration of Fn.
FTIR图谱显示等离子体聚丙烯酸薄膜上保留了丙烯酸单体中的部分羧基官能团。TBO法检测的薄膜表面羧基浓度结果表明：随着有效功率的增大,等离子体聚丙烯酸薄膜表面的羧基浓度减少。接触角测试结果表明表面亲水性随有效功率的增大而减小。同时,等离子体聚丙烯酸薄膜在双蒸水中动态浸泡不同时间后,SEM和AFM观察结果显示随着有效功率的增大,等离子体聚丙烯酸薄膜的稳定性增大。这是由于功率提高,反应能量增大而导致薄膜交联度提高,从而使薄膜更加致密,减少溶胀现象。
为了进一步提高等离子体聚合薄膜的稳定性,本文对功率系列最大羧基浓度的等离子体聚丙烯酸薄膜在150℃和250℃下进行真空热处理,研究了不同真空热处理温度对等离子体聚丙烯酸薄膜性能的影响。结果显示150℃真空热处理对于等离子体聚丙烯酸薄膜稳定性的提高效果与200W下沉积的薄膜基本相同,而前者处理后的薄膜能够保留更多的羧基浓度。
通过FTIR和XPS结果显示纤连蛋白成功地固定在等离子体聚丙烯酸薄膜表面,纤连蛋白修饰的表面能够促进内皮细胞的粘附和增殖,但采用不同纤连蛋白浓度的溶液所制备的纤连蛋白修饰的等离子体聚丙烯酸薄膜表面的内皮细胞增殖效果无显著差异。ELISA检测结果表明固定了纤连蛋白的等离子体聚丙烯酸薄膜表面RGD肽段的暴露情况基本相同,因此Fn蛋白的促内皮化作用主要取决于RGD肽段的量。
综上所述,采用等离子体聚合技术,在医用不锈钢表面制备的等离子体聚丙烯酸薄膜能保留较大浓度的羧基基团。通过与增大射频功率来提高等离子体聚合薄膜的稳定性的途径相比,真空热处理能够同时提高薄膜交联度并有效地保留较高羧基浓度。固定纤连蛋白后的等离子体聚丙烯酸薄膜能够促进内皮细胞的粘附、铺展和增殖。内皮细胞的粘附与生长与样品表面RGD肽段的暴露情况密切相关。
Low temperature plasma polymerization technology was used to deposit plasma polymerized acrylic acid (PPAA) films which contained carboxyl group, and subsequently vacuum thermal treatment was used to improve the stability of PPAA films. Furthermore, reacted with-COOH of the PPAA film the the molecules of fibronectin (Fn) was covalently immobilized on the surface of PPAA. Fourier Transform Infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), atomic force microscopy (AFM), toluidine blue-O(TBO) method and contact-angle measurement were used to investigate the component, structure and physiochemical property of PPAA films and Fn-immobilized PPAA films. The dynamical stability of PPAA films in distilled water was detected. Human umbilical vein endothelial cells (HUVECs) adhesion and proliferation testing in vitro were performed to evaluate the biological properties of Fn-immobilized PPAA films.
FTIR spectra indicated the existence of -COOH and C=O, which suggested that effective retention of the carboxylic functional groups. The results of TBO demonstrated that the concentration of -COOH on PPAA decreased as the effective power increased. Meanwhile, the stability of PPAA increased as the effective power increased. This was because of the enhancement of cross linking degree.
Different temperature of vacuum thermal treatment was used to improve the stability of PPAA films deposited at 50W. The results revealed that the stability of PPAA films after vacuum thermal treatment of 150℃was the same as the PPAA films deposited at 200W, but there was more -COOH concentration on the surface of PPAA films after vacuum thermal treatment of 150℃.
The FTIR and XPS results proved that the molecules of Fn were successfully immobilized on the PPAA's surface. The Fn-immobilized PPAA could enhance the attachment and coverage of HUVECs. The proliferation of HUVECs on the different Fn-immobilized film prepared by three kinds of Fn's concentration were not different from each other in statistics. The results of ELISA showed that exposure of RGD peptide on different Fn-immobilized films were almost the same. Therefore, the exposure of RGD peptide was essential for adhesion and proliferation of the HUVECs on Fn-immobilized PPAA films.
All these results illustrated that plasma polymerization is an effective way to deposit the functional films on stainless steel. Compared to the increase of effective power, vacuum thermal treatment either can enhance stability of the film or keep more functional groups on the film. The immobilization of Fn on the PPAA films can significantly improve adhesion and proliferation of the HUVECs. The exposure of RGD peptide may be related to the changes of configuration of Fn.
引文
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