抗凝血性等离子体聚烯丙胺薄膜的制备及性能研究
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摘要
医用金属材料如不锈钢、钛基合金以及钴基合金由于其优良的力学性能(高强度、抗疲劳)及化学稳定性,在下腔静脉滤器、血管支架等介入器械制造中得到了广泛应用。然而,这些长期植入人体内与血液接触的介入器械,其自身抗凝血性能明显不足。肝素作为一种抗凝试剂被广泛的应用于临床治疗和心血管材料表面抗凝改性。然而,由于医用金属生物材料表面官能团的数量少,使得表面固定的抗凝分子浓度难以满足介入器械实际应用中的抗凝水平要求。因此如何通过表面处理,在金属材料表面形成一定浓度的反应性官能团,并通过官能团有效的固定抗凝分子肝素来提高金属生物材料表面的抗凝血性成为研究难点和重点。
本文以316L医用不锈钢这种常用的与血液接触的金属材料为研究对象,采用脉冲可调制波等离子体聚合技术,以烯丙胺为单体,通过调控脉冲占空比及放电功率在不锈钢表面沉积了一系列含伯胺基官能团的等离子体聚烯丙胺薄膜,并进一步以胺基为反应位点共价固定抗凝生物分子肝素。
傅立叶红外光谱(FTIR)、拉曼光谱及X射线光电子能谱(XPS)表征结果表明:等离子体聚烯丙胺薄膜为类聚合物(polymer-like)结构,低功率及占空比下合成的等离子体聚烯丙胺薄膜能部分保持单体的结构,薄膜伯胺基含量(NH_2/C)最大值为2.43%。薄膜表面固定肝素量达到4.10μg/cm~2。经等离子体聚烯丙胺薄膜改性后的不锈钢表面亲水性得到较大的提高。纳米划痕及薄膜动态稳定性结果表明,薄膜与基底具有良好的结合力,低功率制备的聚合薄膜在水溶液中动态稳定性较差,60W合成的薄膜具有较好的动态稳定性能
针对低功率下等离子体聚烯丙胺薄膜交联化程度低、薄膜水溶液中动态稳定性较差的特点,本文采用真空热处理的方法(真空为1.5×10~(-4)Pa,温度为200℃)对其进行进一步处理。拉曼光谱显示,热处理后,薄膜结构发生较大改变,薄膜中残留的烯键消失,骨架震动成分增多,薄膜交联度得到提高。X射线光电子能谱(XPS)测量结果表明,热处理后薄膜的伯胺基含量只有微量减少。薄膜在水溶液中动态稳定性得到了显著的提高。
体外血液相容性结果表明:等离子体聚烯丙胺薄膜及其肝素化的薄膜几乎无溶血现象;等离子体聚烯丙胺薄膜显示出较强的促内皮细胞粘附和增殖作用;表面肝素化的等离子体聚烯丙胺薄膜对纤维蛋白原的激活起到了一定的抑制作用,其表面具有较少的血小板粘附、集聚和激活,APTT时间由不锈钢的35S延长到50.1S。动物体内实验结果表明,经肝素化等离子体聚烯丙胺薄膜改性的不锈钢,表面覆盖了一层薄而均匀内皮细胞层,对初期急性血栓的产生起到了明显的抑制作用,表现出了良好的抗凝血性能。
The metallic biomaterials such as stainless steel (AISI 316L), tantalum and the shape memory alloy TiNi have been widely used for coronary stent, the peripheral vascular stent and vena cava filter which are of the cardiovascular interventional device due to their excellent mechanical attributes. However, the haemocompatibility of these biomedical metals is insufficient for the long-term antithrombogenic demand in vivo applications. Immobilization of heparin biomolecules on biomaterials is a widely investigated approach to modify surfaces for improving antithrombotic property. Because the metallic biomaterials used for cardiovascular implants possess a paucity of reactive functional groups, biomolecular immobilization of these materials is challenging.
In this paper, 316L stainless steel as a commonly used cardio vascular material is chosen as the materials in our research. Allylamine films with primary amine (-NH_2) were fabricated on medical stainless steel using pulsed plasma polymerization technique. Allylamine films were subsequently covalently immobilized by heparin via their surface primary amine.
The component, structure and properties of the plasma polymerized allylamine (PPAa) films are characterized using Raman laser spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and trifluoromethylbenzaldehyde (TFBA) derivatization process as function of radio frequency (RF) power and pulsed duty cycle (Pdc). The results indicate that PPAa films are polymer-like structure; High content of primary amine was retained in the PPAa film, the highest content of primary amine (NH_2/C) was 2.43% and the concentration of immobilized heparin was 4.10μg/cm~2 which determined by toluidine blue method.
While increasing the RF discharge power and pulsed duty cycle, more and more large size of particles appear in the film, film surface became much rougher, and water contact angle was gradually increased. And our nano-scratch results indicate that these films have a good binding strength to the stainless steel. The complicated change of the weight loss of the films indicated that stability of the films was determined significantly by diversified factors. The PPAa film fabricated under lower RF power displayed bad dynamical stability in the phosphate buffer solution (PBS).
In this paper, in order to improve the PPAa films dynamical stability in aqueous solution, the PPAa films was further treated under 200°C and 1.5×10~(-4)Pa vaccum condition. Raman spectra and XPS results indicate that the configuration of the alkene was disappeared, the component of framework vibration increased significantly, and tiny decreasing content of NH_2. However, the dynamical stability of the films was improved remarkably.
The evaluation of hemocompatibility was carried out including in vitro hemolysis ratio, Alamar Blue assay, fibrinogen adsorption, platelet adhesion, the lactate dehydrogenase (LDH), the glycoproteins granule membrane protein 140 (GMP140), activated partial thromboplastin time (APTT) tests and in vivo implantation into dog's external iliac arteries. The hemo-evaluations indicate that the heparin-immobilized PPAa films had better hemocompatibility.
本文以316L医用不锈钢这种常用的与血液接触的金属材料为研究对象,采用脉冲可调制波等离子体聚合技术,以烯丙胺为单体,通过调控脉冲占空比及放电功率在不锈钢表面沉积了一系列含伯胺基官能团的等离子体聚烯丙胺薄膜,并进一步以胺基为反应位点共价固定抗凝生物分子肝素。
傅立叶红外光谱(FTIR)、拉曼光谱及X射线光电子能谱(XPS)表征结果表明:等离子体聚烯丙胺薄膜为类聚合物(polymer-like)结构,低功率及占空比下合成的等离子体聚烯丙胺薄膜能部分保持单体的结构,薄膜伯胺基含量(NH_2/C)最大值为2.43%。薄膜表面固定肝素量达到4.10μg/cm~2。经等离子体聚烯丙胺薄膜改性后的不锈钢表面亲水性得到较大的提高。纳米划痕及薄膜动态稳定性结果表明,薄膜与基底具有良好的结合力,低功率制备的聚合薄膜在水溶液中动态稳定性较差,60W合成的薄膜具有较好的动态稳定性能
针对低功率下等离子体聚烯丙胺薄膜交联化程度低、薄膜水溶液中动态稳定性较差的特点,本文采用真空热处理的方法(真空为1.5×10~(-4)Pa,温度为200℃)对其进行进一步处理。拉曼光谱显示,热处理后,薄膜结构发生较大改变,薄膜中残留的烯键消失,骨架震动成分增多,薄膜交联度得到提高。X射线光电子能谱(XPS)测量结果表明,热处理后薄膜的伯胺基含量只有微量减少。薄膜在水溶液中动态稳定性得到了显著的提高。
体外血液相容性结果表明:等离子体聚烯丙胺薄膜及其肝素化的薄膜几乎无溶血现象;等离子体聚烯丙胺薄膜显示出较强的促内皮细胞粘附和增殖作用;表面肝素化的等离子体聚烯丙胺薄膜对纤维蛋白原的激活起到了一定的抑制作用,其表面具有较少的血小板粘附、集聚和激活,APTT时间由不锈钢的35S延长到50.1S。动物体内实验结果表明,经肝素化等离子体聚烯丙胺薄膜改性的不锈钢,表面覆盖了一层薄而均匀内皮细胞层,对初期急性血栓的产生起到了明显的抑制作用,表现出了良好的抗凝血性能。
The metallic biomaterials such as stainless steel (AISI 316L), tantalum and the shape memory alloy TiNi have been widely used for coronary stent, the peripheral vascular stent and vena cava filter which are of the cardiovascular interventional device due to their excellent mechanical attributes. However, the haemocompatibility of these biomedical metals is insufficient for the long-term antithrombogenic demand in vivo applications. Immobilization of heparin biomolecules on biomaterials is a widely investigated approach to modify surfaces for improving antithrombotic property. Because the metallic biomaterials used for cardiovascular implants possess a paucity of reactive functional groups, biomolecular immobilization of these materials is challenging.
In this paper, 316L stainless steel as a commonly used cardio vascular material is chosen as the materials in our research. Allylamine films with primary amine (-NH_2) were fabricated on medical stainless steel using pulsed plasma polymerization technique. Allylamine films were subsequently covalently immobilized by heparin via their surface primary amine.
The component, structure and properties of the plasma polymerized allylamine (PPAa) films are characterized using Raman laser spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and trifluoromethylbenzaldehyde (TFBA) derivatization process as function of radio frequency (RF) power and pulsed duty cycle (Pdc). The results indicate that PPAa films are polymer-like structure; High content of primary amine was retained in the PPAa film, the highest content of primary amine (NH_2/C) was 2.43% and the concentration of immobilized heparin was 4.10μg/cm~2 which determined by toluidine blue method.
While increasing the RF discharge power and pulsed duty cycle, more and more large size of particles appear in the film, film surface became much rougher, and water contact angle was gradually increased. And our nano-scratch results indicate that these films have a good binding strength to the stainless steel. The complicated change of the weight loss of the films indicated that stability of the films was determined significantly by diversified factors. The PPAa film fabricated under lower RF power displayed bad dynamical stability in the phosphate buffer solution (PBS).
In this paper, in order to improve the PPAa films dynamical stability in aqueous solution, the PPAa films was further treated under 200°C and 1.5×10~(-4)Pa vaccum condition. Raman spectra and XPS results indicate that the configuration of the alkene was disappeared, the component of framework vibration increased significantly, and tiny decreasing content of NH_2. However, the dynamical stability of the films was improved remarkably.
The evaluation of hemocompatibility was carried out including in vitro hemolysis ratio, Alamar Blue assay, fibrinogen adsorption, platelet adhesion, the lactate dehydrogenase (LDH), the glycoproteins granule membrane protein 140 (GMP140), activated partial thromboplastin time (APTT) tests and in vivo implantation into dog's external iliac arteries. The hemo-evaluations indicate that the heparin-immobilized PPAa films had better hemocompatibility.
引文
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