生物医用聚合物材料表面功能化构建及抗蛋白吸附研究
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摘要
生物聚合物材料以其良好的机械性能、耐磨性和加工性能而被广泛应用于与体液或血液接触的材料。但与生物环境接触时都是以外源性物质的状态存在，不同程度地引起生物体的不良反应（异物反应）。这些反应都与非特异性蛋白质迅速吸附在无保护的材料表面有关，而非特异性蛋白的吸附又严重影响材料的表面物理化学特性。因此，材料表面的功能化构建及对非特异性蛋白吸附的抑制，是聚合物材料生物相容性的重要研究内容。
蛋白质主要是靠疏水作用吸附在材料表面。对于生物医用聚合物材料，材料表面亲/疏水性是影响蛋白质吸附的首要因素。同时，由于蛋白质是带有两性电荷的聚电解质，若材料表面也带有两亲性离子结构或亲水基团，通过富集水化层或空间排斥也可以削弱材料与蛋白的相互作用，抑制非特异性蛋白的吸附。因此，针对表面非特异性蛋白吸附引起的异物反应问题，本论文利用氨等离子体表面改性和活性生物分子接枝技术，在聚合物材料表面引入两亲性离子或亲水的功能化基团，研究表面抗非特异性蛋白吸附的机理，为其在后期临床的广泛应用提供重要理论依据。
采用低温氨等离子体改性技术，将亲水性基团引入疏水性丙烯酸酯和聚甲基丙烯酸甲酯（PMMA）材料表面。表面元素组成及接触角分析表明氨等离子体处理后，材料表面引入含氮的-NH2、-NH3+等极性基团，成功构建了氨基化的材料表面。同时表面也伴随着-COO-的产生，形成两亲性离子结构，亲水性改善。一定程度的等离子体刻蚀对后续研究影响不大，且透光率基本保持不变，优异的光学性能得到保留。但该技术处理的时效性较差。蛋白吸附实验表明，疏水性丙烯酸酯氨基化后的表面蛋白吸附减少，而氨基化的PMMA表面吸附增多，仍需要进一步接枝提高PMMA材料的表面抗蛋白吸附能力。
为了进一步增强表面抗蛋白吸附能力及长效性，首次利用酰胺键将水蛭素多肽结合在氨基化的丙烯酸酯系材料表面。紫外分光光度分析显示在静态吸附下，氨基化处理后的PMMA浸泡在500μg/ml的水蛭素溶液中4h时，吸光值最高，效果最好；表面形貌为规整有序；水蛭素接枝后表面亲水性单纯氨基化的表面要差，这是水蛭素分子中的负电荷中和了材料表面的正电荷导致的，这个推论也与表面能结果一致；表面-NH3+键含量下降而N-C=O键含量增加，证明水蛭素在材料表面接枝成功，表面也形成两性离子结构。通过石英晶体微天平动态吸附模型测试，接枝水蛭素后Fn的吸附迅速减少，且形成的吸附层最为疏松，容易被洗脱，实现了表面抗蛋白吸附功能，性能稳定。
为了验证氨基化改善亲水性技术的普适性，采用氨等离子体表面改性处理PET膜，构建亲水性表面。氨基化后表面亲水性大幅改善，并引入较多的含N基团（-NH2/-NH3+）和-COO-官能团，膜表面形貌没有变化。氨基化的PET表面蛋白吸附明显偏少，说明氨基化技术对于表面疏水的聚合物材料具有普遍适用性。通过氨基化构建的机理分析，表面基团的形成也为后续进一步接枝单体奠定基础。
采用2-甲基丙烯酰氧乙基磷酰胆碱（MPC）在氨基化的PET膜表面构建亲水性生物磷脂层。MPC分子的两亲性离子结构进一步改善了PET表面的亲水性和抗蛋白吸附能力。高分辨XPS图谱和FTIR光谱证明MPC接枝后，亲水性基团如-COOH，-N-C=O、-P-OH及-N+（CH3）3成功接入到材料表面。在接枝10mg/ml MPC时蛋白吸附量最低，表面平整、均一。通过MPC功能化表面作用机理进一步分析，磷脂基团构建的PET表面通过水化层和空间排斥共同作用，减少蛋白质的非特异性吸附。由于MPC接枝稳定，所以MPC构建的PET表面也具有抗非特异性蛋白吸附的长效性。
根据生物医用聚合物材料与表面抗非特异性蛋白、细胞的吸附关系，构建了功能化表面生成模型。并以上述三种聚合物材料为基底进行细胞相容性和动物体内实验研究。几种功能化的表面均不同程度地促进细胞增殖。水蛭素或MPC接枝的材料表面比单纯氨基化的表面抗细胞黏附能力大大提高。动物体内实验结果显示，接枝水蛭素的人工晶状体能够始终保持很好的透明度。对生物医用聚合物材料表面功能化构建及抗蛋白吸附机理进行研究，表明疏水材料表面亲水性和抗蛋白吸附功能化的构建是由于两亲性离子及水化层的存在，能够对也带两性离子的蛋白质起到排斥作用，从而减少非特异性蛋白吸附引起的不良反应，为今后材料在临床植入领域的更广泛应用奠定理论基础。
The biomedical polymer has been widely used for body fluids and blood materials as itsgood mechanical activity and chemical stability. However, it is still limited by thenon-specific protein absorption on its surface. Therefore, in order to improve the proteinresistance of the polymer surface, we use several methods to activate the polymer surface toprostheses with excellent performance. This research should provide the basis for constructionof tissue-engineering application.
Protein adsorption on the material surface is mainly due to hydrophobic effect.Hydrophobic and hydrophilic performance is the primary factor affecting the proteinadsorption for biomedical polymer materials. Foreign body reaction can be caused bynon-specific protein adsorption on surfaces. In this paper, ammonia plasma surfacemodification and active biological molecular grafting techniques have been applied to theacrylate and polyester material to introduce hydrophilic groups. The mechanism of proteinresistantance have also been researched for its widely application in clinical implant field.
In this study, we first use ammonia low-temperature plasma technology to introducehydrophilic groups onto the surface of the hydrophobic acrylic and PMMA surfaces. Theresults showed that the hydrophilicity of the surface was improved most. Surface elementalcomposition analysis showed that the polar groups of-NH2or-NH3+were introduced to thesurfaces, and amination surfaces were constructed. After amination, no obvious surfacescratches and damage were found. The transmittance was not significantly reduced,essentially excellent optical properties were retained. Timeliness results showed that if thehydrophobic recovery would occure. Protein adsorption experiments showed that the proteinadsorption reduced on hydrophobic acrylic surface after amination, but increased on theaminiated PMMA surface. Grafting was further needed on PMMA materials to improveability to resist protein adsorption.
Recombinant Hirudin (rH) peptide was first applied to modify the aminated PMMAsurfaces. After the integration, the contact angle of the surface increased slightly while thesurface had regular morphology. The-NH3+on the surface decreased with the increase of theN-C=O. The PMMA integrated with the hirudin had negative electricity, and the QCM resultsshowed that compared to the original surface and the plasma-treated surface, the surface withhirudin has better protein-resistant activity.
After that, in order to test if the technology had widely used area, we use this plasma technology to treat the PET film, which was widely used for blood vessel prostheses. Theresults showed that the treatment could improve the hydrophilicity of the surface with nochange of the morphology. There were-NH2and-COOH on the surface after treatment. Theprotein-resistant activity of the materials could also be improved. Then, we integrate the MPConto the treated surface. The integration of the MPC could improve the hydrophilicity of thesurface by introducing the-COO-,-N-C=O and-P-OH groups. The FITC results also showedthat the MPC was constructed onto the PET surface. The MPC-graft PET surface was coveredwith molecular layer, and it had the lowest protein adsorption with the MPC concentration of10mg/ml.
According to the relation of nonspecific protein resistantance of biomedical polymermaterials and cell adhesion, the functionalized surface generation model is constructed. Thebiological test results showed that the amino plasma treatment could decrease the adhesion ofthe cells, and the integration of hirudin or MPC could decrease this activity furthermore. Afterimplant into the animal eye, the plasma-treated IOL had fibrosis turbid immediately, while theoriginal IOL and the IOL with hirudin had excellent transparency after1month. Althoughthere was the cell migration to the center on the original IOL, it would not affect thetransparency. The cell experiments results showed that the long chain and the highhydrophilicity or hydrophobic were not benefit for the adhesion of cells.Finally, the surfacefunctionalization mechanism for protein resistance of biomedical polyester materialsconstruction was carried out in-depth analysis. It suggests that hydrophilic surface fornon-specific protein resistance is due to the presence of amphiphilic ion and hydration layer,which rejected the proteins also with zwitterionic electric charges. Thus the reduction ofnon-specific protein absorption caused by adverse reactions.It provides the theoretical basisfor more extensive application of biomedical materials for clinical implantation in the future.
蛋白质主要是靠疏水作用吸附在材料表面。对于生物医用聚合物材料，材料表面亲/疏水性是影响蛋白质吸附的首要因素。同时，由于蛋白质是带有两性电荷的聚电解质，若材料表面也带有两亲性离子结构或亲水基团，通过富集水化层或空间排斥也可以削弱材料与蛋白的相互作用，抑制非特异性蛋白的吸附。因此，针对表面非特异性蛋白吸附引起的异物反应问题，本论文利用氨等离子体表面改性和活性生物分子接枝技术，在聚合物材料表面引入两亲性离子或亲水的功能化基团，研究表面抗非特异性蛋白吸附的机理，为其在后期临床的广泛应用提供重要理论依据。
采用低温氨等离子体改性技术，将亲水性基团引入疏水性丙烯酸酯和聚甲基丙烯酸甲酯（PMMA）材料表面。表面元素组成及接触角分析表明氨等离子体处理后，材料表面引入含氮的-NH2、-NH3+等极性基团，成功构建了氨基化的材料表面。同时表面也伴随着-COO-的产生，形成两亲性离子结构，亲水性改善。一定程度的等离子体刻蚀对后续研究影响不大，且透光率基本保持不变，优异的光学性能得到保留。但该技术处理的时效性较差。蛋白吸附实验表明，疏水性丙烯酸酯氨基化后的表面蛋白吸附减少，而氨基化的PMMA表面吸附增多，仍需要进一步接枝提高PMMA材料的表面抗蛋白吸附能力。
为了进一步增强表面抗蛋白吸附能力及长效性，首次利用酰胺键将水蛭素多肽结合在氨基化的丙烯酸酯系材料表面。紫外分光光度分析显示在静态吸附下，氨基化处理后的PMMA浸泡在500μg/ml的水蛭素溶液中4h时，吸光值最高，效果最好；表面形貌为规整有序；水蛭素接枝后表面亲水性单纯氨基化的表面要差，这是水蛭素分子中的负电荷中和了材料表面的正电荷导致的，这个推论也与表面能结果一致；表面-NH3+键含量下降而N-C=O键含量增加，证明水蛭素在材料表面接枝成功，表面也形成两性离子结构。通过石英晶体微天平动态吸附模型测试，接枝水蛭素后Fn的吸附迅速减少，且形成的吸附层最为疏松，容易被洗脱，实现了表面抗蛋白吸附功能，性能稳定。
为了验证氨基化改善亲水性技术的普适性，采用氨等离子体表面改性处理PET膜，构建亲水性表面。氨基化后表面亲水性大幅改善，并引入较多的含N基团（-NH2/-NH3+）和-COO-官能团，膜表面形貌没有变化。氨基化的PET表面蛋白吸附明显偏少，说明氨基化技术对于表面疏水的聚合物材料具有普遍适用性。通过氨基化构建的机理分析，表面基团的形成也为后续进一步接枝单体奠定基础。
采用2-甲基丙烯酰氧乙基磷酰胆碱（MPC）在氨基化的PET膜表面构建亲水性生物磷脂层。MPC分子的两亲性离子结构进一步改善了PET表面的亲水性和抗蛋白吸附能力。高分辨XPS图谱和FTIR光谱证明MPC接枝后，亲水性基团如-COOH，-N-C=O、-P-OH及-N+（CH3）3成功接入到材料表面。在接枝10mg/ml MPC时蛋白吸附量最低，表面平整、均一。通过MPC功能化表面作用机理进一步分析，磷脂基团构建的PET表面通过水化层和空间排斥共同作用，减少蛋白质的非特异性吸附。由于MPC接枝稳定，所以MPC构建的PET表面也具有抗非特异性蛋白吸附的长效性。
根据生物医用聚合物材料与表面抗非特异性蛋白、细胞的吸附关系，构建了功能化表面生成模型。并以上述三种聚合物材料为基底进行细胞相容性和动物体内实验研究。几种功能化的表面均不同程度地促进细胞增殖。水蛭素或MPC接枝的材料表面比单纯氨基化的表面抗细胞黏附能力大大提高。动物体内实验结果显示，接枝水蛭素的人工晶状体能够始终保持很好的透明度。对生物医用聚合物材料表面功能化构建及抗蛋白吸附机理进行研究，表明疏水材料表面亲水性和抗蛋白吸附功能化的构建是由于两亲性离子及水化层的存在，能够对也带两性离子的蛋白质起到排斥作用，从而减少非特异性蛋白吸附引起的不良反应，为今后材料在临床植入领域的更广泛应用奠定理论基础。
The biomedical polymer has been widely used for body fluids and blood materials as itsgood mechanical activity and chemical stability. However, it is still limited by thenon-specific protein absorption on its surface. Therefore, in order to improve the proteinresistance of the polymer surface, we use several methods to activate the polymer surface toprostheses with excellent performance. This research should provide the basis for constructionof tissue-engineering application.
Protein adsorption on the material surface is mainly due to hydrophobic effect.Hydrophobic and hydrophilic performance is the primary factor affecting the proteinadsorption for biomedical polymer materials. Foreign body reaction can be caused bynon-specific protein adsorption on surfaces. In this paper, ammonia plasma surfacemodification and active biological molecular grafting techniques have been applied to theacrylate and polyester material to introduce hydrophilic groups. The mechanism of proteinresistantance have also been researched for its widely application in clinical implant field.
In this study, we first use ammonia low-temperature plasma technology to introducehydrophilic groups onto the surface of the hydrophobic acrylic and PMMA surfaces. Theresults showed that the hydrophilicity of the surface was improved most. Surface elementalcomposition analysis showed that the polar groups of-NH2or-NH3+were introduced to thesurfaces, and amination surfaces were constructed. After amination, no obvious surfacescratches and damage were found. The transmittance was not significantly reduced,essentially excellent optical properties were retained. Timeliness results showed that if thehydrophobic recovery would occure. Protein adsorption experiments showed that the proteinadsorption reduced on hydrophobic acrylic surface after amination, but increased on theaminiated PMMA surface. Grafting was further needed on PMMA materials to improveability to resist protein adsorption.
Recombinant Hirudin (rH) peptide was first applied to modify the aminated PMMAsurfaces. After the integration, the contact angle of the surface increased slightly while thesurface had regular morphology. The-NH3+on the surface decreased with the increase of theN-C=O. The PMMA integrated with the hirudin had negative electricity, and the QCM resultsshowed that compared to the original surface and the plasma-treated surface, the surface withhirudin has better protein-resistant activity.
After that, in order to test if the technology had widely used area, we use this plasma technology to treat the PET film, which was widely used for blood vessel prostheses. Theresults showed that the treatment could improve the hydrophilicity of the surface with nochange of the morphology. There were-NH2and-COOH on the surface after treatment. Theprotein-resistant activity of the materials could also be improved. Then, we integrate the MPConto the treated surface. The integration of the MPC could improve the hydrophilicity of thesurface by introducing the-COO-,-N-C=O and-P-OH groups. The FITC results also showedthat the MPC was constructed onto the PET surface. The MPC-graft PET surface was coveredwith molecular layer, and it had the lowest protein adsorption with the MPC concentration of10mg/ml.
According to the relation of nonspecific protein resistantance of biomedical polymermaterials and cell adhesion, the functionalized surface generation model is constructed. Thebiological test results showed that the amino plasma treatment could decrease the adhesion ofthe cells, and the integration of hirudin or MPC could decrease this activity furthermore. Afterimplant into the animal eye, the plasma-treated IOL had fibrosis turbid immediately, while theoriginal IOL and the IOL with hirudin had excellent transparency after1month. Althoughthere was the cell migration to the center on the original IOL, it would not affect thetransparency. The cell experiments results showed that the long chain and the highhydrophilicity or hydrophobic were not benefit for the adhesion of cells.Finally, the surfacefunctionalization mechanism for protein resistance of biomedical polyester materialsconstruction was carried out in-depth analysis. It suggests that hydrophilic surface fornon-specific protein resistance is due to the presence of amphiphilic ion and hydration layer,which rejected the proteins also with zwitterionic electric charges. Thus the reduction ofnon-specific protein absorption caused by adverse reactions.It provides the theoretical basisfor more extensive application of biomedical materials for clinical implantation in the future.
引文
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