两亲性蛋白质—高分子杂化体的制备及其自组装性能研究
发布时间：2018-11-13 06:53
【摘要】：蛋白质是一种结构复杂的生物大分子,具有良好的生物相容性,在生物医学材料领域具有广泛的应用。但是,不同环境下的性质差异如溶解性、保留时间、稳定性等又限制了其进一步的应用,对蛋白质进行改性是拓宽其应用范围的有效方法之一。聚甲基丙烯酸甲酯(PMMA)是一种性能稳定的疏水性材料。工业上通过单体的本体聚合和乳液聚合法制备PMMA,所得产物有良好的透光性和加工性能被称为有机玻璃,并在涂料工业、光学材料等领域有广泛应用,同时PMMA有很好的生物相容性而被用于生物医学等领域如作为血液透析的透析膜。因此将合成聚合物PMMA通过表面接枝(grafting to)的方式与蛋白质结合制备两亲性蛋白质-高分子杂化体,与传统的两亲性嵌段共聚物相比,它不仅有自组装特性更兼具生物相容性,有望在智能药物控释领域有良好的应用。本文采用原子转移自由基聚合(ATRP)法制备了分子量可控的端基官能化PMMA,考察了接枝牛血清白蛋白(BSA)合成两亲性BSA-PMMA杂化体的合成条件。通过核磁共振氢谱(1H-NMR)、凝胶渗透色谱(GPC)、变性蛋白质聚丙烯酰胺凝胶电泳(SDS-PAGE)等方法对聚合物及杂化体结构进行了表征。随后,采用动态激光光散射(DLS)、扫描电子显微镜(SEM)、透射电子显微镜(TEM)等方法研究了BSA-PMMA杂化体在溶液中的的自组装行为。首先,利用2-溴代异丁酸和N-羟基琥珀酰亚胺合成ATRP引发剂2-溴代异丁酸N-羟基琥珀酰亚胺酯(NHS-BIBA),以CuBr和N,N,N',N,'N''-五甲基二亚乙基三胺(PMDETA)为催化体系,以丙酮为溶剂,合成了端基官能化聚合物PMMA。通过1H-NMR和GPC对所得引发剂和聚合产物进行表征,结果表明,成功合成了ATRP引发剂和分子量可控的端基官能化的PMMA。其次,研究了由亲水的BSA与疏水的PMMA制备具有两亲性的BSA-PMMA杂化体,确定了构建两亲性BSA-PMMA杂化体的最佳条件。通过变性蛋白聚丙烯酰氨凝胶电泳(SDS-PAGE)测试的结果表明,在丙酮:水=1:25,pH=5的条件下PMMA能够很好地通过表面接枝的方式与BSA结合形成杂化体。最后,通过激光动态散射仪(DLS)、扫描电子显微镜(SEM)、透射电子显微镜(TEM)研究了两亲性BSA-PMMA杂化体在溶液中的自组装行为。DLS测试的结果表明,两亲性BSA-PMMA杂化体在溶液中的自组装形成了胶束,随着杂化体中聚合物分子量或比例的增加自组装胶束粒径增大,胶束溶液的Zeta电势结果为-44.5mv,表明溶液稳定性很好。SEM测试的结果表明,两亲性BSA-PMMA杂化体自组装形成各种复杂的空间结构如囊泡结构(直径约500nm)、棒状结构、膜状结构等。TEM测试的结果表明,两亲性BSA-PMMA杂化体自组装形成的胶束外壁具有流动性。
[Abstract]:Protein is a complex biomolecules with good biocompatibility and is widely used in biomedical materials. However, the differences of properties in different environments, such as solubility, retention time, stability and so on, restrict its further application. Modification of protein is one of the effective methods to broaden its application range. Polymethyl methacrylate (PMMA) is a stable hydrophobic material. In industry, the products obtained by bulk polymerization and emulsion polymerization of monomers have good transmittance and processability, and have been widely used in coating industry, optical materials and other fields. At the same time, PMMA has good biocompatibility and has been used in biomedical fields such as hemodialysis as dialysis membrane. Therefore, amphiphilic protein-polymer hybrids were prepared by surface grafting of PMMA with protein. Compared with traditional amphiphilic block copolymers, it not only has self-assembly properties, but also has biocompatibility. It is expected to have a good application in the field of intelligent drug controlled release. Amphiphilic BSA-PMMA hybrids were prepared by atom transfer radical polymerization (ATRP) and their molecular weight controlled terminal functionalized PMMA, (PMMA,) were prepared. The synthesis conditions of amphiphilic BSA-PMMA hybrids were investigated by grafted bovine serum albumin (BSA) (BSA). The structures of polymers and hybrids were characterized by 1H-NMR and (GPC), denatured protein gel electrophoresis (SDS-PAGE). Then the self-assembly behavior of BSA-PMMA hybrids in solution was studied by means of dynamic laser light scattering (DLS), scanning electron microscope (SEM), transmission electron microscope (TEM) and so on. Firstly, 2-bromoisobutyric acid and N-hydroxysuccinimide were used to synthesize 2-bromoisobutyric acid N-hydroxysuccinimide (NHS-BIBA) initiator. The terminal functionalized polymer (PMMA.) was synthesized by using 'Nishi-pentamethylenediethylenetriamine (PMDETA) as catalyst system and acetone as solvent. The initiators and polymerization products were characterized by 1H-NMR and GPC. The results showed that ATRP initiator and PMMA. with controllable molecular weight were successfully synthesized. Secondly, the preparation of amphiphilic BSA-PMMA hybrids from hydrophilic BSA and hydrophobic PMMA was studied, and the optimal conditions for constructing amphiphilic BSA-PMMA hybrids were determined. The results of polyacrylamide gel electrophoresis (SDS-PAGE) of denatured protein showed that under the condition of acetone: water = 1: 25 (pH = 5), PMMA could bind to BSA by surface grafting to form hybrids. Finally, the self-assembly behavior of amphiphilic BSA-PMMA hybrids in solution was studied by (DLS), scanning electron microscope (SEM),) and transmission electron microscope (TEM) with a dynamic laser scattering instrument. The self-assembly of amphiphilic BSA-PMMA hybrids in solution formed micelles. With the increase of molecular weight or proportion of polymer in the hybrid, the particle size of self-assembled micelles increased, and the Zeta potential of micellar solution was -44.5 MV. The results of SEM test show that the amphiphilic BSA-PMMA hybrids self-assemble into various complex spatial structures, such as vesicle structure (diameter about 500nm), rod structure, membrane structure, etc. The results of TEM test show that, The outer wall of micelle formed by self-assembly of amphiphilic BSA-PMMA hybrids has fluidity.
【学位授予单位】：兰州交通大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O631.1;O629.73
本文编号：2328340
[Abstract]:Protein is a complex biomolecules with good biocompatibility and is widely used in biomedical materials. However, the differences of properties in different environments, such as solubility, retention time, stability and so on, restrict its further application. Modification of protein is one of the effective methods to broaden its application range. Polymethyl methacrylate (PMMA) is a stable hydrophobic material. In industry, the products obtained by bulk polymerization and emulsion polymerization of monomers have good transmittance and processability, and have been widely used in coating industry, optical materials and other fields. At the same time, PMMA has good biocompatibility and has been used in biomedical fields such as hemodialysis as dialysis membrane. Therefore, amphiphilic protein-polymer hybrids were prepared by surface grafting of PMMA with protein. Compared with traditional amphiphilic block copolymers, it not only has self-assembly properties, but also has biocompatibility. It is expected to have a good application in the field of intelligent drug controlled release. Amphiphilic BSA-PMMA hybrids were prepared by atom transfer radical polymerization (ATRP) and their molecular weight controlled terminal functionalized PMMA, (PMMA,) were prepared. The synthesis conditions of amphiphilic BSA-PMMA hybrids were investigated by grafted bovine serum albumin (BSA) (BSA). The structures of polymers and hybrids were characterized by 1H-NMR and (GPC), denatured protein gel electrophoresis (SDS-PAGE). Then the self-assembly behavior of BSA-PMMA hybrids in solution was studied by means of dynamic laser light scattering (DLS), scanning electron microscope (SEM), transmission electron microscope (TEM) and so on. Firstly, 2-bromoisobutyric acid and N-hydroxysuccinimide were used to synthesize 2-bromoisobutyric acid N-hydroxysuccinimide (NHS-BIBA) initiator. The terminal functionalized polymer (PMMA.) was synthesized by using 'Nishi-pentamethylenediethylenetriamine (PMDETA) as catalyst system and acetone as solvent. The initiators and polymerization products were characterized by 1H-NMR and GPC. The results showed that ATRP initiator and PMMA. with controllable molecular weight were successfully synthesized. Secondly, the preparation of amphiphilic BSA-PMMA hybrids from hydrophilic BSA and hydrophobic PMMA was studied, and the optimal conditions for constructing amphiphilic BSA-PMMA hybrids were determined. The results of polyacrylamide gel electrophoresis (SDS-PAGE) of denatured protein showed that under the condition of acetone: water = 1: 25 (pH = 5), PMMA could bind to BSA by surface grafting to form hybrids. Finally, the self-assembly behavior of amphiphilic BSA-PMMA hybrids in solution was studied by (DLS), scanning electron microscope (SEM),) and transmission electron microscope (TEM) with a dynamic laser scattering instrument. The self-assembly of amphiphilic BSA-PMMA hybrids in solution formed micelles. With the increase of molecular weight or proportion of polymer in the hybrid, the particle size of self-assembled micelles increased, and the Zeta potential of micellar solution was -44.5 MV. The results of SEM test show that the amphiphilic BSA-PMMA hybrids self-assemble into various complex spatial structures, such as vesicle structure (diameter about 500nm), rod structure, membrane structure, etc. The results of TEM test show that, The outer wall of micelle formed by self-assembly of amphiphilic BSA-PMMA hybrids has fluidity.
【学位授予单位】：兰州交通大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O631.1;O629.73
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