高分子微球的表面分子嫁接及生化亲和特性
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摘要
亲和高分子乳液微球是一类利用生物分子或基团亲和配位原理合成的功能高分子微球。将具有特定的亲和配位能力的生物分子或基团之一如药物、抗体和DNA固定在高分子乳液微球表面,则可以借助后者的高比表面积高效地识别配位、并吸附分离出目标分子或基团。亲和高分子乳液微球不仅具有高效、稳定、功能多样等特征,而且还具有机械性强、反应灵敏、优异的生物相容性、可循环使用、对环境污染小等特有的优点。因此,在诸如药物快速评价、免疫抗体识别和DNA诊断等领域有很高的应用价值。例如,将药物分子固定在纳米级高分子乳液微球表面,利用亲和微球的生物惰性和高比表面积等优点,高效的从生物组织液中识别、分离、纯化药物受体蛋白质。通过对药物受体蛋白质分析测定及受体蛋白质与药物分子之间相互作用机理研究,可拓宽新药开发思路、缩短药物开发周期。
以疏水性单体苯乙烯St和甲基丙烯酸环氧丙酯GMA为单体,采用无皂乳液聚合的方法制备了表面带环氧基的高分子乳液微球PSG聚(苯乙烯-甲基丙烯酸环氧丙酯)。为降低微球表面的空间位阻效应和壁面效应,采用1,6-已二胺作为空间臂分子,对PSG微球进行表面修饰,获得了端氨基的微球粒子PSGN(PSG-1,6-已二胺)。并采用元素分析法和4-硝基苯甲醛法分析测定了微球表面的氨基浓度。然后采用培美曲塞二钠MTA与PSGN微球表面的氨基进行共价偶联反应,制得具有药物固定的亲和高分子微球。实验结果表明培美曲塞二钠药物分子固定在PSGN微球表面的接枝量主要受微球粒径大小和活泼氨基浓度的影响；通过改变投料比和功能单体多段加料的方式可控制乳液微球粒径大小和表面活泼基团的浓度,进而实现对药物接枝量的控制。另外,实验还考察了体系pH、催化剂比例及用量和温度对MTA偶联反应的影响,获得了药物分子偶联在PSGN微球表面的最佳反应条件。
此外还研究了药物固定高分子微球对蛋白质的吸附特性。采用牛血蛋白质BSA作为模型蛋白质,着重考察了pH、离子强度和不同BSA初始浓度对于蛋白质吸附的影响。结果表明：MTA药物分子固定在微球表面的浓度较高、分布均匀时,微球药物对BSA的吸附动力学较符合一级动力学过程,并且静电引力对于蛋白质吸附具有重要影响。
Affinity polymer latex microsphere is a kind of functional polymer microsphere, which has affinity molecules or groups on its surface to efficiently recognize and separate the target molecules by the high specific surface areas of the microsphere. There are high degree of specific recognition and coordination between affinity molecules and target molecules. These objects mainly include drug-receptor, antibody-antigen, complementary DNA chains, etc. Affinity polymer latex microspheres have the common advantages of functional polymer microspheres, i.e., stable dispersion, large specific surface areas as well as versatile properties. In addition, these smart latex microspheres have several particular features, such as well mechanical performance, sensitivity, excellent biology compatibility, easy to recovery and repetitious to use, friendly to environment. Affinity polymer latex microspheres showed potential application for bioseparation, antibody identification, DNA diagnosis as well as efficient evaluation of medicine. For example, the affinity microspheres hold the bio-inert and high surface area can be used for identification, separation and purification of drug receptor from biological tissue by immobilizing the drug molecules on its surface. Expand the ideals of new drugs development and shorten the drug development cycle by studying the mechanism of drug and drug receptor.
Poly (styrene-co-glycidyl methacrylate) (PSG) latex microspheres with epoxy groups on its surface are prepared by soap-free emulsion polymerization with hydrophobic monomer styrene and glycidyl methacrylate. The PSG microspheres are modified with 1,6-hexanediamine space arm to reduce the steric hindrance and side interaction of the surface and the amino-modified microsphere PSGN was obtained. The content of amino groups on PSGN particle is measured by elemental analysis and 4-nitrobenzaldehyd methods. Then the pemetrexed disodium (MTA) is covalent immobilized on the surface of the PSGN particles through the amidation between the carboxylic acid end group of MTA in acidic medium and the amino groups on the PSGN particle surface. The experimental results suggest MTA immobilization is mainly dependent upon both the particle size and active amino groups on the PSGN surface. The amino group density of the PSGN microsphere surface could be controlled through altering the ratio of feed and the mode of operation. Further, the immobilization of MTA is also controllable. Researches on the influence of pH, temperature and the different ratio and concentration of catalysts on MTA coupling reaction are performed and the optimum reaction conditions are earned.
The adsorption property of BSA protein onto the surface of polymer microsphere drug is emphatically studied. Bovine serum albumin (BSA) is selected as the model protein. The effect of pH, ion concentration and different first BSA concentration on adsorption is emphatically examined. The results showed the higher covalent immobilization concentration and uniform distribution of MTA on the polymer microspheres have the regular adsorption process of BSA protein. The process of BSA protein adsorption onto polymer latex microsphere drug accords with the first order kinetics under ideal condition. Electrostatic attraction plays an important role in the BSA adsorption.
以疏水性单体苯乙烯St和甲基丙烯酸环氧丙酯GMA为单体,采用无皂乳液聚合的方法制备了表面带环氧基的高分子乳液微球PSG聚(苯乙烯-甲基丙烯酸环氧丙酯)。为降低微球表面的空间位阻效应和壁面效应,采用1,6-已二胺作为空间臂分子,对PSG微球进行表面修饰,获得了端氨基的微球粒子PSGN(PSG-1,6-已二胺)。并采用元素分析法和4-硝基苯甲醛法分析测定了微球表面的氨基浓度。然后采用培美曲塞二钠MTA与PSGN微球表面的氨基进行共价偶联反应,制得具有药物固定的亲和高分子微球。实验结果表明培美曲塞二钠药物分子固定在PSGN微球表面的接枝量主要受微球粒径大小和活泼氨基浓度的影响；通过改变投料比和功能单体多段加料的方式可控制乳液微球粒径大小和表面活泼基团的浓度,进而实现对药物接枝量的控制。另外,实验还考察了体系pH、催化剂比例及用量和温度对MTA偶联反应的影响,获得了药物分子偶联在PSGN微球表面的最佳反应条件。
此外还研究了药物固定高分子微球对蛋白质的吸附特性。采用牛血蛋白质BSA作为模型蛋白质,着重考察了pH、离子强度和不同BSA初始浓度对于蛋白质吸附的影响。结果表明：MTA药物分子固定在微球表面的浓度较高、分布均匀时,微球药物对BSA的吸附动力学较符合一级动力学过程,并且静电引力对于蛋白质吸附具有重要影响。
Affinity polymer latex microsphere is a kind of functional polymer microsphere, which has affinity molecules or groups on its surface to efficiently recognize and separate the target molecules by the high specific surface areas of the microsphere. There are high degree of specific recognition and coordination between affinity molecules and target molecules. These objects mainly include drug-receptor, antibody-antigen, complementary DNA chains, etc. Affinity polymer latex microspheres have the common advantages of functional polymer microspheres, i.e., stable dispersion, large specific surface areas as well as versatile properties. In addition, these smart latex microspheres have several particular features, such as well mechanical performance, sensitivity, excellent biology compatibility, easy to recovery and repetitious to use, friendly to environment. Affinity polymer latex microspheres showed potential application for bioseparation, antibody identification, DNA diagnosis as well as efficient evaluation of medicine. For example, the affinity microspheres hold the bio-inert and high surface area can be used for identification, separation and purification of drug receptor from biological tissue by immobilizing the drug molecules on its surface. Expand the ideals of new drugs development and shorten the drug development cycle by studying the mechanism of drug and drug receptor.
Poly (styrene-co-glycidyl methacrylate) (PSG) latex microspheres with epoxy groups on its surface are prepared by soap-free emulsion polymerization with hydrophobic monomer styrene and glycidyl methacrylate. The PSG microspheres are modified with 1,6-hexanediamine space arm to reduce the steric hindrance and side interaction of the surface and the amino-modified microsphere PSGN was obtained. The content of amino groups on PSGN particle is measured by elemental analysis and 4-nitrobenzaldehyd methods. Then the pemetrexed disodium (MTA) is covalent immobilized on the surface of the PSGN particles through the amidation between the carboxylic acid end group of MTA in acidic medium and the amino groups on the PSGN particle surface. The experimental results suggest MTA immobilization is mainly dependent upon both the particle size and active amino groups on the PSGN surface. The amino group density of the PSGN microsphere surface could be controlled through altering the ratio of feed and the mode of operation. Further, the immobilization of MTA is also controllable. Researches on the influence of pH, temperature and the different ratio and concentration of catalysts on MTA coupling reaction are performed and the optimum reaction conditions are earned.
The adsorption property of BSA protein onto the surface of polymer microsphere drug is emphatically studied. Bovine serum albumin (BSA) is selected as the model protein. The effect of pH, ion concentration and different first BSA concentration on adsorption is emphatically examined. The results showed the higher covalent immobilization concentration and uniform distribution of MTA on the polymer microspheres have the regular adsorption process of BSA protein. The process of BSA protein adsorption onto polymer latex microsphere drug accords with the first order kinetics under ideal condition. Electrostatic attraction plays an important role in the BSA adsorption.
引文
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