Steffens G C M, Nothdurft L, Buse G, Thissen H, Höcker H, Klee D
Department of Biochemistry, Institut für Biochemie, Universitätsklinikum RWTH, Aachen, Germany.
Biomaterials. 2002 Aug;23(16):3523-31. doi: 10.1016/s0142-9612(02)00091-1.
The use of graft polymers for the functionalisation of biomaterial surfaces is already widespread. We investigated the adsorptive and covalent binding of a variety of proteins and peptides to poly(D,L-lactide) grafted with polyacrylic acid. Covalent attachment was achieved through coupling of amino groups of the protein/peptide to the carboxyl groups of the graft polymer by using a water-soluble carbodiimide and N-hydroxysuccinimide. Binding densities were determined by automated amino acid analysis after acid hydrolysis of both the poly(D,L-lactide) and the adsorbed and covalently bound proteins. Experiments in the absence and presence of the coupling reagents allow to discriminate between adsorptive and covalent binding. Although the adsorptivc binding is quite substantial in absolute terms, the amount of adsorbed protein is relatively low as compared to the total amount of bound protein. Total binding densities of 20-30 microg/cm2 can easily be achieved. Depending on the concentration and on the properties of the proteins and peptides, between 5% and 80% of the totally bound protein may be physically adsorbed. Densities expressed in molecules/10 nm2 vary from 0.5 molecule fibronectin to 2,000 laminin-peptide molecules: their binding densities clearly correlate with their respective molecular masses. Obviously, the binding densities are governed by their individual three-dimensional space requirements rather than the density of the available carboxyl groups. From the number of carboxyl groups/10 nm2 (18,000-30,000 COOH/10 nm2) the average length of the acrylic acid graft polymer molecules was estimated. Based on the assumption that about 10 copolymer chains can be accommodated on 10 nm2, the average length of the polymer chains, which corresponds to the thickness of the graft phase, is estimated to be 0.5-1 microm. The organisation of the proteins and peptides within the polyacrylic acid phase was further investigated by experiments in which a protein (BSA) and a peptide (Val-Lys) were allowed to react in either a singular, a consecutive or a simultaneous way. Together with XPS and IR-ATR surface characterisation experiments a three-dimensional picture of the arrangement of the immobilised proteins and peptides within the graft polymer phase emerges.
接枝聚合物用于生物材料表面功能化的应用已经十分广泛。我们研究了多种蛋白质和肽与接枝有聚丙烯酸的聚（D，L-丙交酯）的吸附和共价结合。通过使用水溶性碳二亚胺和N-羟基琥珀酰亚胺，将蛋白质/肽的氨基与接枝聚合物的羧基偶联，实现共价连接。在对聚（D，L-丙交酯）以及吸附和共价结合的蛋白质进行酸水解后，通过自动氨基酸分析测定结合密度。在有无偶联剂的情况下进行实验，可以区分吸附结合和共价结合。尽管从绝对值来看吸附结合相当可观，但与结合蛋白质的总量相比，吸附的蛋白质量相对较低。很容易实现20 - 30微克/平方厘米的总结合密度。根据蛋白质和肽的浓度及性质，5%至80%的总结合蛋白可能是物理吸附的。以分子/10纳米²表示的密度从0.5个纤连蛋白分子到2000个层粘连蛋白 - 肽分子不等：它们的结合密度显然与其各自的分子量相关。显然，结合密度由它们各自的三维空间需求决定，而非可用羧基的密度。根据每10纳米²的羧基数量（18,000 - 30,000个COOH/10纳米²）估算了丙烯酸接枝聚合物分子的平均长度。基于每10纳米²可容纳约10条共聚物链的假设，聚合物链的平均长度（对应接枝相的厚度）估计为0.5 - 1微米。通过使蛋白质（牛血清白蛋白）和肽（缬氨酸 - 赖氨酸）以单一、连续或同时的方式反应的实验，进一步研究了聚丙烯酸相内蛋白质和肽的组织情况。结合X射线光电子能谱（XPS）和红外衰减全反射（IR - ATR）表面表征实验，一幅接枝聚合物相内固定化蛋白质和肽排列的三维图像浮现出来。
