Behravesh Esfandiar, Sikavitsas Vassilios I, Mikos Antonios G
Department of Bioengineering, Rice University, MS-142, P.O. Box 1892, Houston, TX 77251, USA.
Biomaterials. 2003 Nov;24(24):4365-74. doi: 10.1016/s0142-9612(03)00338-7.
This study describes a method for the quantification of active ligand surface concentration for bulk-modified hydrogels. Two poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) block copolymers were synthesized with terminal poly(ethylene glycol) (PEG) chains of number average molecular weight 1960 and 5190 g/mol. Hydrogels were synthesized with bulk-modified biotin as a model ligand, making use of a PEG spacer arm with a molecular weight of 3400 g/mol. Bulk concentration of biotin was calculated from the initial concentration of biotin, sol fraction, equilibrium water content, and relative incorporation of the polymers to the hydrogel. Surface concentration of biotin bulk-modified hydrogels was quantified with an enzyme linked immunosorbent assay using mouse monoclonal anti-biotin antibody (IgG), horseradish peroxidase-conjugated anti-mouse IgG, and a chemiluminescent substrate. The larger size of the IgG relative to the mesh size of the hydrogels allowed for the quantification of the active biotin at the surface of the hydrogels. Luminescent imaging was used to qualitatively show the isolation of the horseradish peroxidase-conjugated antibodies to the surface of the bulk-modified hydrogel. The active biotin ligands at the surface of hydrogels synthesized with terminal PEG chains of 1960 g/mol were at the top 7.2 nm while for those synthesized with terminal PEG chains of 5190 g/mol were at the top 4.4 nm of the bulk-modified hydrogel. The relationship between bulk ligand concentration and the active ligand concentration at the surface was dependent on the hydrogel composition. The relative magnitude of the PEG spacer arm of the ligand compared to the PEG block length of the copolymer affected the surface availability of the ligand. The results suggest that steric hindrances caused by mobile PEG chains of the copolymer of molecular weight greater than that of the PEG spacer arm contributed to the decreased surface concentration of ligand. This work relates the bulk concentration of a ligand to its surface concentration, an important parameter for the adhesion, migration, and function of anchorage dependent cells.
本研究描述了一种用于定量本体改性水凝胶中活性配体表面浓度的方法。合成了两种聚（富马酸丙二醇酯 - 共 - 乙二醇）（P(PF - co - EG)）嵌段共聚物，其末端聚乙二醇（PEG）链的数均分子量分别为1960和5190 g/mol。以本体改性生物素作为模型配体合成水凝胶，使用分子量为3400 g/mol的PEG间隔臂。根据生物素的初始浓度、溶胶分数、平衡含水量以及聚合物与水凝胶的相对掺入量计算生物素的本体浓度。使用小鼠单克隆抗生物素抗体（IgG）、辣根过氧化物酶偶联的抗小鼠IgG和化学发光底物，通过酶联免疫吸附测定法定量生物素本体改性水凝胶的表面浓度。IgG相对于水凝胶网孔尺寸更大，这使得能够定量水凝胶表面的活性生物素。使用发光成像定性地显示辣根过氧化物酶偶联抗体在本体改性水凝胶表面的隔离情况。用1960 g/mol末端PEG链合成的水凝胶表面的活性生物素配体位于本体改性水凝胶顶部7.2 nm处，而用5190 g/mol末端PEG链合成的水凝胶表面的活性生物素配体位于本体改性水凝胶顶部4.4 nm处。本体配体浓度与表面活性配体浓度之间的关系取决于水凝胶组成。配体的PEG间隔臂与共聚物的PEG嵌段长度相比的相对大小影响配体的表面可及性。结果表明，分子量大于PEG间隔臂的共聚物的可移动PEG链引起的空间位阻导致配体表面浓度降低。这项工作将配体的本体浓度与其表面浓度联系起来，表面浓度是锚定依赖性细胞粘附、迁移和功能的重要参数。
