Thermosensitive display of ligand molecules on microgel scaffolds to facilitate switchable bioadhe-sion

在生物传感、组织工程、药物输送和其他领域，具有可切换的细胞或病原体相互作用的刺激响应聚合物涂层变得越来越重要。然而，文献中的绝大多数报道都描述了通过在温度或ph等刺激下切换非特异性相互作用（即疏水性或位阻斥力）来改变其对生物系统的亲和力的材料。因此，本项目的广泛目标是建立具有配体部分热敏显示的“智能”微凝胶系统，将特异性相互作用切换到蛋白质受体。最近，我们建立了与各种配体偶联的非反应性水凝胶微凝胶，与蛋白质受体表现出高度特异性的相互作用。这种水凝胶支架的受体结合亲和力可以通过改变共轭配体的多价显示来调节，例如通过改变它们的密度、连接剂化学或聚合物主链。在这个项目中，我们现在将这种方法扩展到以聚（n -异丙基丙烯酰胺）（PNIPAM）为响应性聚合物基质的微凝胶，通过温度刺激可以改变凝胶颗粒的表面积及其配体的显示。此外，我们打算利用PNIPAM在温度变化时疏水性的变化来控制配体的显示。因此，将生物素、甘露糖和透明质酸作为具有不同疏水性的高度相关配体系统的案例研究将耦合到PNIPAM微凝胶上，并通过量热受体结合研究来测试它们的温度依赖性表现。此外，配体在微凝胶内的径向分布将发生变化，形成具有功能化壳和热敏核的微凝胶，以提高它们相互作用的特异性。考虑到微凝胶网络结构的温度依赖性，我们设想配体功能化微凝胶的表面涂层具有捕获和释放病原体的能力。因此，将通过荧光、光散射和力谱测量来研究温度依赖性配体显示、微凝胶力学和微凝胶的生物粘附性。最后，将通过研究大肠杆菌在甘露糖功能化微凝胶膜上的粘附性来测试其积累和释放病原体的能力。总之，本研究建立的微凝胶偶联物可能代表了一种通过温度刺激来控制聚合物支架中配体活性的新方法。本提案主要关注响应凝胶中配体显示的整体可行性、性能和机理等基本问题。作为第一个概念验证应用，项目的最后一部分将集中在材料科学方面和配体共轭PNIPAM微凝胶作为生物活性涂层的应用。

Stimuli-responsive polymer coatings with switchable interaction to cells or pathogens are becoming increasingly important in the context of biosensing, tissue engineering, drug delivery and other areas. However, the great majority of reports in the literature describe materials that change their affinity to biological systems by switching non-specific interactions, i.e. hydrophobicity or steric repulsion upon stimuli like temperature or pH. Therefore, it is the broad aim of this project to establish "smart" microgel systems with thermosensitive display of ligand moieties to switch the specific interaction to protein receptors. Recently, we have established non-responsive hydrogel-based microgels conjugated with various ligands exhibiting highly specific interactions to protein receptors. The receptor binding affinity of such hydrogel scaffolds could be tuned by varying the multivalent display of conjugated ligands, e.g. by changing their density, linker chemistry or polymer backbone. Within this project, we now extend this approach towards microgels with poly(N-isopropylacryalamide) (PNIPAM) as responsive polymer matrix that allow changing surface area of gel particles and their ligand display by means of temperature stimulus. Furthermore, we intend to utilize the shift in hydrophobicity of PNIPAM upon temperature change to control ligand display. Therefore, case studies on biotin, mannose and hyaluronic acid as highly relevant ligand systems with varying degree of hydrophobicity will be coupled to PNIPAM microgels and their temperature dependent presentation will be tested by calorimetric receptor binding studies. In addition, the radial distribution of ligands within the microgel will be varied to form microgels with a functionalized shell and a thermosensitive core in order to improve the specificity of their interaction. Taking the temperature dependence of the microgels’ network structure into consideration, we envision surface coatings of ligand-functionalized microgels with the ability to catch and release pathogens. Therefore, the temperature dependent ligand display, microgel mechanics and bio-adhesion of the microgels will be studied via fluorescence, light scattering and force spectroscopy measurements. Finally, their ability to accumulate and release pathogens will be tested by studying the adhesion of E. coli on mannose functionalized microgel films.Overall, the microgel conjugates to be established in this proposal may represent a new way to control the activity of ligands in a polymer scaffolds by temperature stimulus. This proposal mainly focuses on fundamental questions, i.e. the overall feasibility, performance and mechanistic aspects of ligand display in responsive gels. As a first proof-of-concept application, the last part of the project will concentrate on material science aspects and application of ligand conjugated PNIPAM microgels as bioactive coating.