Novel functionalized polymer/fibrin self-assembling monolayers

新型功能化聚合物/纤维蛋白自组装单层

• 批准号: RGPIN-2014-05626
• 负责人: Hoemann, Caroline
• 金额: $ 1.41万
• 依托单位: École Polytechnique de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=653692
• 关键词: Novel; functionalized; polymer; fibrin; self

The long-term objective of the program is to study the dynamic interactions of chitosan with citrated plasma, a complex and highly relevant biological system, in order to develop new strategies for the fabrication of functionalized polymer/fibrin self-assembling monolayers (SAM). Citrated plasma is a naturally-derived fluid that contains all zymogens, co-factors, and substrates required to process fibrinogen to fibrin, a self-assembling protein complex. We recently discovered that chitosan, a naturally-derived cationic polysaccharide, forms electrostatic complexes with key anionic zymogens (inactive enzyme precursors), and attenuates burst fibrin polymerization. We also determined that exosomes, lipid vesicles shed from activated platelets, increase fibrin branching and reduce fibril diameter. In this project we will test the hypothesis that the kinetics of fibrin self-assembly and final fibrin structure can be controlled by the surface characteristics of purified exosomes and the physicochemical properties of structurally distinct chitosans. **Specific Hypotheses *1. The rate of fibrin self-assembly and final polymerized structure (tensile strength, fibril thickness, compaction, branching) can be controlled by chitosan-zymogen interactions.*2. Chitosan forms complexes with exosomes, with a binding affinity increased by positive charge state (i.e., higher glucosamine content and Mw), and inhibits their pro-coagulant activity. *3. Chitosan/exosome/fibrin SAM with controlled fibrin fiber diameter and incorporated with periodically spaced functional groups can be created and used to control cell behavior. **Specific Objectives*1. Develop a statistical correlative model between time-dependent fibrin self-assembly, thrombin activation, and chitosan structure.*2. Characterize the ability of structurally distinct chitosans to form complexes with different types of exosomes and modulate their ability to induce fibrin fibril branching.*3. Generate hybrid chitosan/exosome/fibrin SAM bearing evenly spaced functional groups and analyze SAM-cell interactions.**For Objective 1, a library of structurally distinct chitosans will be generated, to analyze the effect of chitosan structure on the kinetics of fibrin self-assembly as measured by thrombelastography, a technique similar to rheometry. In objective 2, exosomes will be purified by asymmetric flow field flow fractionation, and characterized for diameter, surface chemistry, charge state, and ability to induce fibrin branching with and without added chitosan. A theoretical model correlating final fibrin fibril diameter with chitosan physicochemical properties and exosome surface chemistry will be generated. In objective 3, we aim to produce SAM with controlled fibrin fiber diameters (40, 100, 250 nm), and functionalized with bioactive factors homogeneously spaced between 1 and 100 µm. SAM will be generated on glass microscope slides using fluorescent components to dissect the molecular interactions. Functionalized SAM will be created by further adding biotin-chitosan and biotin-biomimetic peptides to incorporate avidin-gold nanoparticles or avidin-bioactive factors with optimized spacing. We will then use layer-by-layer SAM to establish controlled gradients of bioactive factors in the matrix. These LBL-SAM will be used to analyze cell migration into the matrix. These versatile self-assembling matrices have a broad array of potential applications in biomaterials, cell science, separation methods, and nanotechnology.

该项目的长期目标是研究壳聚糖与柠檬酸血浆（一个复杂且高度相关的生物系统）的动态相互作用，以开发制造功能化聚合物/纤维蛋白自组装单层（SAM）的新策略。柠檬酸血浆是一种天然衍生的液体，含有将纤维蛋白原加工成纤维蛋白所需的所有酶原、辅助因子和底物，纤维蛋白是一种自组装蛋白质复合物。我们最近发现，壳聚糖，一种天然衍生的阳离子多糖，与关键的阴离子酶原（无活性的酶前体）形成静电复合物，并减弱纤维蛋白的爆发聚合。我们还确定外泌体，脂质囊泡从活化的血小板脱落，增加纤维蛋白分支和减少纤维直径。在这个项目中，我们将测试纤维蛋白自组装动力学和最终纤维蛋白结构可以由纯化外泌体的表面特征和结构不同的壳聚糖的物理化学性质控制的假设。**具体假设*纤维蛋白的自组装速率和最终聚合结构（拉伸强度、纤维厚度、压实度、分支）可以通过壳聚糖-酶原相互作用来控制。壳聚糖与外泌体形成复合物，通过正电荷状态（即更高的葡萄糖胺含量和分子量）增加其结合亲和力，抑制其促凝活性。* 3。壳聚糖/外泌体/纤维蛋白SAM具有可控制的纤维蛋白纤维直径，并结合周期性间隔的官能团，可用于控制细胞行为。* *具体目标* 1。建立时间依赖性纤维蛋白自组装、凝血酶激活和壳聚糖结构之间的统计相关模型。表征结构不同的壳聚糖与不同类型的外泌体形成复合物的能力，并调节其诱导纤维蛋白原纤维分支的能力。生成具有均匀间隔官能团的杂化壳聚糖/外泌体/纤维蛋白SAM，并分析SAM与细胞的相互作用。**对于目的1，将生成一个结构不同的壳聚糖文库，以分析壳聚糖结构对纤维蛋白自组装动力学的影响，这是一种类似于流变学的技术。在目标2中，外泌体将通过不对称流场流动分馏法纯化，并对添加和不添加壳聚糖的外泌体的直径、表面化学、电荷状态和诱导纤维蛋白分支的能力进行表征。最终纤维蛋白原纤维直径与壳聚糖的物理化学性质和外泌体表面化学性质之间的关系将产生一个理论模型。在目标3中，我们的目标是生产具有可控制纤维蛋白纤维直径（40,100,250 nm）的SAM，并具有均匀间隔在1至100 μ m之间的生物活性因子功能化。SAM将在玻璃显微镜载玻片上产生，使用荧光成分来解剖分子相互作用。通过进一步添加生物素-壳聚糖和生物素-仿生肽，以优化间距加入亲和金纳米颗粒或亲和金生物活性因子，形成功能化的SAM。然后，我们将使用一层一层的SAM来建立基质中生物活性因子的可控梯度。这些LBL-SAM将用于分析细胞向基质中的迁移。这些多功能自组装基质在生物材料、细胞科学、分离方法和纳米技术方面具有广泛的潜在应用。