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: $ 1.46万
• 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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=638071
• 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 Objectives1. 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.

该计划的长期目标是研究壳聚糖与柠檬酸等离子体（一种复杂且高度相关的生物系统）的动态相互作用，以开发用于制造功能化聚合物/纤维蛋白自组装单分子膜（SAM）的新策略。柠檬酸盐血浆是一种天然来源的液体，含有将纤维蛋白原加工成纤维蛋白（一种自组装蛋白复合物）所需的所有酶原、辅因子和底物。我们最近发现，壳聚糖，一种天然来源的阳离子多糖，与关键的阴离子酶原（无活性的酶前体）形成静电复合物，并减弱纤维蛋白聚合的爆发。我们还确定了外泌体，从活化的血小板脱落的脂质囊泡，增加纤维蛋白分支并减少原纤维直径。在这个项目中，我们将测试的假设，纤维蛋白自组装和最终的纤维蛋白结构的动力学可以控制的纯化外泌体的表面特性和结构不同的壳聚糖的理化性质。具体假设1。纤维蛋白自组装的速率和最终的聚合结构（拉伸强度、原纤维厚度、压缩、分支）可以通过壳聚糖-酶原相互作用来控制.壳聚糖与外来体形成复合物，其结合亲和力通过正电荷状态（即，较高的葡糖胺含量和Mw），并抑制其促凝血活性。3.可以产生具有受控纤维蛋白纤维直径并掺入周期性间隔的官能团的壳聚糖/外泌体/纤维蛋白SAM，并用于控制细胞行为。具体目标1。建立时间依赖性纤维蛋白自组装、凝血酶活化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。制备具有均匀分布的功能基团的杂合壳聚糖/外泌体/纤维蛋白SAM，并分析SAM-细胞相互作用。目标1：制备结构不同的壳聚糖文库，分析壳聚糖结构对纤维蛋白自组装动力学的影响，如通过血栓弹力图（一种类似于流变仪的技术）测量的。在目标2中，外泌体将通过不对称流场流分级纯化，并表征直径、表面化学、电荷状态和在添加和不添加壳聚糖的情况下诱导纤维蛋白分支的能力。将生成将最终纤维蛋白原纤维直径与壳聚糖物理化学性质和外泌体表面化学相关联的理论模型。在目标3中，我们的目标是生产具有受控纤维蛋白纤维直径（40、100、250 nm）的SAM，并且用均匀间隔在1和100 µm之间的生物活性因子进行功能化。将使用荧光组分在玻璃显微镜载玻片上生成SAM，以分析分子相互作用。通过进一步添加生物素-壳聚糖和生物素-仿生肽，以优化的间距掺入抗生物素蛋白-金纳米颗粒或抗生物素蛋白-生物活性因子，将产生功能化的SAM。然后，我们将使用逐层SAM来建立基质中生物活性因子的受控梯度。这些LBL-SAM将用于分析细胞迁移至基质中。这些多功能的自组装基质在生物材料、细胞科学、分离方法和纳米技术中具有广泛的潜在应用。