Novel functionalized polymer/fibrin self-assembling monolayers

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。

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.