Novel functionalized polymer/fibrin self-assembling monolayers

该计划的长期目标是研究壳聚糖与柠檬酸血浆这一复杂且高度相关的生物体系的动态相互作用，以开发制备功能化聚合物/纤维蛋白自组装单分子层(SAM)的新策略。柠檬酸血浆是一种自然衍生的液体，含有将纤维蛋白原转化为纤维蛋白所需的所有酵素、辅因子和底物，纤维蛋白是一种自组装蛋白质复合体。我们最近发现，壳聚糖是一种天然的阳离子多糖，能与关键的阴离子酶原(非活性酶前体)形成静电络合物，并抑制纤维蛋白的突然聚合。我们还确定，外体，即从激活的血小板中脱落的脂泡，增加了纤维蛋白的分支，减少了纤维直径。在这个项目中，我们将验证这样一个假设，即纤维蛋白自组装和最终纤维蛋白结构的动力学可以由纯化的外切体的表面特征和结构不同的壳聚糖的物理化学性质来控制。具体假设1.纤维蛋白的自组装速率和最终聚合结构(抗拉强度、纤维厚度、紧实度、支化)可以通过壳聚糖-酶原相互作用来控制。壳聚糖与胞外体形成络合物，结合亲和力随着正电荷态(即较高的氨基葡萄糖含量和相对分子质量)的增加而增加，并抑制其促凝血活性。3.具有可控制的纤维纤维直径并结合了周期性间隔的官能团的壳聚糖/外切体/纤维蛋白SAM可以被创建并用于控制细胞行为。具体目标1。建立了依赖时间的纤维蛋白自组装、凝血酶激活和壳聚糖结构之间的统计相关模型。表征不同结构的壳聚糖与不同类型的外切体形成复合体的能力，并调节它们诱导纤维原纤维分支的能力。制备含等间距官能团的壳聚糖/外切体/纤维蛋白杂化自组装膜，并分析自组装膜与细胞的相互作用。对于目标1，我们将构建一个结构不同的壳聚糖库，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，外切体将通过不对称流场流动分离纯化，并对其直径、表面化学、电荷状态以及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进行表征。最终的纤维原纤维直径与壳聚糖的物理化学性质和外切体表面化学之间的理论模型将会被产生。在目标3中，我们的目标是制造纤维直径可控(40、100、250 nm)的SAM，并使用均匀间隔在1至100微米之间的生物活性因子进行功能化。SAM将使用荧光成分在玻璃显微镜载玻片上生成，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，以最佳间距掺入亲和素-金纳米颗粒或亲和素-生物活性因子，将创建功能化的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将用于分析细胞向基质中的迁移。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。

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.