Imaging nanophysical properties of actively transporting bronchial mucus

项目摘要 粘液覆盖在肺上皮上，捕获我们每天吸入的数千种病原体。人类 衬在肺内的支气管上皮（hBE）细胞具有纤毛，通过剪切力推动粘液，这是一种机制， 称为粘膜纤毛转运（MCT）。MCT的作用是清除粘液，提供一个主要的防御， 病原体在呼吸系统疾病，如囊性纤维化（CF）和慢性阻塞性肺病 （COPD），MCT分解，导致慢性感染、气道组织损伤，最终导致发病 and mortality. MCT的这种损失与粘液脱水直接相关（即，增加粘液固体 浓度）。正因为如此，针对气道粘液的水合或稀释的治疗方法正在开发中 在COPD和CF患者中重新建立MCT，尽管它们的疗效甚微。重要的是 对MCT的这种浓度依赖性效应的潜在机制尚不清楚; 粘液性质的变化作为粘液浓度的函数已被广泛研究，但 缺乏在纤毛用于运输粘液的振荡剪切力下测量粘液的方法。我们 假设构成粘液的大分子（粘蛋白）的纳米结构被纤毛修饰， 以浓度依赖的方式产生剪切力，这决定了纤毛剪切力在 粘液层，使MCT。更好地理解这些非均匀的、与剪切相关的性质 将提供所需的洞察力的战略，为发展更有效的粘液稀释疗法。 在这里，我们提出了一种生物分析工具，以图像纳米结构的变化，粘液经历积极的粘膜， 纤毛运输，同时定量MCT。我们已经证明聚乙二醇化金纳米棒 GNRs容易扩散到人气道粘液中，并且使用光学相干断层扫描（OCT）， GNR的光散射提供了GNR扩散速率的精确测量， 与粘液浓度相关。我们将使用GNR的扩散敏感OCT（DS-OCT）来深度分辨 粘液层内的粘液纳米孔隙，从高剪切睫状体周围层（PCL）到无应力空气 边界通过跟踪内源性散射体同时测量粘液流场， 嵌入的微珠将提供剪切应变和MCT速度。我们的方法是首先验证 在平行板剪切系统（PPSS）中进行测量，该系统将受控的纤毛样振荡剪切施加在 熟悉的液体然后，我们将对hBE粘液进行PPSS测量，以确定发病条件 剪切稀化和纳米孔隙度变化。我们的第二个目标将是执行这些相同的测量 在主动运输粘液的体外模型上研究剪切稀化和纳米孔隙度对 MCT。最后，将在应用粘液水合剂以进行研究期间和之后获得测量结果。 治疗期间粘液水合作用和MCT重建的动态效应。这些研究将提供 与体内类似疾病的重要联系，提供了以前无法获得的关于MCT和药物治疗的见解。

Project Summary Mucus coats the lung epithelium and traps thousands of pathogens that we inhale every day. Human bronchial epithelial (hBE) cells lining the lung have cilia that propel mucus via shear forces, a mechanism known as muco-ciliary transport (MCT). MCT acts to clear mucus, providing a primary defense against trapped pathogens. In respiratory diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), MCT breaks down, leading to chronic infection, damage to airway tissues, and ultimately, morbidity and mortality. This loss of MCT is directly associated with mucus dehydration (i.e., increasing mucus solids concentration). Because of this, therapies that target hydrating or thinning airway mucus are being developed to re-establish MCT in patients with COPD and CF, although they are only marginally effective. Importantly, the underlying mechanism for this concentration-dependent effect on MCT is not well understood; bulk rheological changes in mucus properties have been extensively studied as a function of mucus concentration, but there is a lack of methods to measure mucus under oscillatory shear forces that cilia apply to transport mucus. We hypothesize that the nanostructure of the macromolecules (mucins) that comprise mucus is modified by ciliary shear forces in a concentration-dependent way, which dictates how ciliary shear forces are propagated within the mucus layer to enable MCT. A better understanding of these heterogeneous, shear-dependent properties of mucus will provide needed insight into strategies for developing more effective mucus thinning therapies. Here we propose a bioanalytical tool to image nanostructural changes in mucus undergoing active muco- ciliary transport, while simultaneously quantifying MCT. We have already shown that PEGylated gold nanorods (GNRs) readily diffuse into human airway mucus, and using optical coherence tomography (OCT), the dynamic light scattering from GNRs provides an accurate measurement of GNR diffusion rate that is inversely correlated with mucus concentration. We will use diffusion-sensitive OCT (DS-OCT) of GNRs to depth-resolve mucus nanoporosity within the mucus layer, from the high-shear peri-ciliary layer (PCL) to the stress-free air boundary. Simultaneous measurements of the mucus flow field by tracking endogenous scatterers or embedded microbeads will provide shear strain and MCT velocity. Our approach will be to first validate measurements in a parallel-plate shearing system (PPSS) that applies controlled, cilia-like oscillatory shear on well-known fluids. We will then perform PPSS measurements on hBE mucus to establish the onset conditions for shear-thinning and nanoporosity changes. Our second Aim will be to perform these same measurements on an in vitro model of actively transporting mucus to study the role of shear-thinning and nanoporosity on MCT. Finally, measurements will be obtained during and after application of mucus hydrating agents to study dynamic effects of mucus hydration and re-establishment of MCT during treatment. These studies will provide an important link to in vivo-like conditions, providing previously inaccessible insight on MCT and drug therapies.