Development of a method for in situ nanorheology of human airway mucus

描述（由申请人提供）：我们提出开发一种新的纳米流变学平台，以同时原位绘制粘液粘弹性和粘液流动。气道粘液复合粘弹性剪切模量（G*）的增加阻碍粘液清除，并且与包括慢性阻塞性肺病（COPD）和囊性纤维化（CF）的呼吸系统疾病相关。在短期内，这个新平台将提供一个更完整的粘膜纤毛清除的生物物理图像，并实现更好的模型，从而产生新的治疗模式。从长远来看，该平台可以转化为体内粘液监测，用于评估疾病进展和治疗干预。 我们的纳米流变学平台基于使用光学相干断层扫描（OCT）监测的等离子体金纳米棒（GNR）的动态光散射（DLS）。具体地，来自GNR的交叉偏振光散射信号提供关于其旋转扩散速率的信息，其通过广义斯托克斯-爱因斯坦关系与粘液的G* 相关。GNR作为流变学探针是有利的，因为它们的高各向异性和等离子体共振提供了针对内源性粘液光散射体的独特光学信号，并且它们的小尺寸提供了快速旋转DLS信号，这允许更快速地收集流变学数据。重要的是，OCT提供GNR扩散的深度分辨DLS测量，这将使得能够对粘液G* 中的异质性进行空间映射，特别是作为与睫状体层的距离的函数。通过将这些测量与基于OCT的粘液流动跟踪相关联，将提供粘液纤毛清除的更完整的生物物理图像。我们的第一个目标是验证DLS-OCT纳米流变学技术在受控复杂流体对标准珠微观流变学和本体流变学。随后，我们将使用从人支气管上皮（hBE）培养物收获的粘液中的非粘膜粘附GNR来验证该技术，并评价作为%固体的函数的响应。我们的第二个目标是采用DLS-OCT监测正常和CF样hBE模型系统中的流动粘液，使用通过雾化递送的GNR。我们将把G* 的测量值与OCT中斑点跟踪测量的流速作为与睫状体层距离的函数相关联。此外，我们将确定OCT在模拟高渗盐水治疗期间监测睫状体功能的能力，并在这些模型中诱导睫状体活动的塌陷和恢复。 通过完成这项研究，我们将验证DLS-OCT纳米流变学对微观和整体流变学技术，并建立了敏感性映射G* 和定量%固体在流动的粘液系统，这将平衡的技术，为未来的体内研究在小鼠模型的囊性纤维化。

DESCRIPTION (provided by applicant): We propose to develop a novel nanorheology platform to simultaneously map mucus viscoelasticity and mucus flow in situ. Increase in airway mucus complex viscoelastic shear modulus (G*) hinders mucus clearance and is associated with respiratory diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). In the short term, this new platform will provide a more complete biophysical picture of mucociliary clearance and enable better models, leading to new treatment paradigms. In the long term, this platform may be translatable to in vivo mucus monitoring for assessment of disease progression and therapeutic interventions. Our nanorheology platform is based upon dynamic light scattering (DLS) from plasmonic gold nanorods (GNRs) monitored using optical coherence tomography (OCT). Specifically, the cross-polarized light scattering signal from GNRs provides information about their rotational diffusion rate, which is related through a generalized Stokes-Einstein relationship to the G* of the mucus. GNRs are advantageous as rheological probes because their high anisotropy and plasmon-resonance provides a unique optical signal against endogenous mucus light scatterers, and their small size provides a fast rotational DLS signal which allows for more rapid collection of rheological data. Importantly, OCT provides depth-resolved DLS measurements of GNR diffusion, which will enable spatial mapping of heterogeneities in mucus G*, particularly as a function of distance from the periciliary layer. By linking these measurements with OCT- based tracking of mucus flow, it will provide a more complete biophysical picture of mucociliary clearance. Our first aim is to validate the DLS-OCT nanorheology technique in controlled complex fluids against standard bead microrheology and bulk rheology. Subsequently, we will validate this technique using non- muco-adherent GNRs in mucus harvested from human bronchi-epithelial (hBE) cultures, and evaluate the response as a function of % solids. Our second aim is to employ DLS-OCT to monitor flowing mucus in normal and CF-like hBE model systems, using GNRs delivered by nebulization. We will associate measurements of G* with flow rate measured by speckle-tracking in OCT as a function of distance from the periciliary layer. In addition, we will determin the ability for OCT to monitor ciliary function during simulated hypertonic saline therapy and induced collapse and revival of ciliary activity in these models. By the completion of this study we will have validated DLS-OCT nanorheology against micro- and bulk rheology techniques, and established the sensitivity for mapping G* and quantifying % solids in a flowing mucus system, which will poise the technology for future in vivo studies in murine models of cystic fibrosis.