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

描述（由申请人提供）：我们建议开发一种新的纳米流变学平台，同时绘制黏液的粘弹性和黏液的原位流动。气道黏液复合物粘弹性剪切模量（G*）的增加阻碍了黏液的清除，并与包括慢性阻塞性肺疾病（COPD）和囊性纤维化（CF）在内的呼吸系统疾病有关。在短期内，这个新的平台将提供一个更完整的粘液纤毛清除的生物物理图像，并使更好的模型，导致新的治疗范例。从长远来看，该平台可以转化为体内粘液监测，以评估疾病进展和治疗干预措施。我们的纳米流变平台是基于使用光学相干断层扫描（OCT）监测等离子体金纳米棒（gnr）的动态光散射（DLS）。具体来说，来自gnr的交叉偏振光散射信号提供了关于它们的旋转扩散速率的信息，这是通过广义Stokes-Einstein关系与黏液的G*相关的。gnr作为流变探针具有优势，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，允许更快速地收集流变数据。重要的是，OCT提供了GNR扩散的深度分辨DLS测量，这将使粘液G*的异质性的空间映射成为可能，特别是作为与睫周层距离的函数。通过将这些测量与基于OCT的黏液流动跟踪联系起来，它将提供更完整的黏液纤毛清除的生物物理图像。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中与标准珠微流变学和体流变学的对比。随后，我们将在人类支气管上皮（hBE）培养的粘液中使用非黏液粘附的gnr来验证该技术，并评估其作为固体百分比的函数的反应。我们的第二个目标是使用DLS-OCT来监测正常和cf样hBE模型系统中的流动粘液，使用雾化输送的gnr。我们将把G*的测量值与OCT中通过斑点跟踪测量的流速作为与睫状膜周围层距离的函数联系起来。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。通过这项研究的完成，我们将验证DLS-OCT纳米流变学与微流变学和体流变学技术的对比，并建立了在流动粘液系统中绘制G*和定量%固体的灵敏度，这将为未来在小鼠囊性纤维化模型中的体内研究奠定基础。

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.