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，以同时绘制粘液粘弹性和粘液流动的原位图谱。呼吸道粘液复合体粘弹性剪切模数(G*)的增加阻碍粘液清除，并与慢性阻塞性肺疾病(COPD)和囊性纤维化(CF)等呼吸系统疾病有关。在短期内，这个新的平台将提供更完整的粘液纤毛清除的生物物理图像，并使更好的模型成为可能，从而导致新的治疗范式。从长远来看，这一平台可能可用于体内粘液监测，以评估疾病进展和治疗干预。我们的纳米流变学平台基于光学相干断层扫描(OCT)监测的等离子体金纳米棒(GNR)的动态光散射(DLS)。具体地说，来自GNRs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，这是通过广义的Stokes-Einstein关系与粘液的G*相关的。GNR作为流变学探针具有优势，因为它们的高度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的DLS信号，从而允许更快速地收集流变学数据。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，特别是作为到睫状层的距离的函数。通过将这些测量与基于OCT的粘液流动跟踪相联系，它将提供更完整的粘液纤毛清除的生物物理图像。我们的第一个目标是在受控复杂流体中验证DLS-OCT纳米流变学技术是否符合标准微珠流变学和整体流变学。随后，我们将使用从人支气管上皮(HBE)培养获得的粘液中的非粘附性GNRs来验证这项技术，并评估作为固体百分比函数的反应。我们的第二个目标是利用雾化提供的GNRs，使用DLS-OCT来监测正常和类CF型HBe模型系统中流动的粘液。我们将把G*的测量值与OCT中散斑跟踪测量的流速关联起来，作为到睫状层的距离的函数。此外，我们还将确定OCT在模拟高渗盐水治疗过程中监测睫毛功能的能力，以及在这些模型中诱导纤毛活动崩溃和恢复的能力。通过这项研究的完成，我们将验证DLS-OCT纳米流变学相对于微观和整体流变学技术的有效性，并建立了在流动粘液系统中定位G*和定量固体百分比的灵敏度，这将为未来囊性纤维化小鼠模型的体内研究奠定技术基础。 与公共卫生相关：我们建议开发一种新技术，以高分辨率测量呼吸道粘液的机械特性，以便更好地了解呼吸道疾病中的粘液增厚。这与慢性阻塞性肺疾病和囊性纤维化相关，并可能导致更好的方法来诊断和评估疾病进展，以及监测治疗和开发更好的药物输送方法。

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. PUBLIC HEALTH RELEVANCE: We propose to develop a new technology to measure the mechanical properties of airway mucus with high resolution in order to better understand mucus thickening in respiratory diseases. This has relevance for chronic obstructive pulmonary disease and cystic fibrosis, and may lead to better methods to diagnose and assess disease progression, as well as for monitoring therapy and developing better methods for drug delivery.