Nanoparticle properties and alveolar epithelial barrier/transport functions

描述（由申请人提供）：吸入超细（纳米）颗粒与心血管、肺和血液学不良反应、颗粒在血管和终末器官的定位以及易感人群发病率和死亡率增加有关。由于颗粒被吸入，它们最可能的进入体循环的途径是穿过肺的肺泡上皮。尽管由于纳米技术的发展和应用，纳米颗粒的应用有望显著增加，但纳米颗粒损伤和/或运输到肺泡上皮的机制尚不清楚。根据我们关于具有明确物理化学特性的几类纳米粒子（由聚苯乙烯、二氧化硅和金属（氧化物）组成）的肺损伤/摄取/贩运的初步数据，以及最近关于吸入超细空气污染物颗粒对健康影响的报告，我们假设纳米颗粒与肺泡上皮细胞之间的相互作用i)可以破坏正常的肺泡上皮细胞稳态并诱导细胞特性和肺泡上皮屏障功能的变化，ii)为纳米颗粒通过经上皮易位途径进入体循环提供主要入口，iii)高度依赖于纳米颗粒的物理化学特性。利用聚苯乙烯、二氧化硅和金属（氧化物）纳米颗粒在体外模型（包括我们已经建立的大鼠或人肺泡上皮细胞的原代培养单层）和大鼠肺中，我们将通过研究以下四个主要目标来验证这些假设：1)纳米颗粒对肺泡上皮主动和被动屏障特性的影响；2)纳米颗粒在肺泡上皮细胞中的内化、命运和作用；3)纳米颗粒在肺泡上皮内的体外转运；4)纳米颗粒在体内大鼠肺中的内化和运输，体内与体外对远端呼吸上皮损伤/摄取/运输的相关性。此外，我们将利用在可渗透过滤器和巨大单层囊泡上重建的人工脂质双分子层的简化模型来确定纳米颗粒进入/退出肺泡上皮细胞时被动机制（例如扩散）和/或破坏脂质双分子层的作用。本文提出的研究结果将深入了解具有特定物理化学性质的纳米颗粒进入/穿过肺泡上皮的细胞毒性和内化/运输机制。我们的主要目标是获得纳米颗粒与肺泡上皮相互作用的新信息，以帮助了解吸入人造纳米颗粒和环境空气污染物超细颗粒对肺的影响，为由此产生的有害影响的管理指明方向，并改进纳米颗粒的设计，以实现更安全和更有效的生物医学应用（例如，肺部药物/基因传递）。

DESCRIPTION (provided by applicant): Inhalation of ultrafine (nano)particles has been associated with adverse cardiovascular, pulmonary and hematologic effects, localization of particles in blood vessels and end organs, and increased morbidity and mortality in susceptible populations. Since the particles are inhaled, their most likely route of entry into the systemic circulation is across the alveolar epithelium of the lung. Although utilization of nanoparticles due to expansion of the science and application of nanotechnology is expected to markedly increase, the mechanisms by which nanoparticles injure and/or are transported into/across alveolar epithelium are not well known. Based on our preliminary data on lung injury/uptake/trafficking of several classes of nanoparticles (composed of polystyrene, silica and metal (oxides)) with defined physicochemical characteristics and recent reports on health effects of inhaled ultrafine air pollutant particulates, we hypothesize that interactions between nanoparticles and alveolar epithelial cells i) can disrupt normal alveolar epithelial cell homeostasis and induce changes in cellular properties and alveolar epithelial barrier function, ii) provide the primary portal of entry for nanoparticles into the systemic circulation via transepithelial translocation pathways, and iii) are highly dependent on physicochemical properties of the nanoparticles. Utilizing defined polystyrene, silica and metal (oxide) nanoparticles in vitro models (including our well-established primary cultured monolayers of rat or human alveolar epithelial cells) and rat lungs in vivo, we will test these hypotheses by investigating the following four major aims: 1) nanoparticle effects on active and passive barrier properties of alveolar epithelium; 2) internalization, fate and effects of nanoparticles in alveolar epithelial cells; 3) trafficking of nanoparticles across alveolar epithelium in vitro; and 4) nanoparticle internalization and trafficking in rat lungs in vivo, correlating injury to/uptake into/trafficking across distal respiratory epithelium in vivo vs. in vitro. In addition, we will utilize simplified models of artificial lipid bilayers reconstituted on permeable filters and giant unilamellar vesicles to determine the role(s) of passive mechanisms (e.g., diffusion) and/or disruption of lipid bilayers in nanoparticle entry into/exit from alveolar epithelial cells. Findings from the investigations proposed herein will provide insights into cytotoxicity and mechanisms of internalization/trafficking of nanoparticles with defined physicochemical properties into/across the lung alveolar epithelium. Our major objective is to obtain new information on nanoparticle interactions with alveolar epithelium in order to help understand effects on the lung of inhaled manufactured nanoparticles and environmental air pollutant ultrafine particulates, point directions for management of resultant deleterious effects, and lead to improved design of defined nanoparticles for safer and more efficient biomedical applications (e.g., pulmonary drug/gene delivery). PUBLIC HEALTH RELEVANCE: Inhalation of ultrafine ambient pollutant particles (<100 nm) may be associated with adverse cardiovascular and pulmonary effects, resulting in increased morbidity and mortality in susceptible populations. The mechanisms by which these nanoparticles injure and/or are transported into/across alveolar epithelium lining distal airspaces of the lung are not well understood, although it is known that they can affect barrier properties of alveolar epithelium and be internalized by/translocated across alveolar epithelial cells. We will use three classes of defined nanoparticles (comprised of polystyrene, silica or metal (oxide)) to determine interactions with both in vitro and in vivo models of the alveolar barrier in order to help prevent injury from inhaled nanoparticles and design nanoparticles for biological applications (e.g., drug/gene delivery).