Biomimetic Microsystem for High Throughput Evaluation of Engineered Nanomaterials

描述（由申请人提供）：工程纳米材料（ENM）具有可引起不良健康影响的独特特性。由于ENM的体积小且有可能在空气中扩散，因此吸入暴露于ENM可能导致过敏性气道疾病的发病率增加和/或恶化。该项目的总体目标是开发ENM的多级毒性测试平台，包括小鼠过敏性气道疾病的体内测量，T细胞活化的体外测量，ENM与模拟细胞膜的双层脂质膜（BLM）相互作用的高通量测量，以及ENM分子特性的计算机预测。ENM具有促进过敏性气道疾病的佐剂样特性的总体假设将使用五个特定目的（SA）进行测试。SA1是合成表征良好的ENM，具有可控的官能团，催化氧化还原反应或激活膜受体。SA2是测定ENM对小鼠气道变应性致敏和哮喘样疾病的辅助作用潜力。SA3是为了确定ENM对树突状细胞诱导的CD4+和CD8+ T细胞的激活和效应功能的影响。SA4是测定ENM对合成双分子层脂膜的直接影响。SA5是开发和验证能够将ENM物理化学特性与其在动物、细胞和膜水平上的生物和毒理学效应联系起来的数学模型，以进行健康风险评估。生物可降解的聚丙炔醇纳米颗粒将被合成并被化学基团（脂多糖（LPS）和醌）包裹，以产生可能刺激免疫反应的ENM。脂多糖与细胞膜上的受体蛋白结合，通过内吞作用触发细胞摄取，醌类可以触发氧化还原反应，包括免疫细胞产生活性氧。将采用多层次的方法来确定在ENM中添加LPS和醌是否通过(1)增加小鼠免疫系统对抗原卵白蛋白的反应，(2)增加树突状细胞对卵白蛋白的激活，以及(3)修改ELM与BLM的分子相互作用来增加ENM促进气道疾病的能力。小鼠将通过吸入暴露于ENM，并对过敏性气道疾病的严重程度进行组织病理学、形态计量学和生化评估。树突状细胞将暴露在ENM中，并使用荧光辅助流式细胞术测量其激活T细胞的能力。BLM将沉积在电极上并暴露在ENM中。ENM和BLM之间产生的相互作用将在高通量模式下使用循环伏安法和电阻抗谱进行测量。将开发理论模型来描述ENM的分子特性及其与细胞成分的相互作用。这些模型将用于分析实验数据，并有助于阐明ENM诱导毒性作用的机制。

DESCRIPTION (provided by applicant): Engineered nanomaterials (ENM) have unique properties that can cause adverse health effects. Due to their small size and potential for airborne dispersion, inhalation exposure to ENM might contribute to the increased incidence and/or exacerbation of allergic airway disease. The overall goal of this project is to develop a multi- level toxicity testing platform for ENM that includes in vivo measurement of allergic airway disease in mice, in vitro measurement of T cell activation, high-throughput measurement of ENM's interactions with bilayer lipid membranes (BLM) that mimic cell membranes, and in silico prediction of ENM's molecular properties. The overarching hypothesis that ENM possess adjuvant-like properties that promote allergic airway disease will be tested using five specific aims (SA). SA1 is to synthesize well-characterized ENM having controlled functional groups that catalyze redox reactions or activate membrane receptors. SA2 is to determine the adjuvant potential of ENM on allergic airway sensitization and asthma-like disease in mice. SA3 is to determine the effects of ENM on dendritic cell-induced activation and effector function of CD4+ and CD8+ T cells. SA4 is to measure the direct effects of ENM on synthetic bilayer lipid membranes. SA5 is to develop and validate mathematical models that can correlate ENM physicochemical properties with their biological and toxicological effects at the animal, cell, and membrane levels for health risk assessment. Biodegradable poly(propargyl glycolide) nanoparticles will be synthesized and coated with chemical groups (lipopolysaccharide (LPS) and quinone) to generate ENM likely to stimulate the immune response. LPS bind to receptor proteins on cell membranes and trigger cellular uptake by endocytosis, and quinones can trigger oxidation-reduction reactions, including production of reactive oxygen species by immune cells. A multi-tiered approach will be used to determine whether addition of LPS and quinone to ENM increases the ENM's ability to promote airway disease by (1) increasing the murine immune system's response to the antigen ovalbumin, (2) increasing T cell activation by dendritic cells in response to ovalbumin, and (3) modifying the ELM's molecular interactions with BLM. Mice will be exposed to the ENM by inhalation, and severity of allergic airway disease will be histopathologically, morphometrically and biochemically assessed. Dendritic cells will be exposed to the ENM, and their ability to activate T cells will be measured using fluorescence assisted flow cytometry. BLM will be deposited on electrodes and exposed to the ENM. The resulting interactions between the ENM and BLM will be measured in a high-throughput mode using cyclic voltammetry and electrical impedance spectroscopy. Theoretical models will be developed that describe the molecular properties of the ENM and their interactions with cellular components. These models will be used to analyze the experimental data and help elucidate mechanisms by which ENM induce toxic effects. PUBLIC HEALTH RELEVANCE: This project will provide fundamental insight into how a nanoparticle's physical and chemical properties determine its ability to enhance allergic airway disease like asthma. This insight will aid in setting health and safety standards for engineered nanomaterials, provide new high- throughput methods for nanoparticle detection and safety screening, and facilitate design of new nanomaterials that simultaneously meet safety standards and exhibit desirable performance properties needed for commercial applications.