Biomimetic Microsystem for High Throughput Evaluation of Engineered Nanomaterials

描述(由申请人提供)：工程纳米材料(ENM)具有独特的特性，可能会导致不利的健康影响。由于ENM体积小，有可能在空气中扩散，吸入ENM可能会增加过敏性呼吸道疾病的发病率和/或加重。该项目的总体目标是开发ENM的多层次毒性测试平台，包括在体小鼠过敏性呼吸道疾病的测量，体外T细胞激活的测量，高通量测量ENM与模拟细胞膜的双层脂膜(BLM)的相互作用，以及ENM分子性质的电子预测。ENM具有促进过敏性呼吸道疾病的佐剂样属性的首要假设将使用五个特定目标(SA)进行测试。SA1的目的是合成具有良好特性的ENM，这些ENM具有催化氧化还原反应或激活膜受体的受控官能团。SA2是为了确定ENM对小鼠过敏性呼吸道致敏和哮喘样疾病的佐剂潜力。SA3是为了确定ENM对树突状细胞诱导的CD4和CD8T细胞的激活和效应功能的影响。SA4是用来测量ENM对合成双层类脂膜的直接影响。SA5将开发和验证数学模型，这些模型可以将ENM的物理化学性质与其在动物、细胞和膜水平上的生物和毒理学效应联系起来，用于健康风险评估。将合成可生物降解的聚炔丙基乙交酯纳米颗粒，并在其表面包裹化学基团(脂多糖和苯二酚)，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，而苯二酚可以触发氧化还原反应，包括免疫细胞产生活性氧物种。将使用多层次方法来确定在ENM中加入内毒素和苯醌是否通过以下方式增加ENM促进呼吸道疾病的能力：(1)增加小鼠免疫系统对卵白蛋白抗原的反应；(2)增加树突状细胞对卵白蛋白反应的T细胞激活；(3)改变ELM与BLM的分子相互作用。小鼠将通过吸入方式暴露于ENM，过敏性呼吸道疾病的严重程度将进行组织病理学、形态计量学和生化评估。树突状细胞将暴露在ENM中，并将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。由此产生的ENM和BLM之间的相互作用将以高通量模式使用循环伏安法和阻抗谱进行测量。将开发描述ENM的分子性质及其与细胞成分相互作用的理论模型。这些模型将被用来分析实验数据，并帮助阐明ENM诱导毒性效应的机制。 与公共卫生相关：该项目将提供有关纳米颗粒的物理和化学特性如何决定其增强哮喘等过敏性呼吸道疾病的能力的基本见解。这一见解将有助于为工程纳米材料设定健康和安全标准，为纳米颗粒检测和安全筛选提供新的高通量方法，并促进同时满足安全标准和展示商业应用所需的理想性能的新纳米材料的设计。

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.