Characterization Methodologies & Proteomics to Assess Carbon Nanotube Exposure

表征方法

• 批准号: 8324820
• 负责人: Somenath Mitra
• 金额: $ 7.9万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8324820
• 关键词: Address; Affect; Animals; Biological; Biological Availability; Biological Markers; Biological Models; Carbon; Carbon Nanotubes; Cell Line; Cell physiology; Cells; Characteristics; Chemicals; Collaborations; Data; Deglutition; Detection; Development; Down-Regulation; Drug Delivery Systems; Electrophoresis; Elements; Engineering; Epithelial Cells; Escalator; Extracellular Protein; Food Chain; Gastrointestinal tract structure; Goals; Health; Hepatic; Hepatocyte; Human; Image; Indiana; Individual; Industrial Waste; Injury; Institutes; Intestines; Label; Lead; Liver; Maps; Mass Spectrum Analysis; Measurement; Methodology; Methods; Microscopy; Mining; Modeling; Molecular; Molecular Profiling; Monitor; Morphology; Mus; Nanotechnology; New Jersey; Organelles; Particle Size; Pathway interactions; Plants; Polymers; Post-Translational Protein Processing; Proliferating; Proteins; Proteomics; Public Health; Raman Spectrum Analysis; Research; Research Project Grants; Residual state; Resources; Rivers; Roentgen Rays; Route; Safety; Scanning Electron Microscopy; Spectrometry; Spectrum Analysis; Techniques; Technology; Thermogravimetry; Time; Toxicology; Universities; Up-Regulation; Validation; Water; Water Pollutants; Water consumption; aqueous; base; drinking water; exposed human population; improved; in vitro Model; kidney cell; light scattering; medical schools; nanomaterials; nanotoxicology; novel; protein expression; public health relevance; research study; response; tool; two-dimensional; zeta potential

DESCRIPTION (provided by applicant): The objective of this project is to develop reliable and reproducible exposure assessment methodologies and protein expression-based methods aimed at the development of biological/ toxicological response endpoints that can serve potential biomarkers for well-characterized engineered nanomaterials (ENMs). While raw, unrefined, and hydrophobic carbon nanotubes tend to settle out of aqueous media/environs, water dispersible, functionalized carbon nanotubes (f-CNTs) will contaminate water resources. Large quantities of these derivatized forms of CNTs will be manufactured as they find diverse applications ranging from polymer composites to drug delivery. Accordingly, one of the major potential routes of human exposure is alimentary, occurring via consumption of water or products from plants and animals that have taken up the CNTs through food-chain dynamics. Reliable and reproducible methods to assess exposure and biological response/toxicological endpoints for well-characterized engineered nanomaterials (ENMs) must be developed. It is hypothesized that highly dispersible, water soluble carbon nanotubes alter protein expression in intestinal and liver cells as biological/toxicological endpoints of exposure and injurious effect in the gastrointestinal tract. These effects will be evident as intra- and/or extracellular protein differences, detectable by proteomic analysis and can be rendered as a panel of protein biomarker candidates. This has lead to the following aims: Specific Aim 1. f- CNTs will be synthesized, characterized, and analytical methodologies for exposure assessment developed. Composition, morphology and size of the functionalized CNTs will be assessed, as will residual impurities in an unrefined, comparator group. CNT-induced changes in cellular function and protein expression will be distinguished among physico-chemical characteristics of the individual CNTs. This will be done by element mapping analysis, X-ray energy dispersive spectrometry (EDS) in scanning electron microscopy (SEM), thermogravimetry analysis (TGA) and Raman Spectroscopy. Specific Aim 2. The effects of f-CNT exposure on colonic and hepatic epithelial cell protein expression profiles will be assessed to discover potential protein biomarkers of exposure/effect. Protein expression data will be obtained by two complementary methods: two- dimensional electrophoresis (2-DE) and label-free quantitative mass spectrometry (LFQMS). This will enable the analysis of relevant organelle, compartment, and pathway-specific responses to f-CNT exposure and identification of biological response/toxicological endpoints of ENM exposure. This project combines complementary expertise in nanotechnology and nanotoxicology using novel applications of emerging analytical techniques to study a relevant yet previously ignored target for water dispersible CNTs, the gut-liver axis. The results of this project will establish reproducible ENM characterization methodologies and lay the groundwork for the validation of individual biomarkers or biomarker panels that can reliably detect and predict biological response to select model ENMs, thereby promoting the safe use of these rapidly proliferating materials and protecting the public. PUBLIC HEALTH RELEVANCE: This project will develop reproducible Engineered Nanomaterial (ENM) characterization methodologies and generate protein biomarker candidates of biological/toxicological effect in cells of the digestive tract. These studies will lay the groundwork for the validation of individual biomarkers or biomarker panels that can reliably detect and predict biological response to well-characterized water dispersible ENMs, thereby promoting the safe use of these rapidly proliferating materials and protecting the public.

描述（由申请人提供）：该项目的目标是开发可靠且可重复的暴露评估方法和基于蛋白质表达的方法，旨在开发生物/毒理学响应终点，为特征良好的工程纳米材料（ENM）提供潜在的生物标志物。虽然原始、未精炼和疏水性碳纳米管往往会从水性介质/环境中沉淀出来，但水分散性、功能化碳纳米管 (f-CNT) 会污染水资源。这些衍生形式的碳纳米管将被大量生产，因为它们具有从聚合物复合材料到药物输送等多种应用。因此，人类接触碳纳米管的主要潜在途径之一是食物，即通过消耗通过食物链动力学吸收碳纳米管的植物和动物的水或产品而发生。必须开发可靠且可重复的方法来评估充分表征的工程纳米材料（ENM）的暴露和生物反应/毒理学终点。据推测，高度分散的水溶性碳纳米管会改变肠和肝细胞中的蛋白质表达，作为胃肠道中暴露和有害影响的生物/毒理学终点。这些影响将通过细胞内和/或细胞外蛋白质差异而变得明显，可通过蛋白质组分析检测到，并可呈现为一组蛋白质生物标志物候选物。这导致了以下目标： 具体目标 1. 将合成、表征 f-CNT，并开发用于暴露评估的分析方法。将评估功能化碳纳米管的组成、形态和尺寸，以及未精炼的比较组中的残留杂质。碳纳米管引起的细胞功能和蛋白质表达的变化将在各个碳纳米管的物理化学特征之间进行区分。这将通过元素图分析、扫描电子显微镜 (SEM) 中的 X 射线能量色散光谱 (EDS)、热重分析 (TGA) 和拉曼光谱来完成。具体目标 2. 将评估 f-CNT 暴露对结肠和肝上皮细胞蛋白质表达谱的影响，以发现暴露/影响的潜在蛋白质生物标志物。蛋白质表达数据将通过两种互补方法获得：二维电泳（2-DE）和无标记定量质谱（LFQMS）。这将能够分析 f-CNT 暴露的相关细胞器、区室和途径特异性反应，并识别 ENM 暴露的生物反应/毒理学终点。该项目结合了纳米技术和纳米毒理学方面的互补专业知识，利用新兴分析技术的新颖应用来研究水分散性碳纳米管的相关但以前被忽视的目标——肠肝轴。该项目的结果将建立可重复的 ENM 表征方法，并为验证单个生物标志物或生物标志物组奠定基础，这些生物标志物或生物标志物组可以可靠地检测和预测对选定模型 ENM 的生物反应，从而促进这些快速增殖材料的安全使用并保护公众。 公共健康相关性：该项目将开发可重复的工程纳米材料（ENM）表征方法，并生成消化道细胞中生物/毒理学效应的候选蛋白质生物标志物。这些研究将为验证单个生物标志物或生物标志物组奠定基础，这些生物标志物或生物标志物组可以可靠地检测和预测对特征良好的水分散性 ENM 的生物反应，从而促进这些快速增殖材料的安全使用并保护公众。