Characterization Methodologies & Proteomics to Assess Carbon Nanotube Exposure

表征方法

• 批准号: 7938767
• 负责人: Somenath Mitra
• 金额: $ 59.9万
• 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/7938767
• 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; 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; 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和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/毒理学反应终点，可以作为表征良好的工程纳米材料（enm）的潜在生物标志物。虽然原始的、未精制的和疏水的碳纳米管倾向于从水介质/环境中沉淀出来，但水分散的、功能化的碳纳米管（f-CNTs）将污染水资源。这些衍生形式的碳纳米管将被大量生产，因为它们有多种应用，从聚合物复合材料到药物输送。因此，人类接触碳纳米管的主要潜在途径之一是通过食物，通过食物链动力学消耗吸收碳纳米管的水或动植物产品。必须开发可靠和可重复的方法来评估具有良好特征的工程纳米材料（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改变了肠道和肝细胞中的蛋白质表达，作为胃肠道暴露和损伤效应的生物学/毒理学终点。这些影响将明显表现为细胞内和/或细胞外蛋白质的差异，通过蛋白质组学分析可以检测到，并且可以呈现为一组蛋白质生物标志物候选物。这导致了以下目标：f-碳纳米管将被合成、表征，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、形态和大小，以及未精制比较物组中的残留杂质。碳纳米管诱导的细胞功能和蛋白质表达的变化将根据单个碳纳米管的物理化学特性进行区分。这将通过元素映射分析、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。蛋白表达数据将通过两种互补的方法获得：二维电泳（2-DE）和无标记定量质谱（LFQMS）。这将有助于分析f-CNT暴露的相关细胞器、隔室和通路特异性反应，并确定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的互补专业知识，使用新兴分析技术的新应用来研究水分散碳纳米管的相关但以前被忽视的靶点——肠-肝轴。该项目的结果将建立可重复的ENM表征方法，并为个体生物标记物或生物标记物面板的验证奠定基础，这些生物标记物或生物标记物面板可以可靠地检测和预测对选择模型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。