Characterization Methodologies & Proteomics to Assess Carbon Nanotube Exposure

表征方法

• 批准号: 8078623
• 负责人: Somenath Mitra
• 金额: $ 6.06万
• 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/8078623
• 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）：本项目的目的是开发可靠且可重现的暴露评估方法和基于蛋白质表达的方法，旨在开发生物学/毒理学反应终点，这些终点可作为充分表征的工程纳米材料（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/环境中沉降出来，但水分散性的、官能化的碳纳米管（f-CNT）将污染水资源。大量的这些衍生形式的碳纳米管将被制造，因为它们发现从聚合物复合材料到药物输送的各种应用。因此，人类接触的主要潜在途径之一是食物，通过消耗水或植物和动物的产品发生，这些植物和动物通过食物链动态吸收了CNT。必须开发可靠和可重复的方法来评估暴露和生物反应/毒理学终点的良好表征的工程纳米材料（ENM）。据推测，高度分散的水溶性碳纳米管改变了肠和肝细胞中的蛋白质表达，作为胃肠道中暴露和损伤效应的生物学/毒理学终点。这些效应将是明显的，因为通过蛋白质组学分析可检测到的细胞内和/或细胞外蛋白质差异，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：具体目标1。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。将评估官能化CNT的组成、形态和尺寸，以及未精制的对照组中的残留杂质。CNT诱导的细胞功能和蛋白质表达的变化将在单个CNT的物理化学特征之间进行区分。这将通过元素图谱分析、扫描电子显微镜（SEM）中的X射线能量色散光谱法（EDS）、热重分析（TGA）和拉曼光谱法完成。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/效应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（LPEMS）。这将使相关的细胞器，隔室，和特定的f-CNT暴露和识别ENM暴露的生物反应/毒理学终点的途径的反应的分析。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，研究水分散性CNT的相关但以前被忽视的目标，即肠道-肝脏轴。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。 公共卫生相关性：该项目将开发可重复的工程纳米材料（ENM）表征方法，并在消化道细胞中产生生物/毒理学效应的蛋白质生物标志物候选物。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表征良好的水分散性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。