Understanding the impact of engineered nanoparticles on the lysosome-autophagy system

过去十年见证了纳米技术产业的快速发展：目前市场上有超过1000种含有纳米粒子（NPs）的消费产品，每天都有各种新型纳米材料被开发出来。因此，纳米技术产业的工人、消费者，以及最终的环境本身，都不可避免地会接触到经过改造的纳米粒子。由于其独特的性质，包括其纳米级尺寸，大表面积，化学成分和反应性，NPs与生物组分和系统相互作用，其中许多也在纳米级水平上运作。毫不奇怪，越来越多的人担心这些相互作用可能对人类产生有害影响。目前，毒理学研究通常用于评估与暴露于新工程纳米材料相关的风险。然而，即使根据标准的体内和体外活力测定，NPs不被认为是有毒的，它们仍然可能显著改变细胞生理学。关于NPs的细胞摄取有丰富的信息，细胞内化NPs的速率可以通过NP的大小、电荷和表面性质来控制。然而，这些物理化学性质对其他细胞过程的影响尚未以系统的方式进行研究。例如，众所周知，在内化后，大多数NPs会引起细胞清除机制的反应。事实上，该项目团队最近发现，许多NPs激活自噬，这是消除细胞废物的主要分解代谢途径。虽然自噬的激活可能导致废物清除的增强，但它也可以诱导细胞死亡程序的激活。此外，初步研究表明，特定电荷和组成的NPs可以激活自噬反应，但也会损害介导降解的细胞成分，最终阻断自噬通量。该项目团队最近开发了一套分析工具来监测自噬通量和细胞模型系统，以测量自噬底物的积累，包括蛋白脂和蛋白聚集体。因此，该团队在研究NPs对自噬系统的影响方面处于独特的地位。该项目的总体目标是将NPs的物理化学性质与其诱导的自噬反应的性质进行映射，最终生成设计规则，以在自噬系统界面处设计具有所需性质的NPs。为了实现这一目标，我们建议追求三个研究目标：1)确定激活自噬的NPs的物理化学性质；2)将NPs的物理化学性质与自噬激活相关的生物相容性和生物不良细胞反应联系起来；3)确定调控NPs自噬反应的细胞网络。智力优势：本研究的结果将详细描述NPs对自噬系统的影响。该项目将确定NP的物理化学性质，即几何参数、电荷和组成，对自噬激活相关的下游效应的影响，即NP和其他自噬底物的清除或自噬相关的细胞死亡。该项目还将确定调控NPs自噬反应的基因网络和np诱导自噬的细胞标志物，从而为评估NPs对细胞生理学的影响提供重要的预测工具。更广泛的影响：这项研究将为研究NPs对生物系统的影响提供一个实验框架。将开发和验证专门设计的报告系统，以将NPs的物理化学性质与细胞反应及其潜在的分子机制联系起来。反过来，了解NPs对细胞生理学的影响将有助于开发安全的纳米材料，并将使设计具有可调特性的第三代NPs成为可能，这些NPs可以与生物系统相结合。最后，提出的项目将为细胞和分子生物学的研究提供新的工具。该研究项目还将提供广泛的教育和培训机会，其总体目标是增加STEM中代表性不足群体的数量和多样性。具体来说，这项研究计划将被利用1)为高中生提供研究和指导机会，2)改善大学生的教学。

CBET - 1336053 The last decade has witnessed rapid growth in the nanotechnology industry: more than 1,000 consumer products containing nanoparticles (NPs) are currently in the market place, and a variety of newly engineered nanomaterials are developed every day. As a result, workers in the nanotechnology industry, consumers, and ultimately the environment itself inevitably come in contact with engineered NPs. Because of their unique properties, including their nanoscale size, large surface area, chemical composition, and reactivity, NPs interact with biological components and systems, many of which also operate at the nanoscale level. Not surprisingly, growing concerns have been raised over the possibility that these interactions could have deleterious effects on humans. Currently, toxicology studies are typically conducted to assess the risk associated with exposure to newly engineered nanomaterials. However, even when NPs are not deemed toxic based on standard in vivo and in vitro viability assays, they might still significantly alter cell physiology. A wealth of information is available about the cellular uptake of NPs, and the rate at which cells internalize NPs can be controlled with respect to NP size, charge, and surface properties. However, the impact of these physicochemical properties on other cellular processes has not been investigated in a systematic fashion. It is known, for instance, that upon internalization, most NPs elicit the reaction of cellular clearance mechanisms. Indeed, the project team recently found that a number of NPs activate autophagy, the main catabolic pathway that eliminates cellular waste. While activation of autophagy may lead to enhanced clearance of waste material, it can also induce activation of cell death programs. Moreover, preliminary studies indicate that NPs of specific charege and composition can activate the autophagic response but also impair cellular components that mediate degradation, ultimately blocking autophagic flux.The project team recently developed a set of analytical tools to monitor the autophagic flux and cell model systems to measure the accumulation of autophagic substrates, including proteolipid and proteinaceous aggregates. The team is thus uniquely positioned to investigate the impact of NPs on the autophagic system. The overarching goal of the proposed project is to map the physicochemical properties of NPs with the nature of the autophagic response that they induce, ultimately generating the design rules to engineer NPs with the desired properties at the interface with the autophagy system. To achieve this goal, we propose to pursue three research objectives: 1) Identify the physicochemical properties of NPs that activate autophagy; 2) link the physicochemical properties of NPs to biocompatible and bioadverse cellular responses associated with autophagy activation; and 3) identify the cellular network that regulates the autophagic response to NPs.Intellectual Merit :Results from this study will provide a detailed characterization of the impact of NPs on the autophagy system. The project will establish the effect of NP physicochemical properties, namely geometric parameters, charge, and composition, on downstream effects associated with activation of autophagy, namely clearance of NPs and other autophagic substrates or autophagy-associated cell death. The project will also identify the gene network that regulates the autophagic response to NPs and cellular markers of NP-induced autophagy, thus providing important predictive tools to assess the impact of NPs on cell physiology.Broader Impacts :This study will generate an experimental framework for investigating the impact of NPs on biological systems. Specially designed reporter systems will be developed and validated to link the physicochemical properties of NPs to cellular responses and their underlying molecular mechanisms. Understanding the impact of NPs on cell physiology, in turn, will contribute to the development of safe nanomaterials and will enable the design of third-generation NPs with tunable properties where these NPs interface with biological systems. Finally, the proposed project will provide novel tools for cellular and molecular biology studies. This research program will also provide broadly reaching educational and training opportunities with the overall goal of increasing the number and diversity of underrepresented groups in STEM. Specifically, this research program will be leveraged 1) to provide research and mentoring opportunities for high school students, and 2) to improve teaching and learning for college students.