Investigating Mechanism of Intracellular Rotational Transport with Optical T

描述(申请人提供)：随着工程纳米颗粒在医学(诊断和治疗)、电子产品、化妆品和纺织品中的使用增加，以及对此类纳米颗粒毒性的相应认识，迫切需要开发方法来评估它们的毒性，特别是在导致氧化应激和不利的长期健康影响的低浓度下。巨噬细胞是肺中主要的防御细胞，负责摄取、降解和清除外来颗粒，所有这些生物物理过程都依赖于完整的细胞骨架。由于纳米颗粒介导的细胞骨架氧化对这些生物物理过程的损害，通过减少颗粒清除和炎症而导致毒性。这种炎症和相关的活性氧物种的过度产生进一步氧化RNA/DNA/蛋白质，导致纤维化、突变和肺癌。为了研究有毒纳米颗粒对巨噬细胞摄取和运输的影响，我们开发了一种新的技术来跟踪吞噬细胞磁性调制光学纳米探针(MagMOON)的细胞内运输。这些MagMOON是微米大小的示踪粒子，一个半球包覆着黄金，产生与取向相关的散射和荧光信号。通过强度变化跟踪旋转传输，即使在低放大倍数下(例如内窥镜检查)，也可以同时分析许多颗粒。由Miller博士(Co-I)开创的一项相关技术的结果表明，细胞内旋转速度是体内巨噬细胞健康的一个强有力的指标，而在体外，有毒纳米颗粒会削弱这种旋转速度。然而，米勒博士的技术只测量了数百万个细胞群的平均传输媒介旋转扩散速率，无法定位粒子或观察单个粒子和细胞的运动。该方案的目的是通过对磁光开关的光学跟踪，将生物物理旋转方法扩展到单个粒子和局部区域。单颗粒研究将为NP诱导的细胞骨架氧化和细胞内转运提供更详细的机制模型。我们的中心假设是，颗粒物质组成的差异通过ROS的产生影响细胞内吞噬小体的运输。这些局部的细胞骨架氧化，与次级信使的氧化和抗氧化剂的耗尽相结合，导致全球细胞骨架损伤和功能障碍、DNA损伤和细胞死亡。我们将通过研究纳米颗粒组成对单个磁性示踪剂颗粒在巨噬细胞中运动和运输的影响来验证这一假设。我们还将利用我们的检测方法来研究NAC等ROS清除剂的保护作用。通过光学跟踪组织模体中的MagMOON，我们还可以展示最终在动物模型中进行示踪颗粒较少的体内检测的可行性，并通过内窥镜和透皮X射线激发的光学发光(XEOL)成像来定位颗粒。这项拟议的研究具有重要意义，因为该项目开发的技术将在检测和了解纳米毒性方面具有重要应用。 公共卫生相关性：这项研究项目开发了一种新的生物物理工具，用于评估单个细胞内工程和环境纳米颗粒的毒性。这项研究与公众健康有关，因为我们开发了一种新的生物分析成像技术来测量纳米颗粒介导的细胞骨架功能障碍和对免疫系统细胞的损害(毒性)。具体地说，我们测量了广泛使用并对人类健康具有重要影响的氧化铜和二氧化钛的毒性。

DESCRIPTION (provided by applicant): With the increased use of engineered nanoparticles in medicine (diagnostics and therapeutics), electronics, cosmetics, and textiles and the corresponding awareness of the toxicity from such nanoparticles, there is a critical need to develop methods to evaluate their toxicity, especially at low concentrations that cause oxidative stress and adverse long-term health effects. Macrophages are the primary defense cells in the lung responsible for uptake, degradation and clearance of foreign particles, all biophysical processes which rely upon an intact cytoskeleton. Impairment of these biophysical processes due to nanoparticle-mediated cytoskeletal oxidation contributes to toxicity via reduced particulate clearance and inflammation. This inflammation and associated overproduction of reactive oxygen species further oxidize RNA/DNA/proteins, leading to fibrosis, mutagenesis and lung cancer. To study how macrophage uptake and transport are affected by toxic nanoparticles, we have developed a novel technique to track intracellular transport of phagocytosed magnetically modulated optical nanoprobes (MagMOONs). These MagMOONs are micron sized tracer particles with one hemisphere coated by gold creating an orientation-dependent scattering and fluorescence signal. Tracking rotational transport via intensity changes allows analysis of many particles simultaneously even at low magnification (e.g. endoscopy). Results from a related technique pioneered by Dr. Miller (Co-I) demonstrate that intracellular rotation rate is a powerful indicator for macrophage health in vivo, and that rotatio is impaired by toxic nanoparticles in vitro. However, Dr. Miller's technique measures only the average transport-mediated rotational diffusion rate from ensembles of millions of cells, and is unable to localize particles or observe motion of individual particles and cells. The objective of this proposal is to extend the biophysical rotation methods to single particles and localized regions via optical tracking of the MagMOONs. Single particle studies will provide a more detailed mechanistic model for intracellular transport and NP- induced cytoskeletal oxidation. Our central hypothesis is that differences in particulate matter composition affect intracellular phagosome transport via ROS generation. These local cytoskeletal oxidations, in concert with the oxidation of secondary messengers and the depletion of antioxidants, cause global cytoskeletal damage and dysfunction, DNA damage, and cell death. We will test this hypothesis by studying the effect of nanoparticle composition on the motion and transport of single magnetic tracer particles in macrophages. We will also use our assay to study the protective effects of ROS scavengers such as NAC. By optically tracking MagMOONs in tissue phantoms we can also show feasibility for eventual in vivo detection in animal models with fewer tracer particles and localization of the particles in the lungs through endoscopy and transdermal X- ray excited optical luminescence (XEOL) imaging. The proposed research is significant because the technique developed in this project will have important applications for detecting and understanding nano-toxicity. PUBLIC HEALTH RELEVANCE: This research project develops a novel biophysical tool for evaluating the toxicity of engineered and environmental nanoparticles within individual cells. The proposed research is relevant to public health because we develop a novel bioanalytical imaging technique to measure nanoparticle mediated cytoskeletal dysfunction and damage (toxicity) on immune system cells. Specifically, we measure toxicity of copper oxide and titanium dioxide which are widely used and have important ramifications for human health.