Nanoparticle Transport to Oocytes and Toxicological Consequences in Fathead Minnows

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。[05:3707]越来越多的证据表明，许多纳米材料是通过鱼鳃或消化道被鱼类吸收的。一旦内化，纳米材料就有可能进入血液，在那里它们可能对包括卵巢在内的内脏器官构成威胁。新出现的文献和目前应用的初步证据表明，纳米材料可能能够进入卵母细胞，潜在地影响胚胎发育。有必要了解纳米材料运输到卵母细胞的基本机制，以及随后自然生物过程的改变。目前的理解是，纳米材料的生理行为在很大程度上受到吸附的蛋白质层的影响，这些蛋白质层是在暴露于生物介质几秒钟后形成的。繁殖活跃的雌鱼血清中蛋黄前体蛋白卵黄原蛋白含量高（高达20毫克/毫升）。这种蛋白质是一种大的磷脂糖蛋白，具有通过各种管道与纳米材料结合的能力，包括高负电荷区域和高疏水性区域。卵黄原蛋白天然靶向于发育中的卵母细胞，并通过卵黄原蛋白特异性受体进入这些卵母细胞。本项目的总体假设是卵黄原蛋白结合的纳米材料将被导入发育中的卵母细胞的卵黄颗粒中，并最终与溶酶体融合。此外，一旦到达这个隔室，某些纳米材料就会溶解并渗入细胞，对发育中的胚胎造成毒性。为了解决这些假设，研究人员有三个具体目标：(1)确定表面修饰对鱼卵巢培养中纳米材料的摄取、细胞分布和保留的影响。(2)研究了雄性和雌性黑头鱼等离子体对纳米材料蛋白表面涂层的影响。(3)测定纳米颗粒在体内的分布和生殖毒性。为了实现这些目标，他们将使用发光纳米粒子模型，即染料掺杂二氧化硅和量子点，这些量子点的尺寸分布和表面特性都经过精心选择，可以详细分析这些材料在体外和体内的运动。提出的研究将提供关键的见解纳米颗粒的性质在其转运到卵母细胞的作用。预计这一信息在开发鱼类纳米毒性预测模型以及减轻纳米材料暴露于环境的潜在不良后果的方法方面非常重要。在粒子科学、工程和生物学的交叉领域对研究生、本科生和高中生进行跨学科培训将是研究计划的重要组成部分。东南研究生教育和教授联盟（SEAGEP），相关的本科生研究经验（REU）项目，以及佛罗里达大学建立的少数民族奖学金项目将被利用，以确保研究项目的多样性。研究结果将发表在同行评议的期刊上，并在国家和国际会议上传播。研究人员还计划与国家纳米技术资源合作，如亚利桑那州立大学的社会纳米技术中心和莱斯大学的ICON，向科学界和教育界以及整个社会传播研究成果。学生们还将参加公共推广项目（例如，佛罗里达大学的工程博览会），为他们提供一个渠道，让公众了解纳米科学的兴奋和环境管理的重要性。佛罗里达大学的格雷厄姆公共政策中心将参与确保研究结果适当地传达给政策制定者。这项拟议的研究将提供介导纳米颗粒运输到卵母细胞的因素的基础知识，并提供可能导致鱼类胚胎改变的机制的见解。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).0853707DenslowGrowing evidence suggests that many nanomaterials are taken up into fish species through the gills or digestive track. Once internalized, there is a potential for nanomaterials to cross into the blood stream, where they could pose a risk to internal organs including the ovary. Emerging literature and preliminary evidence in the current application suggest that nanomaterials may be capable of entering oocytes, potentially impacting embryonic development. There is a need to understand fundamental mechanisms that enable nanomaterial transport to oocytes, and the subsequent alterations of natural biological processes. It is currently understood that the physiological behavior of nanomaterials is largely impacted by adsorbed proteinaceous layers, which form upon seconds of exposure to biological media. Female fish that are actively reproducing have high serum levels (up to 20 mg/ml) of the egg yolk precursor protein, vitellogenin. This protein is a large phospholipoglycoprotein that has the capacity to bind to nanomaterials through various conduits, including areas of high negative charge and areas of high hydrophobicity. Vitellogenin is naturally targeted to developing oocytes and is imported into these oocytes via a vitellogenin-specific receptor. The overall hypothesis of this project is that vitellogenin-bound nanomaterials will be imported into yolk granules in developing oocytes and eventually merge with lysosomes. Furthermore, upon reaching this compartment certain nanomaterials could dissolve and leach into the cell causing toxicity for developing embryos. To address these hypotheses, the researchers have 3 specific aims: (1) Determine the effect of surface modification on uptake, cellular distribution and retention of nanomaterials in fish ovarian cultures. (2) Characterize the protein surface coating of nanomaterials exposed to male and female fathead minnow plasma. (3) Determine the distribution and reproductive toxicity of nanoparticles in vivo. To achieve these aims they will use model luminescent nanoparticulates, namely dye doped silica and quantum dots of carefully chosen size distributions and surface properties that will enable detailed analysis of the movement of these materials in vitro and in vivo. The proposed research will provide critical insights into the role of nanoparticulate properties on their translocation to oocytes. This information is anticipated to be highly important in the development of predictive models for nanotoxicity in fish species, and methods to mitigate potentially adverse outcomes of nanomaterial exposure to the environment. Cross-disciplinary training of graduate, undergraduate, and high school students at the interface between particle science and engineering and biology will be an essential part of the research program. The Southeast Alliance for Graduate Education and the Professoriate (SEAGEP), related Research Experience for Undergraduates (REU) programs, and established minority fellowship programs at the University of Florida will be leveraged to ensure diversity in the research program. Research results will be published in peer-reviewed journals and disseminated at national and international meetings. The investigators also plan to collaborate with national nanotechnology resources such as the Center for Nanotechnology in Society at Arizona State University and ICON at Rice to disseminate research results to the scientific and educational communities, and to society at large. The students will also participate in public outreach programs (e.g., UF's Engineering Fair)providing a conduit for them to engage the general public about the excitement of nanoscience and the importance of environmental stewardship. UF's Graham Center for Public Policy will be engaged to ensure that research findings are appropriately communicated to policy makers. The proposed research will provide fundamental knowledge of factors that mediate nanoparticle transport to oocytes, and offer insights on mechanisms that may lead to alterations in fish embryos.