Translocation, biological fate, stability, and effective dose of engineered nanomaterials for nanosafety studies

1530790(Heinz)这项建议涉及工程纳米材料(NMS)的体外和体内命运研究。将生产出形状、尺寸和表面性质可控的氧化锌、金和聚乳酸纳米颗粒以及石墨烯。NMS的细胞内定位、动力学和聚集状态将首先通过生物物理技术在体外进行研究。离子束显微镜将用于确定氧化锌和金纳米颗粒的细胞内剂量。将进行毒理学研究，并与囊内剂量相关。生物日冕的稳定性和日冕中蛋白质的构象状态将在细胞内进行研究。将进一步对NMS进行放射性标记以用于活体成像，并将开发双重放射性标记策略。正电子发射断层扫描和单光子发射计算机断层扫描将用于研究放射性标记的NMS在不同给药途径的小鼠体内的生物分布和归宿。通过双核素标记，研究纳米材料的体内稳定性。将进行各种纳米材料的合成，包括金属和金属氧化物纳米颗粒，基于聚合物的PLGA纳米颗粒，以及石墨烯和石墨烯氧化物纳米材料。这些材料将以不同的方式进行表面功能化，并将探索在核心和日冕中引入双重放射性标记，以便能够在合作者进行的体外和体内研究中跟踪NM的位置。对NMS的表征包括Zeta电位测量、DLS、NTA、ATR-FTIR、TEM和微观扫描透射式离子显微镜。还将通过分子模拟来探索纳米颗粒与其电晕之间的特定相互作用，从而跟踪表面化学对纳米材料的配体堆积、结合强度和团聚的影响。通过引导分子动力学模拟的进一步测试旨在了解通过细胞膜的转运过程，并探索纳米粒子电晕的稳定性。随着实验信息的获得，将在模型中探索与选定的多肽和蛋白质的特定相互作用，以合理地在特定器官和组织中积累。将使用全原子和粗粒模拟相结合的方法来探索从纳米到微米的长度尺度。在这个项目中产生的知识将对理解纳米材料在细胞和生物水平上的行为至关重要，最终目标是将毒性降至最低。这一目标对于化妆品、医疗和医药产品以及人类接触到的纳米材料增强的新结构材料的未来发展至关重要。各种系统的合成、建模和测试也有助于揭示设计纳米材料以将毒性降至最低的方法。纳米材料稳定性研究还将解决核心周围涂层的稳定性、核心本身的稳定性以及纳米材料的聚集程度等问题。在该项目中，我们提出了一种复杂且史无前例的标签策略组合，这将使PI能够通过高灵敏度、非侵入性的成像技术(如PET或SPECT)来解决这些问题。表面涂层的稳定性可以解释为什么相同的纳米材料核心在用不同的涂层功能化时，甚至当相同的涂层以不同的方式连接到纳米材料表面时，会有不同的毒性。涂层和纳米材料的稳定性与其分布的关系可以解释特定的毒理学反应，并提供对毒理学反应的时间框架的理解。纳米材料的稳定性可以成为一个新的毒理学终点，并成为风险评估的基础。最终，结果将有助于对与所调查材料相关的风险进行量化评估。拟议的努力还将包括通过工程职业生涯日和在大学实验室的实践研究经验对博士生进行培训，并为高中生开展外联活动

1530790(Heinz)This proposal deals with in vitro and in vivo fate studies of engineered nanomaterials (NMs). ZnO, Au and PLGA NPs and Graphene will be produced with controlled shape, size and surface properties. The intracellular localization, dynamics and state of aggregation of the NMs will be first studied in vitro by a combination of biophysical techniques. Ion Beam Microscopy will be applied to determine intracellular dose of ZnO and Au NPs. Toxicological studies will be performed and related to the intracelular dose. The stability of the bio corona and conformational state of proteins in the corona will be studied intracelularly. NMs will be further radiolabelled for in vivo imaging and strategies for dual radiolabelling will be developed. Positron Emission Tomography and Single Photon Emission Computed Tomography will be applied to study the biodistribution and fate of radiolabelled NMs in small rodents following different administration routes. By dual radio labelling the in vivo stability of NMs will be investigated.The synthesis of various nanomaterials (NM) will be carried out, including metal and metal oxide nanoparticles, polymeric PLGA based nanoparticles, as well as graphene and graphene oxide nanomaterials. The materials will be surface-functionalized in different ways, and the introduction of dual radiolabels in the core and in the corona will be explored to be able to track the location of the NM during in vitro and in vivo studies by collaborators. Characterization of the NMs involves zeta potential measurements, DLS, NTA, ATR-FTIR, TEM, and micro scanning transmission ion microscopy. The specific interactions between the nanoparticles and their corona will also be explored by molecular simulation, which allows to track the effects of surface chemistry on ligand packing, binding strength, and agglomeration of the nanomaterials. Further tests by steered molecular dynamics simulation aim at understanding the translocation process through cell membranes and probing the stability of the nanoparticle corona. Specific interactions with selected peptides and proteins will be explored in models to rationalize accumulation in specific organs and tissues as experimental information becomes available.A combination of all-atom and coarse-grain simulations will be employed to explore length scales from nanometers to micrometers.The knowledge generated in this project will be essential to understand the behavior of nanomaterials at cellular and organism levels with the ultimate goal to minimize toxicity. This objective is of paramount importance for future developments in cosmetics, medical and pharmaceutical products, as well as for new structural materials enhanced by nanoscale materials to which humans are exposed. The synthesis, modeling, and testing of various systems also helps uncover ways in which nanomaterials may be designed to minimize toxicity. Nanomaterial stability studies will also tackle issues such as the stability of the coating around the core, the stability of the core itself as well as the degree of aggregation of the nanomaterials. In the project we propose a complex and unprecedented combination of labelling strategies, which will allow the PI to address these issues through highly sensitive, non-invasive imaging techniques such as PET or SPECT. The stability of surface coating can explain why the same nanomaterial core can have different toxicity when functionalized with different coating or even when the same coating is linked to the nanomaterial surface in different ways. Coating and nanomaterial stability in relation with their distribution can explain specific toxicological responses as well as provide understanding on the time frame of the toxicological response. The nanomaterial stability can become a novel toxicological end point and be fundamental in risk assessment. Ultimately, the results will contribute to a quantitative evaluation of risks associated with the materials investigated. The proposed effort will also include the training of PhD students and outreach activities for High School students through Engineering Career Days and hands-on research experiences in university laboratories