Health effects of manufactured nanoparticles (NP): molecular and cellular biology and toxicology (NanoBioTox)

该项目旨在促进我们对纳米颗粒(NPs)与呼吸道蛋白质、组织和细胞相互作用的理解。在前一次供资期间，我们开发了一套具有明确定义的特定性质的核动力源工具包。我们已经从动力学和平衡的角度分析了模型蛋白质与我们的NPs的结合。通过体外研究，我们已经证明了NP的表面性质决定了它们在细胞内的摄取和最终目的地，以及细胞毒效应。我们进一步提供了证据表明，在体内，NP的表面属性，包括蛋白质的吸附，同样影响它们在体内的分布。基于这些结果，我们计划继续我们的研究，包括旨在阐明NPs被细胞摄取后的命运的实验，特别是关于包裹在NPs上的“蛋白质日冕”。我们的工作安排在三个主要领域。第一种方法(“NP工具箱”)旨在合成和表征水溶性功能纳米粒。修饰NPs周围的蛋白质冠层将发挥重要作用。第二个主题(“NP-蛋白质复合体”)集中于NP与蛋白质和体液中其他生物分子的相互作用。将特别关注上皮衬里液，一种含有糖蛋白和肺表面活性物质成分的水液层，以及位于肺内表面的气液界面的连续的肺表面活性物质膜。我们将量化与NPs结合的关键蛋白质的动力学和平衡行为，作为我们NPs的特定物理和化学性质的函数。此外，我们还将详细研究蛋白质冠层的结构。蛋白质是以明确的取向还是随机的方式吸附在纳米颗粒上？蛋白质的结构在吸附时是保守的，还是被吸附的蛋白质朝着变性构象发展？在第三个领域(“NP-细胞相互作用和细胞反应”)中，我们研究了NPs与体外肺组织细胞、体外肺细胞及其细胞器的相互作用。此外，还将研究蛋白质电晕的影响。细胞摄取NP后，蛋白质冠层的结构会被保留吗？最后，我们将研究吸附的蛋白质是否以及如何影响NPs引起的毒性效应。

This project aims to advance our understanding of the interactions of nanoparticles (NPs) with proteins, tissue and cells of the respiratory tract. In the previous funding period, we have developed a toolkit of NPs with well-defined, specific properties. We have analyzed the binding of model proteins to our NPs with respect to kinetics and equilibrium. With in-vitro studies, we have demonstrated that NP surface properties govern their uptake by and their final destination inside cells, and also cytotoxic effects. We further provided evidence that, in vivo, NP surface properties, including protein adsorption, likewise affect their distribution within the body. Based on these results, we plan to continue our studies by including experiments aimed at elucidating the fate of NPs after their uptake by cells, specifically, with regard to the “protein corona” enshrouding the NPs. Our work is structured in three major areas. The first one ("NP toolbox") aims at the synthesis and characterization of water-soluble functional NPs. Modification of the protein corona around NPs will play a major role. The second topic ("NP-protein complexes") focuses on NP interactions with proteins and other biomolecules of body fluids. Special attention will be devoted to the epithelial lining fluid, an aqueous liquid layer containing glycoproteins and pulmonary surfactant components, and to the continuous pulmonary surfactant film at the air-liquid interface of the internal lung surface. We will quantify kinetic and equilibrium behavior of key proteins binding to NPs as a function of the specific physical and chemical properties of our NPs. Moreover, we will examine the structure of the protein corona in detail. Do proteins adsorb to NPs in well defined orientations or randomly? Is the protein structure conserved upon adsorption, or do adsorbed proteins develop toward denatured conformations? In the third area ("NP-cell interactions and cellular responses") we study the interaction of NPs with cells from lung tissue ex vivo, lung cells in vitro and their organelles. Also here, the influence of the protein corona will be investigated. Will the structure of the protein corona be preserved after NP uptake by the cell? Finally, we will investigate if and how adsorbed proteins affect toxic effects elicited by NPs.