Impact of the protein corona on metal oxide nanoparticle-induced toxicity mechanisms

除了在工业产品中普遍使用纳米颗粒（NPs）之外，它们的生物医学应用也在扩大。虽然这两个发展将导致人类越来越多地接触纳米粒子，我们目前的机制知识的过程中的纳米生物界面仍然是支离破碎的。NP在与所有生物环境接触时吸附生物分子，形成所谓的NP-生物分子冠。重要的是，我们和其他人表明，特别是蛋白质冠不仅严重影响纳米毒理学，而且影响纳米生物医学应用的成功和安全。因此，生物系统大多不面对原始制造的，而是冠状涂层的转化的NP。此外，冠蛋白如何影响纳米生物界面的潜在分子机制尚未解决。我们发现，关注高度可控的NP模型结合系统分析是将观察到的效应与不同的NP特征相关联的关键。因此，我们将研究金属氧化物纳米颗粒（MOx_NPs）不仅代表了工业和生物医学相关的纳米颗粒类别，而且也是一个很好的模型系统。实际上，MOx_NP的物理化学特性。因此，它们与其他金属的受控掺杂允许控制它们的反应性，并因此将反应性与生物效应相关联。然而，蛋白质冠的相关性迄今尚未研究。基于我们以前的工作，我们预计冠也是一个关键因素MOx_NP触发（不良）生物效应，通过影响氧化应激，（亚毒性）信号，细胞摄取，和细胞内溶解过程。因此，将受控MOx_NPs合成和表征与最先进的分析方法相结合，例如多参数高含量分析、基于质谱的蛋白质组学以及（共培养）体外暴露细胞模型，该项目的科学目标是：分析生物分子冠对具有增量PdO-/Fe-掺杂的MOx_NP的物理化学性质（尺寸、表面电荷、胶体稳定性、老化）的影响。目标二。通过定量蛋白质组学分析MOx_NPs蛋白质冠。目标3.采用系统的高含量细胞为基础的分析，以确定蛋白质冠对生命力的影响，信号传导，和氧化应激反应在人类暴露细胞模型。目标4。生物信息学相关性分析和鉴定与MOx_NPs纳米结构-活性关系（nanoSAR）相关的蛋白质冠特征。目标5。通过分级分离和功能评价实验，在实验上鉴定与MOx_NPs诱导的（病理性）生物学效应有因果关系的关键冠蛋白。所产生的知识不仅将提高我们对纳米生物界面基本过程的理解，而且可能随后被利用来合理设计具有改进的功效、安全性和生物相容性的纳米材料。

Besides the prevalent use of nanoparticles (NPs) in industrial products, also their biomedical applications are expanding. Although both developments will result in an increasing exposure of humans to NPs, our current mechanistic knowledge of processes at the nano-bio interface is still fragmented. NPs adsorb biomolecules upon contact with all biological environments forming the so called NP-biomolecule corona. Importantly, we and others showed that particularly the protein corona critically impacts not only nanotoxicology but also the success and safety of nanobiomedical applications. Thus, biological systems are mostly not facing pristine manufactured but rather corona-coated transformed NPs. Moreover, the underlying molecular mechanisms how corona proteins affect the nano-bio interface are not yet resolved. We showed that focusing on highly controllable NP models combined with systematic analysis is key to correlate observed effects with distinct NP characteristics. Hence, we will investigate metal oxide NPs (MOx_NPs) not only representing an industrially and biomedical relevant category of NPs but also an excellent model system. Indeed the MOx_NPs physico-chemical characteristics. Hence, their controlled doping with other metals allows to control their reactivity and thus, to correlate reactivity with biological effects. However, the relevance of the protein corona was not studied so far. Based on our previous work we expect that the corona is also a key factor for MOx_NPs-triggered (adverse) biological effects by influencing oxidative stress, (subtoxic) signalling, cellular uptake, and intracellular dissolution processes. Consequently, combining controlled MOx_NPs synthesis and characterization with state-of-the-art analytical methods, such as multi-parametric high-content analysis, mass-spectrometry based proteomics together with (co-culture) in vitro exposure cell models, the scientific objectives of the project are: Aim 1. Analysing the impact of the biomolecule corona on the physico-chemical properties (size, surface charge, colloidal stability, aging) of MOx_NPs with incremental PdO-/Fe-dopings. Aim 2. Profiling of the MOx_NPs protein coronas by quantitative proteomics. Aim 3. Employing systematic high-content cell-based analysis to determine the impact of the protein corona on vitality, signaling, and oxidative stress responses in human exposure cell models. Aim 4. Bioinformatic correlation analysis and identification of protein corona signatures correlating with the MOx_NPs nano structure-activity relationships (nanoSARs). Aim 5. Experimentally identify key corona proteins causally involved in MOx_NPs-induced (patho)biological effects by fractionation and functional evaluation experiments. The generated knowledge will not only improve our understanding of basic processes at the nano-bio interface but may subsequent exploited to rationally design nanomaterials with improved efficacy, safety, and biocompatibility.