Di Santo Riccardo, Digiacomo Luca, Quagliarini Erica, Capriotti Anna Laura, Laganà Aldo, Zenezini Chiozzi Riccardo, Caputo Damiano, Cascone Chiara, Coppola Roberto, Pozzi Daniela, Caracciolo Giulio
Nanodelivery Lab, Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.
Department of Chemistry, Sapienza University of Rome, Rome, Italy.
Front Bioeng Biotechnol. 2020 May 25;8:491. doi: 10.3389/fbioe.2020.00491. eCollection 2020.
The protein corona (PC) that forms around nanomaterials upon exposure to human biofluids (e.g., serum, plasma, cerebral spinal fluid etc.) is personalized, i.e., it depends on alterations of the human proteome as those occurring in several cancer types. This may relevant for early cancer detection when changes in concentration of typical biomarkers are often too low to be detected by blood tests. Among nanomaterials under development for diagnostic (IVD) testing, Graphene Oxide (GO) is regarded as one of the most promising ones due to its intrinsic properties and peculiar behavior in biological environments. While recent studies have explored the binding of single proteins to GO nanoflakes, unexplored variables (e.g., GO lateral size and protein concentration) leading to formation of GO-PC in human plasma (HP) have only marginally addressed so far. In this work, we studied the PC that forms around GO nanoflakes of different lateral sizes (100, 300, and 750 nm) upon exposure to HP at several dilution factors which extend over three orders of magnitude from 1 (i.e., undiluted HP) to 10. HP was collected from 20 subjects, half of them being healthy donors and half of them diagnosed with pancreatic ductal adenocarcinoma (PDAC) a lethal malignancy with poor prognosis and very low 5-year survival rate after diagnosis. By dynamic light scattering (DLS), electrophoretic light scattering (ELS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nano liquid chromatography tandem mass spectrometry (nano-LC MS/MS) experiments we show that the lateral size of GO has a minor impact, if any, on PC composition. On the other side, protein concentration strongly affects PC of GO nanoflakes. In particular, we were able to set dilution factor of HP in a way that maximizes the personalization of PC, i.e., the alteration in the protein profile of GO nanoflakes between cancer vs. non-cancer patients. We believe that this study shall contribute to a deeper understanding of the interactions among GO and HP, thus paving the way for the development of IVD tools to be used at every step of the patient pathway, from prognosis, screening, diagnosis to monitoring the progression of disease.
纳米材料暴露于人体生物流体（如血清、血浆、脑脊液等）后形成的蛋白质冠层（PC）是个性化的，即它取决于人类蛋白质组的变化，就像在几种癌症类型中发生的那样。这可能与早期癌症检测相关，因为典型生物标志物浓度的变化通常过低，难以通过血液检测来检测。在正在开发用于诊断（IVD）测试的纳米材料中，氧化石墨烯（GO）因其固有特性和在生物环境中的特殊行为而被视为最有前途的材料之一。虽然最近的研究探索了单一蛋白质与GO纳米片的结合，但导致在人血浆（HP）中形成GO-PC的未探索变量（如GO横向尺寸和蛋白质浓度）迄今为止仅得到了少量关注。在这项工作中，我们研究了不同横向尺寸（100、300和750 nm）的GO纳米片在暴露于HP时在几个稀释因子下形成的PC，这些稀释因子从1（即未稀释的HP）到10跨越了三个数量级。HP取自20名受试者，其中一半是健康供体，另一半被诊断患有胰腺导管腺癌（PDAC），这是一种预后不良且诊断后5年生存率极低的致命恶性肿瘤。通过动态光散射（DLS）、电泳光散射（ELS）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和纳升液相色谱串联质谱（nano-LC MS/MS）实验，我们表明GO的横向尺寸对PC组成的影响很小（如果有影响的话）。另一方面，蛋白质浓度强烈影响GO纳米片的PC。特别是，我们能够以一种最大化PC个性化的方式设置HP的稀释因子，即癌症患者与非癌症患者之间GO纳米片蛋白质谱的变化。我们相信这项研究将有助于更深入地理解GO与HP之间的相互作用，从而为开发在患者诊疗路径的每个步骤（从预后、筛查、诊断到监测疾病进展）中使用的IVD工具铺平道路。
