The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

中文翻译：

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通过 AFM 纳米机械成像对 EV-脂蛋白混合物进行颗粒分析

脂蛋白（LP）和细胞外囊泡（EV）的广泛重叠的物理化学性质是分离和表征这些普遍存在的生物脂质纳米颗粒的主要障碍之一。我们在此介绍基于原子力显微镜 (AFM) 的定量形态测定法在混合 LP 和 EV 样品的快速纳米机械筛选中的应用。该方法可以在几个小时内确定数百个单独纳米物体的直径和机械刚度。获得的直径与通过冷冻电子显微镜（cryo-EM）测量的直径定量一致；为每个物体分配特定的纳米力学读数可以同时区分共隔离的 EV 和 LP，即使它们具有重叠的尺寸分布。EV 和所有类别的 LP 都具有直径和刚度的特定组合的特征，因此可以根据化学计量比、表面积和体积来估计它们在 EV/LP 混合样品中的相对丰度。作为一个附带发现，我们展示了特定 LP 类别的机械行为如何与冷冻电镜揭示的独特结构特征相关。所描述的方法是无标签的、单步的并且执行相对较快。重要的是，它可用于分析由于整体平均、对小颗粒敏感性低或两者兼而有之的原因，用几种现有技术评估非常困难的样品，从而为快速评估 EV/LP 分离株的纯度提供了非常有用的工具包括血浆和血清衍生的制剂。