细胞外囊泡可扩展生物力学特性的高含量、定量 AFM 分析,Nanoscale

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细胞外囊泡可扩展生物力学特性的高含量、定量 AFM 分析
Nanoscale ( IF 5.8 ) Pub Date : 2021-3-17 , DOI: 10.1039/d0nr09235e
Salvatore Andrea Gazze ₁ , Samantha J Thomas , Jetzabel Garcia-Parra , David W James , Paul Rees , Victoria Marsh-Durban , Randolph Corteling , Deyarina Gonzalez , R Steven Conlan , Lewis W Francis

Affiliation

细胞外囊泡 (EV) 作为天然生物分子穿梭机及其诊断和治疗潜力被广泛研究。这种兴趣呈指数级增长凸显了对电动汽车表征的高度稳健和可重复的方法的需求。在这里，我们优化了定量纳米机械工具，并展示了通过原子力显微镜 (AFM) 筛选 EV 种群的优势。我们的高内涵信息学分析工具可供 EV 社区使用，用于广泛、标准化地确定结构稳定性。超速离心 (UC) 和超声处理是分别用于 EV 隔离和加载的常用机械技术，用于展示优化的 PeakForce-定量纳米力学 (PF-QNM) 分析的实用性。工业规模生产的电动汽车在暴露于这些常用技术后表现出生化和生物力学变化。UC 导致物理尺寸略有增加，并且 EV 粘附减少，同时 CD63 含量减少。超声处理的 EV 表现出 CD81 水平显着降低、尺寸减小、杨氏模量增加和粘附力降低。这些生物力学和生化变化突出了 EV 样品制备技术对与 EV 细胞摄取和生物功能相关的关键特性的影响。PF-QNM 提供了关于电动汽车在纯化和下游加工后的结构信息的重要附加信息，分析工具将确保电动汽车社区对 AFM 数据分析的一致性，

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High content, quantitative AFM analysis of the scalable biomechanical properties of extracellular vesicles

Extracellular vesicles (EVs) are studied extensively as natural biomolecular shuttles and for their diagnostic and therapeutic potential. This exponential rise in interest has highlighted the need for highly robust and reproducible approaches for EV characterisation. Here we optimise quantitative nanomechanical tools and demonstrate the advantages of EV population screening by atomic force microscopy (AFM). Our high-content informatics analytical tools are made available for use by the EV community for widespread, standardised determination of structural stability. Ultracentrifugation (UC) and sonication, the common mechanical techniques used for EV isolation and loading respectively, are used to demonstrate the utility of optimised PeakForce-Quantitative Nano Mechanics (PF-QNM) analysis. EVs produced at an industrial scale exhibited biochemical and biomechanical alterations after exposure to these common techniques. UC resulted in slight increases in physical dimensions, and decreased EV adhesion concurrent with a decrease in CD63 content. Sonicated EVs exhibited significantly reduced levels of CD81, a decrease in size, increased Young's modulus and decreased adhesive force. These biomechanical and biochemical changes highlight the effect of EV sample preparation techniques on critical properties linked to EV cellular uptake and biological function. PF-QNM offers significant additional information about the structural information of EVs following their purification and downstream processing, and the analytical tools will ensure consistency of analysis of AFM data by the EV community, as this technique continues to become more widely implemented.

更新日期：2021-03-17

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