Background

Extracellular vesicles (EVs), nano-sized particles released by cells, exhibit inherent heterogeneity, posing challenges for precise classification. This study introduces a method utilizing the coffee ring effect for size-based separation analysis, driven by the outward flow during droplet evaporation with Marangoni flow resulting from a surface tension gradient.

Results

The controlled separation of nanoparticles based on size was applied to characterize EV and virus-like particle (VLP) samples. Tetraspanin markers exhibited distinct distribution patterns in dried droplets, with CD63-single-positive particles being smaller than those positive for CD9, CD81, or gag. Additionally, using previously developed fluorescence nanoparticle tracking analysis and transmission electron microscopy, single vesicle analysis validated the size disparities in CD-positive particles. RNA analysis through the coffee ring effect and fluorescent NTA revealed differential patterns in EVs and VLPs, providing different insights into RNA packaging.

Significance

This multifaceted approach enhances understanding EV heterogeneity, emphasizing the potential influence of cellular origin and biogenesis pathways on particle characteristics.

中文翻译：

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基于大小的细胞外囊泡分离研究 Tetraspanins 和 RNA 之间的关系

 背景

细胞外囊泡 （EV） 是细胞释放的纳米级颗粒，表现出固有的异质性，对精确分类构成挑战。本研究介绍了一种利用咖啡环效应进行基于尺寸的分离分析的方法，该方法由液滴蒸发过程中的向外流动驱动，表面张力梯度产生马兰戈尼流。

 结果

基于大小的纳米颗粒受控分离用于表征 EV 和病毒样颗粒 （VLP） 样品。Tetraspanin 标志物在干燥的液滴中表现出不同的分布模式，CD63 单阳性颗粒小于 CD9 、 CD81 或 gag 阳性颗粒。此外，使用先前开发的荧光纳米颗粒跟踪分析和透射电子显微镜，单囊泡分析验证了 CD 阳性颗粒的大小差异。通过咖啡环效应和荧光 NTA 进行的 RNA 分析揭示了 EV 和 VLP 的不同模式，为 RNA 包装提供了不同的见解。

 意义

这种多方面的方法增强了对 EV 异质性的理解，强调了细胞起源和生物发生途径对颗粒特性的潜在影响。