Department of Physics , Chalmers University of Technology , 41296 Gothenburg , Sweden.
Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition , University of Gothenburg , 40530 Gothenburg , Sweden.
Langmuir. 2018 Jul 24;34(29):8522-8531. doi: 10.1021/acs.langmuir.7b04214. Epub 2018 Jul 11.
Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index ( n ≈ 1.35) compared with the LD EVs ( n ≈ 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.
细胞外囊泡 (EVs) 因其在各种生物过程中发挥的关键作用以及在临床诊断中的生物标志物和药物输送及基因治疗应用中的有效载体的潜在用途而引起了越来越多的关注。然而，它们的充分利用取决于将其分类为不同亚群的可能性，而这一任务又依赖于识别其独特生物分子和物理特征的有效手段。由于 EV 样品的高度异质性，这些信息仍然难以捉摸，因此需要新的和补充的表征方案来帮助扩展不同 EV 特征的库。在这项工作中，我们使用具有单 EV 分辨率的表面敏感波导散射显微镜来表征根据其浮力密度差异预先分离的两种类似大小的 EV 子集。出乎意料的是，散射强度分布表明，高密度 (HD) 群体的散射强度平均比低密度 (LD) 群体低三倍。通过进一步用自插入脂质膜染料标记 EV 样品，可以在单粒子水平上同时测量和关联 EV 的散射和荧光强度。标记的 HD 样品不仅表现出较低的荧光和散射强度，而且与 LD EVs（ n ≈ 1.38）相比，有效折射率 ( n ≈ 1.35) 也较低，表明 HD EVs 中的脂质和蛋白质含量确实较低。尽管相似大小的 EV 密度梯度分离通常与生物分子含量的差异有关，但根据这些观察结果，我们建议这种分离更能反映溶质穿透包围 EV 的脂质膜的能力，也就是说，两个梯度带更可能是由于膜通透性的差异，而不是 EV 中生物分子含量的差异。
