PDF(Pubmed)
Abstract：
Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling.
摘要：
血浆中的细胞外囊泡(EV)被认为是疾病的潜在生物标志物。虽然血浆很容易获得，由于这种体液的生物学复杂性，在单颗粒水平上对电动汽车的分析仍然具有挑战性。除了电动汽车，血浆含有不同类型的脂蛋白颗粒（LPP），数量超过电动汽车的数量级，并且在生物物理特性如尺寸方面部分重叠，密度和分子组成。因此,在EV隔离期间，LPP通常是共同隔离的。此外，已在纯化的EV制剂中观察到物理EV-LPP复合物。由于LPP的共同隔离或关联可能会影响基于EV的分析和生物标志物概况分析，我们通过使用无标记原子力显微镜研究了生物样品中EV-LPP复合物的存在和形成，低温电子层析成像和光学捕获粒子的同步瑞利和拉曼散射分析以及基于荧光的高灵敏度单粒子流式细胞术。此外，我们使用纯化的肿瘤细胞系衍生的EV在不同类别的纯化人LPP中的体外刺入实验,评估了在存在LPP的情况下对流式细胞仪分析的影响.基于正交单颗粒分析技术，我们证明了EV-LPP复合物可以在生理条件下形成。此外，我们表明，在基于荧光的流式细胞仪EV分析染色的LPP，以及EV-LPP协会，可以影响定量和定性的EV分析。最后,我们证明了电动汽车所在的生物流体的胶体基质会影响它们的浮力密度，尺寸和/或折射率(RI)，这可能会对下游EV分析和EV生物标志物分析产生影响。
