Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma.
Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma.
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作者：
Woo HK;Cho YK;Lee CY;Lee H;Castro CM;Lee H
细胞外囊泡（EV）携带从亲本细胞遗传的信息，在疾病诊断中具有重要的潜力。然而，在血液中，EV的数量超过低密度脂蛋白（LDL）的104倍，但大小和密度相似。这些基本的缺点通常会导致LDL溢出到EV分离株中，从而混淆检测结果。我们假设EV可以根据电荷与LDL进一步分离：EV和LDL具有不同的脂质组成，这可能导致不同的表面电荷密度。为了验证这一假设，我们对EV和LDL的表面电荷进行了建模和定量，并使用这些信息通过离子交换色谱法将EV与LDL最佳分离。研究方法：我们构建了增强型双模式色谱（eDMC）装置，其进行i）尺寸排阻以去除小于EV和LDL的颗粒，以及ii）在酸性洗脱中进行阳离子交换以保留LDL长于EV。通过分析分离的EV的产率和纯度来评价与仅尺寸排阻相比的eDMC的性能。结果如下：通过在不同缓冲液pH下测量和建模zeta电位，我们估计了EV（-6.2mC/m2）和LDL（-3.6mC/m2）的表面电荷密度，揭示了EV比LDL带更多的负电荷。此外，EV和LDL之间的电荷差异在弱酸性条件下（pH = 6.4）最大。通过应用这些发现，我们优化了eDMC操作，以直接从血浆中富集EV，在30分钟内耗尽>99.8%的LPP。最大限度地减少LDL污染改善了EV分子测定中的分析信号，包括单囊泡成像，批量蛋白质测量和mRNA检测。结论：这些发展将促进双模分离的转化价值-一种用于从血浆样品中分离EV的快速、无设备和无偏置的方式。
Extracellular vesicles (EVs) carry information inherited from parental cells, having significant potential for disease diagnosis. In blood, however, EVs are outnumbered >104-fold by low density lipoproteins (LDLs), yet similar in size and density. These fundamental disadvantages often cause LDL spillover into EV isolates, thus confounding assay results. We hypothesized that EVs can be further separated from LDLs based on electric charge: EVs and LDLs have different lipid composition, which can lead to differential surface charge densities. To test this hypothesis, we modeled and quantified the surface charge of EVs and LDLs, and used the information to optimally separate EVs from LDLs via ion-exchange chromatography. Methods: We built an enhanced dual-mode chromatography (eDMC) device which performed i) size-exclusion to remove particles smaller than EVs and LDLs and ii) cation-exchange in an acidic elution to retain LDLs longer than EVs. The performance of the eDMC, in comparison to size-exclusion only, was evaluated by analyzing the yield and purity of the isolated EVs. Results: By measuring and modeling zeta potentials at different buffer pH, we estimated surface charge densities of EVs (-6.2 mC/m2) and LDLs (-3.6 mC/m2), revealing that EVs are more negatively charged than LDLs. Furthermore, the charge difference between EVs and LDLs was maximal at a weak acidic condition (pH = 6.4). By applying these findings, we optimized eDMC operation to enrich EVs directly from plasma, depleting >99.8% of LPPs within 30 min. Minimizing LDL contamination improved analytical signals in EV molecular assays, including single vesicle imaging, bulk protein measurements, and mRNA detection. Conclusions: These developments will promote the translational value of the dual-mode separation - a fast, equipment-free, and non-biased way for EV isolation from plasma samples.