Novel Quantification of Extracellular Vesicles with Unaltered Surface Membranes Using an Internalized Oligonucleotide Tracer and Applied Pharmacokinetic Multiple Compartment Modeling.
Novel Quantification of Extracellular Vesicles with Unaltered Surface Membranes Using an Internalized Oligonucleotide Tracer and Applied Pharmacokinetic Multiple Compartment Modeling.
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DOI：
10.1007/s11095-021-03102-z
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发表时间：
2021-10
影响因子：
3.7
通讯作者：
Benson EA
中科院分区：
文献类型：
--
作者：
De Luca T;Stratford RE Jr;Edwards ME;Ferreira CR;Benson EA
我们开发了一种标记小细胞外囊泡（sEV）而不破坏内源性配体的方法。使用标记的sEV给予清醒的大鼠，我们开发了一个多室药代动力学模型，以确定潜在的差异处置的sEV从三种不同的细胞类型。用非同源寡核苷酸标记粗sEV，并使用商业试剂从细胞培养基中分离。颈静脉导管用于将EV引入清醒大鼠（n = 30）并收集血液样品。利用数字PCR以允许在宽动态范围内进行定量。采用一阶条件估计-扩展最小二乘法（FOCE ELS）的非线性混合效应分析估计具有相关动物内变异性的群体水平参数。86.5% ± 1.5%（平均值±标准误）的EV颗粒在45-195 nm的尺寸范围内，并证明了内体来源的蛋白质和脂质标志物。掺入的寡核苷酸在血液中是稳定的，并且在五个半衰期内是可检测的。数据最好用三室模型描述，其中一个从中央室消除。我们进行了基于观察的模拟后验预测评价与预测校正的视觉预测检查。协变量和bootstrap分析确定了影响外周血容量（V2和V3）和清除率（Cl 3）的细胞类型。我们的方法依赖于已建立的实验室技术，可以针对细胞外囊泡的药代动力学处置的各种生物学问题进行定制，并将为EV摄取和靶向治疗背景下研究EV配体-受体相互作用提供补充方法。在线版本包含补充材料，可通过10.1007/s11095-021-03102-z获取。
We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation – extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45–195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics. The online version contains supplementary material available at 10.1007/s11095-021-03102-z.
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