Li Bingxuan, Yu Fei
Key Laboratory of Molecular Pharmacology and Drug Evaluation, School of Pharmacy, Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China.
Pharmaceutics. 2025 May 27;17(6):699. doi: 10.3390/pharmaceutics17060699.
Exosomes are nanoscale extracellular vesicles actively secreted by cells that play critical roles in disease biomarker discovery, drug delivery, and direct therapeutic applications. However, in vivo pharmacokinetic (PK) studies of exosomes remain limited, hindering their clinical translation. Due to their complex and heterogeneous composition, most existing PK methods rely on chemical or genetic labeling, which may alter their native behavior and complicate accurate analysis. To address this challenge, we developed a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to investigate the PK of naive exosome-based therapeutic modalities. Exosomes were isolated from rat plasma using size exclusion chromatography (SEC) and quantified using multiple reaction monitoring (MRM) targeting specific exosomal peptides as surrogate analytes. Following intravenous administration of exosomes via the tail vein, plasma concentrations were determined by peptide peak areas, and PK parameters were calculated using a non-compartmental model. The method was rigorously validated for specificity, linearity, sensitivity, and reproducibility. Its feasibility was further confirmed by successfully characterizing the PK profile of HEK 293F-derived exosomes in rats. This analytical strategy enables direct, label-free quantification of exosomes in plasma and provides a robust platform for advancing exosome-based drug development and translational research.
外泌体是细胞主动分泌的纳米级细胞外囊泡，在疾病生物标志物发现、药物递送和直接治疗应用中发挥着关键作用。然而，外泌体的体内药代动力学（PK）研究仍然有限，这阻碍了它们的临床转化。由于其复杂且异质的组成，大多数现有的PK方法依赖于化学或基因标记，这可能会改变它们的天然行为并使准确分析变得复杂。为了应对这一挑战，我们开发了一种无标记液相色谱-串联质谱（LC-MS/MS）方法来研究基于天然外泌体的治疗方式的PK。使用尺寸排阻色谱（SEC）从大鼠血浆中分离出外泌体，并使用靶向特定外泌体肽作为替代分析物的多反应监测（MRM）进行定量。通过尾静脉静脉注射外泌体后，通过肽峰面积测定血浆浓度，并使用非房室模型计算PK参数。该方法在特异性、线性、灵敏度和重现性方面经过了严格验证。通过成功表征大鼠中HEK 293F衍生外泌体的PK谱，进一步证实了其可行性。这种分析策略能够直接、无标记地定量血浆中的外泌体，并为推进基于外泌体的药物开发和转化研究提供了一个强大的平台。
