Cho Young-Woo, Cho Mi Young, Yoon Jaehyeon, Hong Da Eun, Lee Ju-Young, Park Hye Sun, Lee Hyunseung, Hong Kwan Soo, Won-Kyu Lee, Saehae Choi, Song Suk-Gil, Noh Young-Woock
Division of Drug Safety Evaluation, NDDC, Osong Medical Innovation Foundation, Cheongju, South Korea.
College of Pharmacy, Chungbuk National University, Cheongju, South Korea.
J Extracell Vesicles. 2024 Jul;13(7):e12489. doi: 10.1002/jev2.12489.
Recently, extracellular vesicles (EVs) have been developed as therapeutic targets for various diseases. Biodistribution is crucial for EVs intended for therapeutic purposes because it can determine the degree of on- and off-target effects. This study aimed to explore techniques to evaluate the biodistribution of unmodified EVs. We devised a novel quantitative polymerase chain reaction (qPCR)-based assay to detect unmodified EVs by targeting mitochondrial deoxyribonucleic acid (mtDNA), a constituent of EVs. We focused on specific mtDNA regions that exhibited homologous variations distinct from their rodent mtDNA counterparts to establish this analytical approach. Herein, we successfully designed primers and probes targeting human and rodent mtDNA sequences and developed a highly specific and sensitive qPCR method. Furthermore, the quantification range of EVs isolated from various cells differed based on the manufacturer and cell source. IRDye 800CW-labelled Expi293F EV mimetics were administered to the animals via the tail vein to compare the imaging test and mtDNA-qPCR results. The results obtained from imaging tests and mtDNA-qPCR to investigate EV biodistribution patterns revealed differences. The results revealed that our newly developed method effectively determined the biodistribution of unmodified EVs with high sensitivity and reproducibility.
最近，细胞外囊泡（EVs）已被开发为多种疾病的治疗靶点。生物分布对于用于治疗目的的EVs至关重要，因为它可以决定靶向和非靶向效应的程度。本研究旨在探索评估未修饰EVs生物分布的技术。我们设计了一种基于新型定量聚合酶链反应（qPCR）的检测方法，通过靶向EVs的组成成分线粒体脱氧核糖核酸（mtDNA）来检测未修饰的EVs。我们专注于特定的mtDNA区域，这些区域表现出与其啮齿动物mtDNA对应物不同的同源变异，以建立这种分析方法。在此，我们成功设计了靶向人类和啮齿动物mtDNA序列的引物和探针，并开发了一种高度特异性和灵敏的qPCR方法。此外，从不同细胞中分离的EVs的定量范围因制造商和细胞来源而异。通过尾静脉向动物注射IRDye 800CW标记的Expi293F EV模拟物，以比较成像测试和mtDNA-qPCR结果。成像测试和mtDNA-qPCR用于研究EV生物分布模式的结果显示存在差异。结果表明，我们新开发的方法能够以高灵敏度和可重复性有效地确定未修饰EVs的生物分布。
