An in vitro lipid-mixing assay to investigate the fusion between small extracellular vesicles and endosome
An in vitro lipid-mixing assay to investigate the fusion between small extracellular vesicles and endosome
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体外脂质混合试验研究小细胞外囊泡与内体之间的融合
DOI：
10.1016/j.ab.2023.115130
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发表时间：
2023
影响因子：
2.9
通讯作者：
Kumiko Sakai-Kato
中科院分区：
文献类型：
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作者：
Arisa Takeda;Asuka Tachibana;Hiroki Nagumo;Kumiko Sakai-Kato
小的细胞外囊泡（sEV）如外来体可以有效地将核酸递送到受体细胞的胞质溶胶中。然而，随后与内体融合的分子机制还没有很好地理解。在这项研究中，我们开发了一种使用内体模拟阴离子脂质体的体外脂质混合试验，以研究sEV与内体之间的融合。我们观察到sEV和阴离子脂质体之间的颗粒数比、脂质体的直径和缓冲液pH对于融合活性都是重要的。此外，我们优化了脂质体脂质组合物，并证明了将阴离子脂质双（单油酰甘油）磷酸盐和胆固醇是重要的有效和可靠的融合。Ourin体外试验表明，pH值的降低增加了融合活性。此外，这表明融合活性的这种pH依赖性增加主要是由于sEV的变化。sEV具有比模拟外泌体的物理化学性质的人工脂质体更大的融合活性。这些结果与先前的体内研究结果一致，支持我们系统的生理相关性。本研究为进一步阐明sEV与内涵体融合的分子机制提供了重要平台。
Small extracellular vesicles (sEVs) such as exosomes can efficiently deliver nucleic acids into the cytosol of recipient cells. However, the molecular mechanism of the subsequent fusion with an endosome is not well understood. In this study, we developed anin vitrolipid-mixing assay using an endosomal-mimicking anionic liposome to investigate the fusion between sEVs and endosomes. We observed that the particle number ratio between the sEVs and the anionic liposomes, the diameter of the liposomes, and the buffer pH were all important for fusion activity. Furthermore, we optimized the liposomal lipid composition and demonstrated that incorporating the anionic lipid bis(monooleoylglycero) phosphate and cholesterol was important for efficient and reliable fusion. Ourin vitroassay suggested that a decrease in pH increased the fusion activity. Additionally, it was suggested that this pH-dependent increase in the fusion activity was predominantly due to a change in the sEVs. sEVs possess a larger fusion activity than artificial liposomes that mimic the physicochemical properties of exosomes. These results are consistent with those of previousin vivostudies, supporting the physiological relevance of our system. This study provides an important platform for further research to clarify the molecular mechanisms of fusion between sEVs and endosomes.