Sato Yuya, Zhang Weixu, Baba Teruhiko, Chung Ung-Il, Teramura Yuji
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Cellular and Molecular Biotechnology Research Institute (CMB), National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
Regen Ther. 2024 Jul 29;26:533-540. doi: 10.1016/j.reth.2024.07.006. eCollection 2024 Jun.
Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)-induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl--glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with Tat-PEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEG-lipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, Tat-PEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EV-liposome hybrids.
细胞外囊泡（EVs）是人体内细胞间通讯的天然载体。通过将其与脂质体等人工纳米载体结合用于药物递送，从而模拟和利用细胞外囊泡已引起了广泛关注。然而，目前用于操控细胞外囊泡以促进其与脂质体融合的技术有限；现有的聚乙二醇（PEG）诱导融合技术在融合方面效率极低。在我们之前的研究中，我们证明了由Tat肽（YGRKKRRQRRR）偶联的聚（乙二醇） - 磷脂（Tat - PEG - 脂质）可诱导膜融合，其中Tat肽和脂质结构域有助于膜附着以及随后细胞与脂质体之间的融合。这种方法对于形成细胞外囊泡和脂质体杂种很有前景。在本研究中，我们旨在使用Tat - PEG - 脂质融合细胞外囊泡和脂质体。我们从HEK293T细胞培养基中分离并表征了细胞外囊泡，并使用具有不同脂质链长度的Tat - PEG - 脂质处理由1,2 - 二棕榈酰 - sn - 甘油 - 3 - 磷酸胆碱和胆固醇（摩尔比1:1）组成的细胞外囊泡与脂质体的混合物。在此，我们使用壬酰基（C9）、十二酰基（C12）和肉豆蔻酰基（C14）基团作为带有5 kDa PEG链的脂质锚定基团。动态光散射分析表明，添加Tat - PEG - 脂质（尤其是C14、C12，其次是C9）后，细胞外囊泡与脂质体混合物的表观尺寸大幅增加。用于分析反应过程的荧光共振能量转移、共聚焦激光扫描显微镜和透射电子显微镜显示，膜融合发生在细胞外囊泡与脂质体之间，而非它们的聚集体之间。Tat - PEG - 脂质的短脂质结构域有效地诱导了膜融合以及杂种细胞外囊泡和脂质体的形成。因此，Tat - PEG - 脂质（C9和C12）可能是诱导膜融合以制备细胞外囊泡 - 脂质体杂种的有前途的候选物。
