Huang Tianwei, Sato Yuya, Kuramochi Akiko, Ohba Yoshio, Sano Masayuki, Miyagishi Makoto, Tateno Hiroaki, Wadhwa Renu, Kawasaki Kazunori, Uchida Takeyuki, Ekdahl Kristina N, Nilsson Bo, Chung Ung-Il, Teramura Yuji
Department of Bioengineering, 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. 2023 Jan 11;22:90-98. doi: 10.1016/j.reth.2022.12.007. eCollection 2023 Mar.
Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.
外泌体（直径30 - 200纳米）是细胞分泌的细胞外囊泡的一种亚型，含有DNA、微小RNA（miRNA）和蛋白质。外泌体有望作为一种在疾病治疗中递送药物或功能性miRNA的手段，具有重要价值。然而，尽管外泌体具有内在的递送功能，但其递送效果仍不够理想。细胞穿膜肽（CPPs）是一类短肽家族，可促进细胞对分子和囊泡的摄取。我们之前报道过，用与聚乙二醇共轭磷脂（PEG - 脂质）共轭的CPPs修饰细胞和脂质体，通过CPPs诱导黏附，可用于基于细胞的分析和收获脂质体。在本研究中，我们旨在使用Tat肽（YGRKKRRQRRR） - PEG - 脂质调节外泌体表面，以改善其向内皮细胞的细胞内递送。我们从HEK 293 T细胞培养上清中分离并鉴定了外泌体。使用异硫氰酸荧光素标记将具有不同间隔分子量和脂质类型的Tat共轭PEG - 脂质掺入外泌体中，以优化固定在外泌体表面的Tat - PEG - 脂质数量。将用Tat - PEG - 脂质修饰的外泌体与人脐静脉内皮细胞（HUVECs）孵育以研究相互作用。通过流式细胞术在HUVECs内高度检测到外泌体表面掺入的与5 kDa PEG和C16脂质共轭的Tat。对照组的HUVECs中荧光可忽略不计。因此，Tat - PEG - 脂质可在外泌体表面进行修饰，改善外泌体的细胞内递送。
