Yamaguchi Satoshi, Kisaka Miho, Higashi Kotaro, Ishijima Ayumu, Azuma Takashi, Nakagawa Keiichi, Shibasaki Yoshikazu, Sakuma Ichiro, Okamoto Akimitsu
Department of Chemistry & Biotechnology, The University of Tokyo, Tokyo, Japan.
Department of Precision Engineering, The University of Tokyo, Tokyo, Japan.
Biotechnol J. 2023 Jul;18(7):e2300018. doi: 10.1002/biot.202300018. Epub 2023 May 7.
Ultrasound-guided protein delivery is promising for site-specific control of cellular functions in the deep interior of the body in a noninvasive manner. Herein, we propose a method for cytosolic protein delivery based on ultrasound-guided intracellular vaporization of perfluorocarbon nano-droplets. The nano-droplets were conjugated with cargo proteins through a bio-reductively cleavable linker and introduced into living cells via antibody-mediated binding to a cell-surface receptor, which gets internalized through endocytosis. After the cells were exposed to ultrasound for endosomal escape of proteins, the ultrasound-responsive cytosolic release of a cargo enzyme was confirmed by visualizing the hydrolysis of the fluorogenic substrate using confocal microscopy. Moreover, a significant decrease in cell viability was achieved via the release of a cytotoxic protein in response to ultrasound treatment. The results of this study provide the proof of a principle that protein-conjugated nano-droplets can be used as carriers in ultrasound-guided cytosolic delivery of proteins.
超声引导下的蛋白质递送有望以非侵入性方式对身体深部内部的细胞功能进行位点特异性控制。在此，我们提出了一种基于全氟碳纳米液滴超声引导下细胞内汽化的胞质蛋白质递送方法。纳米液滴通过生物可还原裂解连接子与负载蛋白偶联，并通过抗体介导与细胞表面受体结合引入活细胞，该受体通过内吞作用内化。在细胞暴露于超声以实现蛋白质的内体逃逸后，通过共聚焦显微镜观察荧光底物的水解，证实了负载酶的超声响应性胞质释放。此外，通过响应超声处理释放细胞毒性蛋白，实现了细胞活力的显著降低。本研究结果提供了一个原理证明，即蛋白质偶联纳米液滴可作为蛋白质超声引导胞质递送的载体。
