Stickney Zachary, Losacco Joseph, McDevitt Sophie, Zhang Zhiwen, Lu Biao
Department of Bioengineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA CA95053, USA.
Biochem Biophys Res Commun. 2016 Mar 25;472(1):53-9. doi: 10.1016/j.bbrc.2016.02.058. Epub 2016 Feb 18.
Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell-cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.
表面展示技术是一种新兴的关键技术，可用于展示功能性蛋白质以实现靶向药物递送和治疗。尽管存在多种技术，但仍缺乏理想的哺乳动物表面展示系统。外泌体是促进细胞间通讯的细胞外囊泡，可被设计成用于细胞特异性递送的纳米穿梭载体。在本研究中，我们报告了一种新型外泌体表面展示技术的开发，该技术利用哺乳动物细胞分泌的纳米囊泡及其跨膜蛋白四跨膜蛋白。通过构建一组荧光报告基因，用于在四跨膜蛋白的两个选定位点上对外泌体进行内表面和外表面展示，我们通过基因转染和体内荧光监测证明了外泌体展示的成功。随后，我们通过证明报告蛋白预期的细胞内分区进入亚细胞区室以及人HEK293细胞中外泌体的分泌，验证了我们的系统。最后，我们建立了稳定的工程细胞，以利用这个强大系统的能力进行持续生产、分泌和摄取展示的外泌体，同时对人类细胞生物学的影响最小。总之，我们的工作为外泌体的潜在应用铺平了道路，包括外泌体追踪和成像、靶向药物递送以及外泌体介导的疫苗和治疗。
