Meyer Conary, Losacco Joseph, Stickney Zachary, Li Lingxuan, Marriott Gerard, Lu Biao
Department of Bioengineering, Santa Clara University, Santa Clara.
Crown College, University of California at Santa Cruz, Santa Cruz.
Int J Nanomedicine. 2017 Apr 18;12:3153-3170. doi: 10.2147/IJN.S133430. eCollection 2017.
Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells.
外泌体是细胞衍生的纳米囊泡，有望作为将治疗药物细胞内递送至哺乳动物细胞的活体载体。然而，由于缺乏将治疗性蛋白质加载到外泌体中并促进其被靶细胞摄取的有效方法，这种潜力受到了削弱。在这里，我们展示了水泡性口炎病毒糖蛋白（VSVG）如何通过假型化机制将蛋白质货物加载到外泌体上并提高其递送能力。通过将一组荧光和发光报告基因与VSVG融合，我们通过基因转染和荧光追踪展示了VSVG融合蛋白成功靶向并整合到外泌体中。我们随后通过对VSVG及其参与最终从转染的HEK293细胞释放的内体/外泌体进行活细胞成像来验证我们的系统。我们表明，外泌体的VSVG假型化不会影响外泌体的大小或分布，并且全长VSVG和没有胞外结构域的VSVG都显示整合到外泌体膜中，这表明蛋白质加载不需要胞外结构域。最后，与加载没有胞外结构域的VSVG的外泌体相比，用全长VSVG假型化的外泌体在更大程度上被多种受体细胞类型内化，证实了胞外结构域在细胞嗜性中的作用。总之，我们的工作引入了一种新的基因编码假型化平台，通过基于外泌体的载体将治疗性蛋白质加载并增强其细胞内递送至靶细胞。
