Department of Chemistry, Stanford University, Stanford, California 94305, United States.
Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin 13353, Germany.
J Am Chem Soc. 2023 May 10;145(18):10220-10226. doi: 10.1021/jacs.3c01145. Epub 2023 Apr 26.
The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms, but existing approaches are limited by immunogenicity, lack of cell-type-specificity, or their multi-component nature. Here we explore inactivated botulinum neurotoxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain can be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3 to 55 kDa in size, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a mixed culture. These explorations suggest that BoNT may be a versatile platform for targeted protein and peptide delivery into mammalian cells.
将蛋白质和肽递送到活哺乳动物细胞的细胞质中是一项具有重大影响的技术，无论是对于基础科学还是医学领域。天然的细胞穿透蛋白毒素已被证明是一种有前途的蛋白质递送平台，但现有的方法受到免疫原性、缺乏细胞类型特异性或其多组分性质的限制。在这里，我们探索了失活的肉毒神经毒素（BoNT）作为一种蛋白质递送平台。我们使用细胞质中分裂荧光素酶重组作为内体逃逸和细胞质递送的读数，表明用纳米体替代其天然受体结合域的 BoNT 嵌合体可以选择性地靶向表达纳米体匹配表面标志物的细胞。我们使用嵌合 BoNT 将一系列大小为 1.3 至 55 kDa 的货物递送到细胞中，并证明了正交货物在混合培养物中不同细胞群体中的选择性递送。这些探索表明，BoNT 可能是一种将蛋白质和肽靶向递送到哺乳动物细胞的多功能平台。
