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. 2019 Oct 28;10:4906. doi: 10.1038/s41467-019-12922-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Shuttle peptide design and protein delivery to airway epithelia. a Amino acid sequences of shuttle peptides. Sequences aligned to highlight structural similarities. Cationic residues are highlighted in blue; hydrophobic residues are in gray. Remaining residues are in green. b Indel% in primary NK cells following Cas12a RNP delivery targeting NKG2A gene with indicated shuttle peptide ([Cas12a]: 1.33 µM; [crRNA]: 2.0 µM). Results quantified 48 h after delivery (mean ± SE; n = 3 independent experiments). c Shuttle peptide-mediated delivery of nuclear-targeted GFP protein to human airway epithelia (HAE) and tracheal explants. Left panel: Schematic of primary HAE grown at air–liquid interface. ASL: airway surface liquid. Right panel: photomicrograph of well-differentiated primary HAE culture demonstrating pseudostratified columnar epithelium. Hematoxylin and eosin stain, ×40 magnification. d Efficiency of GFP delivery by different shuttle peptides. Peptides applied at four concentrations (5, 10, 15, or 20 μM); GFP concentrations 10 or 20 μM. Left graph: Cells were transduced with indicated peptide and GFP combinations and GFP quantified by fluorescence reader. Results mean ± SE; n = 4 donors. *P < 0.05, **P < 0.005, ***P < 0.001, by one-way ANOVA with Tukey’s multiple comparison test. Right panels: Representative en face views of epithelial sheets following GFP delivery at the highest peptide:GFP concentration (20 μM:20 μM), ×20 magnification. Formulations were applied for 15 min. e GFP localization in multiple epithelial cell types following shuttle peptide-mediated delivery. HAEs were transduced for 15 min with GFP protein and S10 peptide. Left panel: confocal image (XZ) shows co-localization of GFP and specific marker of ciliated cells (α-tubulin, red, indicated by white arrows), non-ciliated cells (white arrowheads), F-actin (phalloidin stain, gray), nuclei (DAPI, blue). Right panel: co-localization of GFP and goblet cell marker (Muc5AC, red, indicated by white arrowhead), nuclei (DAPI, blue), and F-actin (phalloidin stain, gray). n ≥ 6 different donor epithelia; ×64 magnification. f Shuttle peptide delivery of GFP to human tracheal explants. S10 (20 µM) and GFP protein (20 µM) applied to tracheal explant for 1 h. Left panel: both ciliated (α-tubulin, red, white arrowhead) and non-ciliated (white arrow) cells were transduced. Right panel: non-ciliated goblet cells were transduced (Muc5AC, red, white arrows); ×40 magnification. Data underlying this Figure are provided as Source Data file