Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

• Published in: Nature communications 2021-06, Vol.12 (1), p.3721-3721, Article 3721
• Main Authors: Teo, Serena L. Y., Rennick, Joshua J., Yuen, Daniel, Al-Wassiti, Hareth, Johnston, Angus P. R., Pouton, Colin W.
• Format: Article
• Language: English
• Subjects: 13/109; 13/44; 14/10; 14/5; 631/1647/1888; 631/61/2297; 631/80/2023; 82/80; Assaying; Cell Line, Tumor; Cell Membrane - metabolism; Cell membranes; Cell Survival - drug effects; Cell-Penetrating Peptides - metabolism; Cytosol; Cytosol - metabolism; Drug delivery; Endosomes - metabolism; Gene Expression; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Humans; Luciferases - chemistry; Luminescent Measurements - methods; Mass Spectrometry; multidisciplinary; Nucleic acids; Peptides; Proteins; Recombinant Proteins; Science; Science (multidisciplinary); Sensitivity; Sensitivity and Specificity
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-23997-x

Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol. Our understanding of cytosolic delivery is hindered by existing methods for quantification which suffer from being indirect and showing low sensitivity. Here the authors report a SLEEQ (Split Luciferase Endosomal Escape Quantification) assay to assess cytosolic delivery of cell-penetrating peptides.