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

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Abstract

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.

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Teo, S. L. Y., Rennick, J. J., Yuen, D., Al-Wassiti, H., Johnston, A. P. R., & Pouton, C. W. (2021). Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-23997-x

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