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.
Highlights
Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids
Our results show a linear correlation with luminescence down to 5 pM of HiBiT proteins were adjusted accordingly (HiBiT) for the split NanoLuciferase assay, which is more than four orders of magnitude more sensitive than the detection limit for split green fluorescence protein (GFP)
It is challenging to compare images with such highly contrasting intensities, and it is challenging to observe weak cytosolic signal, as the bright punctate fluorescence in the endosomes overwhelms any diffuse cytosolic signal. Techniques such as the chloroalkane penetration assay[37], fluorescence correlation spectroscopy (FCS)[28] and NanoClick[38] have been developed and while they provide a way of measuring the amount of material that has reached the cytosol, they do not measure the efficiency of endosomal escape
Summary
Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. 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 Biological therapies such as peptides, proteins and nucleic acids have emerged as promising approaches for combating a wide variety of infectious, immunological and genetic disorders[1,2]. Co-incubation of materials of interest with calcein, a small membrane-impermeable dye that appears punctate when sequestered within endosomal/ lysosomal compartments, is often another method used to overcome the requirement for fluorescent labelling This approach does not provide a direct measurement of cargo escape. Split green fluorescence protein (GFP) was developed as an endosomal escape probe that allows cytosolic signal to be distinguished from sequestered signal[15,16,17] This approach provides a direct quantification of cytosolic delivery, but is limited by poor sensitivity. Concentrations above 10 μM are required to generate a measurable signal or to observe endosomal escape, but these concentrations are significantly higher than therapeutically and clinically relevant doses for most biological materials
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