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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. [ABSTRACT FROM AUTHOR]

In this article, the microstructures of Zn-55Al-1.6Si coating spangles on steel were examined using optical microscopy and the crystallographic orientations of the spangles analyzed using X-ray diffraction (XRD) and electron backscatter diffraction (EBSD). It was found that α-Al dendrite arms forming the spangles grew along straight lines with uniform size, uniform dendrite arm spacing, and in most sectors in directions parallel to their border lines. It was also found that (111) and (100) were the most common orientations in the coating. The likely preferred dendrite arm growth directions for the spangles having the (111) orientation were [110] or [112], whereas both [100] and [110] were possible growth directions when the spangles had (100) orientations. The [320] directions suggested in the literature were unlikely to be the growth directions in any of the spangles examined. [ABSTRACT FROM AUTHOR]

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells. Determining the trafficking of intracellular material is commonly done by colocalisation analysis using microscopy. Here the authors monitor trafficking of select cargo by measuring the conversion of quenched SNAP-tag substrates by subcellularly-localised SNAP-tag and detection by flow cytometry. [ABSTRACT FROM AUTHOR]