Abstract
Recent progress with chemistry tools to deliver into living cells has seen a shift of attention from counterion-mediated uptake of cell-penetrating peptides (CPPs) and their mimics, particularly the Schmuck cation, toward thiol-mediated uptake with cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs), here exemplified by asparagusic acid. A persistent challenge in this evolution is the simultaneous and quantitative detection of cytosolic delivery and cytotoxicity in a high-throughput format. Here, we show that the combination of the HaloTag-based chloroalkane penetration assay (CAPA) with automated high-content (HC) microscopy can satisfy this need. The automated imaging of thousands of cells per condition in multiwell plates allows us to obtain quantitative data on not only the fluorescence intensity but also on the localization in a very short time. Quantitative and statistically relevant results can be obtained from dose–response curves of the targeted delivery to selected cells and the cytotoxicity in the same experiment, even with poorly optimized cellular systems.
Highlights
The effective delivery of substrates of free choice into cells with minimal endosomal capture on the one hand and a minimal toxicity on the other remains one of the grand challenges in science [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]
This difference revealed that even with fully optimized stable HGM cell lines, whole-cell analyses, such as flow cytometry, of the chloroalkane penetration assay (CAPA) contain a small but nonnegligible error that can be removed with the HC CAPA
To quantify the impact of the HC CAPA on the detection of the cytosolic delivery, the newly introduced peptide-based cyclic oligochalcogenides (COCs) 23 was evaluated for the transport of a model protein with the new analytical improvements
Summary
The effective delivery of substrates of free choice into cells with minimal endosomal capture on the one hand and a minimal toxicity on the other remains one of the grand challenges in science [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17].
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