How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides

• Published in: Biochimica et biophysica acta. Biomembranes Vol. 1861; no. 9; pp. 1533 - 1545
• Main Authors: Seisel, Quentin, Pelletier, François, Deshayes, Sébastien, Boisguerin, Prisca
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-09-2019; Elsevier
• Subjects: Animals; Biological Transport; Cell Membrane - metabolism; Cell Membrane Permeability - drug effects; Cell-penetrating peptides; Cell-Penetrating Peptides - chemistry; Cell-Penetrating Peptides - metabolism; Cellular Biology; CHO Cells; Cricetulus; Endocytosis; Fluorescence; Life Sciences; Protein Transport; Quantification; Quenching; Spectrometry, Fluorescence - methods; Tryptophan; Tryptophan - chemistry; Uptake; Fluorescence; Tryptophan; Quantification; Cell-penetrating peptides; Uptake; Quenching; Cell-Penetrating Peptides
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2019.06.011

Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a series of TAMRA-labeled conjugates formed of either cationic or amphipathic CPPs covalently coupled to a decamer peptide, we observed important differences in internalization levels between both methods.
We partly explained these discrepancies by analyzing the effect of buffer conditions (pH, detergents) and peptide sequence/structure on TAMRA dye accessibility. Based on this analysis, we calculated a correction coefficient allowing a better coherence between both methods. However, an overestimated signal was still observable for both amphipathic peptides using the spectroscopic detection, which could be due to their localization at the cell membrane. Based on several in vitro experiments modeling events at the plasma membrane, we hypothesized that fluorescence of peptides entrapped in the membrane bilayer could be quenched by the tryptophan residues of close transmembrane proteins. During cell lysis, cell membranes are disintegrated liberating the entrapped peptides and restoring the fluorescence, explaining the divergences observed between flow cytometry and spectroscopy on lysates.
Overall, our results highlighted major biases in the fluorescently-based quantification of internalized fluorescently-labeled CPP conjugates, which should be considered for accurate uptake quantification.
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•Comparison of cell-penetrating peptide (CPP) cellular uptake measured by flow cytometry or fluorescence spectroscopy.•CPP sequence (cationic versus amphipathic), secondary structure and cell membrane interactions affect the quantification of cellular uptake.•Uptake evaluation of CPPs should always be confirmed with different methods to avoid the overestimation of the results.