Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

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Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantificationScientific Reports 6, 36938 (2016)

 

Since their discovery twenty years ago, cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have been described as promising drug delivery systems. There are increasing numbers of successful applications of CPPs/PTDs in vivo. However, one of the limitations to their wide and diverse application is the diversity of their uptake pathways. The CPP and its cargo may end up free in the cytosol and reach their biological target only following translocation, but remain confined in intracellular vesicles after endocytosis, unless subsequent events such as endosomal rupture occur. Along with the development of CPPs/PTDs as vectors to carry various macromolecules for targeted cellular therapies, engineering new CPPs/PTDs with optimized transport and internalization capacities would greatly benefit from the understanding of cell entry mechanisms that still remain subject of controversy in the field7. In particular, one should understand how these peptides cross cell membranes. The translocation pathway differs from endocytosis because it still occurs at low temperature (<12 °C), although with lower efficiency due to decreases in cell membrane fluidity and dynamics.

 

The study herein directly addresses the issue of the quantification and the mechanisms of cell entry, by providing reliable and robust methods to detect total CPPs/PTDs inside cells. Measurement of the total amount of internalized species irrespective of its localization, cytosolic or vesicular, is an important task since it would encompass all uptake pathways. Indeed, CPP internalization and subsequent intracellular traffic are highly dynamic processes, and quantitative snapshots of CPPs/PTDs internalization in restricted areas of the cells, might only provide an incomplete picture of CPP functionality. Therefore, quantitative measurements of local and total peptide in cells are complementary and both informative on CPP/PTD efficacy.

 

 

To study the internalization of CPPs /PTDs, fluorescence-based techniques, such as flow cytometry or fluorescence microscopy imaging are predominant in the litterature. These techniques allow monitoring CPP accumulation and for imaging, its intracellular distribution. These techniques rely on the conjugation of a fluorochrome or a biotin to the studied CPP sequence. Irrespective of the nature of the tag added to track their fate, most CPPs strongly interact with the cell surface generating a pool of membrane-bound peptides usually defined as high membrane- or cell surface binding peptides, which cannot be eliminated by saline washings12–14. The cell-surface fraction of these peptides may represent from 10 up to 100 times the quantity of internalized peptides as we previously reported. Therefore, to measure accurately the amount of intracellular peptides, whatever the protocol and method used, one needs to distinguish the internalized from the cell surface bound peptides.

 

Pour en savoir plus, consultez le communiqué de presse associé à cet article : Mesurer l’efficacité d’un vecteur biologique !

 

 

Résumé: 

Scientific Reports 6, 36938 (2016)

 

The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

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Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

 

Françoise Illien, Nicolas Rodriguez, Mehdi Amoura, Alain Joliot, Manjula Pallerla, Sophie Cribier, Fabienne Burlina & Sandrine Sagan

 

Scientific Reports 6, 36938 (2016)

 

DOI: 10.1038/srep36938