title: Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification.
abstract: 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 >6.108for 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.
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Illien
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rodriguez
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Amoura
authors.affiliation: Center for Interdisciplinary Research in Biology, Collège-de-France, PSL Research University, 11 place M. Berthelot, Paris, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Joliot
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Manjula
authors.initials: M
authors.lastname: Pallerla
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Cribier
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Burlina
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005 Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism*; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence
chemical_list: D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; C414409:penetratin