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 >6.108 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.

Cell Penetrating Peptides, Novel Vectors for Gene Therapy

“…Although the exact transduction mechanism of CPPs remains elusive, extensive work from multiple investigators has shed considerable light. There is evidence both for mechanisms that are non-endocytic/energy independent or endocytic/energy-dependent [41]. The broad range of cells that are readily transducible by non-specific CPPs such as Tat suggests a role for ubiquitously shared cellular structures such as surface binding to plasma membrane phospholipids or, in particular proteoglycans through electrostatic interactions, as a first step towards cell entry [42,43].…”

Membrane Active Peptides and Their Biophysical Characterization

“…FC gives a statistically relevant and quantitative signal of fluorescence very quickly and allows sensitive detection. The technique provides both qualitative and quantitative information [159,250,252].…”

“…This information may then be used to retrieve information on the molecular mode of action, target, and specificity of the AMPs [157,[254][255][256]. In addition to addressing the mechanism of cell entry, FC is an especially reliable and robust method in the investigation of CPP internalization and subsequent intracellular trafficking to quantify the fraction of internalized CPPs [159].…”

Neue Modalitäten für schwierige Zielstrukturen in der Wirkstoffentwicklung

“…Wenn jede Modalitätu rsprünglich aus einem anderen chemischen Raum kommt, stellt die Kombination und Vereinigung von Modalitäten eine Integration von chemischen Räumen dar. Die Verwendung fluoreszenzmarkierter Moleküle in Kombination mit Zellsortierung (FACS) ist momentan weithin üblich, [241] was aber die unmarkierten Versionen dieser Moleküle außer Acht lässt. Dies konnte beispielsweise anhand der Proteinund Peptidfusion mit Fc-Fragmenten gezeigt werden.…”