posted on 2019-12-02, 15:45authored byAndrea Barba-Bon, Yu-Chen Pan, Frank Biedermann, Dong-Sheng Guo, Werner M. Nau, Andreas Hennig
The membrane transport mechanisms of cell-penetrating
peptides
(CPPs) are still controversial, and reliable assays to report on their
internalization in model membranes are required. Herein, we introduce
a label-free, fluorescence-based method to monitor membrane transport
of peptides in real time. For this purpose, a macrocyclic host and
a fluorescent dye forming a host–dye reporter pair are encapsulated
inside phospholipid vesicles. Internalization of peptides, which can
bind to the supramolecular host, leads to displacement of the dye
from the host, resulting in a fluorescence change that signals the
peptide uptake and, thus, provides unambiguous evidence for their
transport through the membrane. The method was successfully validated
with various established CPPs, including the elusive peptide TP2,
in the presence of counterion activators of CPPs, and with a calixarene-based
supramolecular membrane transport system. In addition, transport experiments
with encapsulated host–dye reporter pairs are not limited to
large unilamellar vesicles (LUVs) but can also be used with giant
unilamellar vesicles (GUVs) and fluorescence microscopy imaging.