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, fluorescencebased 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.
The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes1–4. To remedy this, charged amphiphilic molecules have been classically used as carriers3,5. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis6,7. Here we show that globular dodecaborate clusters, and prominently B12Br122−, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146–4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions8–12. We demonstrate that B12Br122− affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies.
A new selective chromo-fluorogenic probe for Fe(3+), Cr(3+) and Al(3+) is reported. Detection limits are in the μM range and the fluorogenic sensing ability could be observed by the naked eye when illuminated with UV-light. No response is observed with divalent cations.
Phosphorylation and dephosphorylation of peptides by kinases and phosphatases is essential for signal transduction in biological systems, and many diseases involve abnormal activities of these enzymes. Herein, we introduce amphiphilic calixarenes as key components for supramolecular, phosphorylation-responsive membrane transport systems. Dye-efflux experiments with liposomes demonstrated that calixarenes are highly active counterion activators for established cell-penetrating peptides, with EC values in the low nanomolar range. We have now found that they can even activate membrane transport of short peptide substrates for kinases involved in signal transduction, whereas the respective phosphorylated products are much less efficiently transported. This allows regulation of membrane transport activity by protein kinase A (PKA) and protein kinase C (PKC), as well as monitoring of their activity in a label-free kinase assay.
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