Supramolecular Tandem Membrane Assays

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Membrane transport of organic molecules plays a crucial role in the discovery and delivery of small-molecule drugs as well as in the mechanism of action of antimicrobial peptides and cell-penetrating peptides (CPPs). The measurement of the actual transport of these compounds across the lipid bilayer membrane remains nevertheless a major experimental challenge. We have recently shown that liposome-encapsulated supramolecular reporter pairs (consisting of a fluorescent dye and a macrocyclic receptor molecule) qualify to unambiguously detect membrane transport of organic molecules and peptides by a simple and fluorescence-based method. Within this proposal, we will comprehensively establish the utility and applicability of this method.In detail, we will encapsulate supramolecular reporter pairs in liposomes of varying size and membrane composition in order to investigate membrane transport of selected organic molecules and peptides by absorption and fluorescence spectroscopic methods. This includes spectroscopic characterizations, time-resolved measurements, including stopped-flow techniques for rapid kinetics, as well as fluorescence microscopy with giant unilamellar vesicles (GUVs) as well as live cells.The supramolecular tandem membrane assays to be developed herein will be utilized to follow membrane transport of small, unlabeled molecules and CPPs. This will afford membrane permeability coefficients and activation energies for membrane transport of small, organic molecules as experimental benchmarks, for example, for computational studies. The adaptation to parallel artificial membrane transport assays (PAMPA) will demonstrate potential future applications in high-throughput screening in the pharmaceutical industry. We will also establish liposome-encapsulated supramolecular reporter pairs as key components in advanced supramolecular sensing systems, in which the membrane transport process improves the detection limit and selectivity of supramolecular sensor systems, thereby enabling orders of magnitude sensitivity enhancements and the discrimination of peptide sequences.