A scanning ion conductance microscopy assay to investigate interactions between cell penetrating peptides and pore-suspending membranes

Abstract

Scanning ion conductance microscopy (SICM) provides a technique for the investigation of surface topography as well as the local ion conductance of a surface without any mechanical contact between probe and sample. This avoidance of interaction is particularly advantageous for the examination of highly flexible structures such as lipid membranes, e.g. living cells or artificial membranes. Pore-suspending membranes (PSMs) as a model system combine high stability with close mimicking of natural structures with respect to lateral mobility and the existence of aqueous compartments on both sides of the bilayer. A major field of research focuses on the interaction of lipids or other membrane constituents with peptides, in recent years notably cell penetrating peptides (CPPs). Among most prominent examples are melittin as the major venom component of the honey bee Apis mellifera and penetratin as the third helix of the Drosophila melanogaster Antennapedia homeodomain. Generalised protocols for the preparation of solvent-free PSMs are reported. Giant unilamellar vesicles (GUVs) of various lipid compositions were spread on porous silicon nitride (Si3N4) substrates which had been functionalised with cholesterylpolyethylenoxy thiol (CPEO3, hydrophobically) or with mercaptoethanol (ME, hydrophilically). Lipid compositions comprised purely zwitterionic phosphatidylcholine (PC) lipids as well as mixtures of PC lipids with cholesterol and PC lipids with phosphatidylserine (PS) lipids. Successful spreading was proven by means of confocal laser scanning microscopy (CLSM) and SICM imaging. The main part of this thesis dealt with the development and application of CPP titration assays based on both the CLSM and SICM techniques aiming at the elucidation of the influence of substrate functionalisation and membrane composition on interactions of melittin and penetratin with PSMs. The CLSM assay was performed with melittin on PSMs of every lipid composition on both hydrophobically and hydrophilically functionalised substrates and with penetratin on PSMs of every lipid composition on hydrophilically functionalised substrates. Rupturing of bilayers on hydrophilically functionalised substrates was observed at comparable concentrations of 1–3 µM for both peptides. Comparison of experiments on hydrophilically and hydrophobically functionalised substrates reveals three times higher melittin concentrations in the former case. On both functionalisation types, a cholesterol content of 10% resulted in an increase in melittin concentration sufficient for membrane rupturing, while 20% PS lipids resulted in a decrease. SICM experiments were performed with melittin on PC/cholesterol PSMs on hyrophobically and on hydrophilically functionalised substrates as well as with pure PC PSMs on hydrophilically functionalised substrates. There was not any significant difference found for membrane rupturing inducing peptide concentrations. These were of the same order as found in CLSM experiments. Prior to rupturing, an increase in pore depth hinting at an increase in membrane permeability was observed.