Assay development

A variety of experiments involving pore-suspending membranes has been conducted demonstrating the potential advantage of this relatively recent improvement in membrane model system development. Fluorescence microscopy was utilised for the investigation of lipid domains within PSMs [Orth et al. 2012], electrophysiology was employed for the characterisation of ion channels [Gaßmann et al. 2009] and atomic force microscopy was used to evaluate bilayer mechanics [Mey et al. 2009; Kocun et al. 2011].

A wide range of assays has been conducted to elucidate mellitin membrane interactions. In one major part of these assays, melittin was applied to erythrocytes. Typical experiments addressed the quantification of lytic activity by means of centrifugation or filtration assays.

Melittin was added to a cell culture and the amount of haemoglobin in the supernatant was determined after centrifugation or filtration, respectively [DeGrado et al. 1982; Katsu et al.

1989; Oren and Shai 1997; Raghuraman and Chattopadhyay 2005]. Further assays included the quantification of inhibition of cell growth via cell density determination [Oren and Shai 1997] or colony counting as well as straightforward optical microscopy to monitor morphological changes [Katsu et al. 1989]. A second major part dealt with the investigation of interactions of melittin with membrane model systems, predominantly small or large unilamellar vesicles (SUVs or LUVs, respectively). Typical experiments include release assays to identify concentrations at which pores are formed or vesicles are lysed, to determine the size of these pores or to address questions with regard to mechanism or kinetics of melittin

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membrane interactions [Schwarz et al. 1992; Ohki et al. 1994; Benachir and Lafleur 1995;

Benachir et al. 1997; Ladokhin et al. 1997; Allende and McIntosh 2003; Allende et al. 2005;

van den Bogaart et al. 2008]. The effect of melittin membrane interactions on both the peptide and the bilayer as well as the peptide’s affinity for membranes were investigated in a number of binding assays, e.g. by means of X-ray scattering to quantify membrane thicknesses, of phosphorous nuclear magnetic resonance (NMR) or fluorimetric analysis of the tryptophan residue’s environment to investigate peptide orientation [Batenburg et al.

1988; Ghosh et al. 1997; Lee et al. 2001; Sheynis et al. 2003; Raghuraman and Chattopadhyay 2004; Lee et al. 2008]. Solid supported membranes (SSMs) were utilised to investigate membrane permeabilisation by means of impedance spectroscopy or to visualise the effect of melittin on lipid bilayers by means of AFM [Steinem et al. 2000], to examine the affinity of melittin for membranes of varying compositions or solve mechanistic questions by means of surface plasmon resonance (SPR) [Lee et al. 2001; Papo and Shai 2002].

In this study, assays based on CLSM and SICM were developed for the investigation of the interactions of the membrane active peptides melittin and penetratin with pore-suspending membranes. The main advantages of SICM are that imaging omits mechanical contact between probe and sample and that membrane permeability is an intrinsic parameter of the method. It is thus comparable with the impedance spectroscopy and AFM study on SSMs [Steinem et al. 2000] but provides the utilisation of PSMs as a further enhancement. Another advantageous feature is that rupturing of pores is observed individually and as part of a statistical ensemble by means of both CLSM and SICM which distinguishes these assays from e.g. vesicle leakage [Schwarz et al. 1992; Ohki et al. 1994; Benachir and Lafleur 1995;

Benachir et al. 1997; Ladokhin et al. 1997; Allende and McIntosh 2003; Allende et al. 2005;

van den Bogaart et al. 2008] or single GUV experiments [Mally et al. 2007; Lee et al. 2008].

5.2 Preparation of pore-suspending membranes

Membranes of various lipid compositions were desired, i.e. bilayers consisting of pure phosphocholine lipids, membranes of phosphocholine lipids with phosphoserine lipid content as well as those of phosphocholines and cholesterol. In addition to these three variations, membranes on both hydrophilically as well as hydrophobically functionalised substrates were intended. To this end, generalised protocols for the preparation of pore-suspending membranes were needed.

Since model membranes aimed at mimicking natural situations as closely as possible, GUV spreading was preferred to painting. The drawback of the latter – residual solvent near the rims – was circumvented, and the drawback of the former – impossibility of the formation of electrically insulating membranes – did not hamper the experiments in this study as they do not require insulation but sufficiently large membrane patches only.

A variety of spreading strategies has been developed. One possibility is the application of vesicle solution on non functionalised substrates such as silicon oxide (SiO2) [Mey et al. 2009]

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or silicon nitride (Si3N4) [Kresák et al. 2009]. A more frequently applied strategy is the coverage of the substrate rims with gold by means of evaporation. Thiolipids or analogues thereof are chemisorbed and form a monolayer on which vesicles can be spread. Early functionalisations aimed at utilisation of electrostatic interactions between negatively charged 3-mercaptopropionic acid (MPA) and vesicles comprising positively charged lipids such as N,N-dimethyl-N,N-dioctadecylammonium bromide (DODAB) [Hennesthal and Steinem 2000; Steltenkamp et al. 2006]. More recent approaches utilised hydrophobic or hydrophilic interactions where gold covered surfaces were further functionalised with octane thiol (OT) or cholesterylpolyethylenoxy thiol (CPEO3) in case of the former [Mey et al. 2009]

and mercaptoethanol (ME) in the latter case [Kocun et al. 2011]. Another strategy circumvents the metal coverage by direct modification of silicon [Heinemann and Schwille 2011] or aluminium oxide substrates [Lazzara et al. 2011a; Lazzara et al. 2011b] with silanes.

The principal difference between the latter strategies of gold evaporation and silanisation is that the metal is evaporated only on top of the covered substrate while simple silanisation does not discriminate between pore rims and interior such that the pore interior may be covered with lipids rather than bilayers suspending the pore. A more sophisticated approach, however, does lead to differently functionalised substrate surfaces [Lazzara et al. 2011b].

In this study, existing protocols for the preparation of specific PSMs on silicon nitride substrates were taken as starting points for the generalisation. These starting points included thiol chemistry, i.e. the use of Ti, Au and CPEO3 for hydrophobic functionalisation [Mey et al.

2009], while ME was chosen for hydrophilic functionalisation based on a study by Kocun et al.

[Kocun et al. 2011]. The evaporation procedure results in sufficiently good discrimination of pore rims and interior such that spreading and PSM formation was achieved. Application of the same functionalisation strategy for both hydrophobically and hydrophilically functionalised substrates appears preferable with respect to comparability of results.

Variation of sucrose concentration inside GUVs, pore diameters, spreading duration, the buffer system and its Ca²⁺ buffer content as well as the spreading temperature resulted in a successfully applicable spreading protocol for CPEO3 functionalised porous substrates of all desired membrane types, i.e. pure PC membranes, PC membranes with PS content and those with cholesterol content. Similarly, slight variations of parameters mentioned above, resulted in successful preparation of PSMs on ME functionalised porous substrates. Thus the developed protocols provide fast and reliable access to the complete range of membrane varieties intended for CLSM and SICM investigations.

A caveat has to be considered for PSMs prepared on CPEO3 functionalised substrates. Due to hydrophobic substrate surfaces, persistence of air bubbles within the pores has to be considered, such that lipid monolayers on air would be achieved instead of bilayers separating two aqueous compartments. In order to prevent air bubbles from remaining in pores, the substrate was covered with ethanol to reduce the surface tension and induce gas emission. After extensive exchange of ethanol against buffer, the sample was carefully handled such that air contact was excluded. Complete removal of gas from the pores, however, cannot be guaranteed by careful handling alone.

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