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Trodusquemine is an aminosterol known to prevent the binding of misfolded protein oligomers to cell membranes and to reduce their toxicity in a wide range of neurodegenerative diseases. Its precise mechanism of action, however, remains unclear. To investigate this mechanism, we performed confocal microscopy, fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) measurements, which revealed a strong binding of trodusquemine to large unilamellar vesicles (LUVs) and neuroblastoma cell membranes. Then, by combining quartz crystal microbalance (QCM), fluorescence quenching and anisotropy, and molecular dynamics (MD) simulations, we found that trodusquemine localises within, and penetrates, the polar region of lipid bilayer. This binding behaviour causes a decrease of the negative charge of the bilayer, as observed through ζ potential measurements, an increment in the mechanical resistance of the bilayer, as revealed by measurements of the breakthrough force applied with AFM and ζ potential measurements at high temperature, and a rearrangement of the spatial distances between ganglioside and cholesterol molecules in the LUVs, as determined by FRET measurements. These physicochemical changes are all known to impair the interaction of misfolded oligomers with cell membranes, protecting them from their toxicity. Taken together, our results illustrate how the incorporation in cell membranes of sterol molecules modified by the addition of polyamine tails leads to the modulation of physicochemical properties of the cell membranes themselves, making them more resistant to protein aggregates associated with neurodegeneration. More generally, they suggest that therapeutic strategies can be developed to reinforce cell membranes against protein misfolded assemblies.

This perspective paper provides some illustrative examples on the interplay between information gathered on planar supported lipid bilayers (SLB) and unilamellar lipid vesicles (ULV) to get an integrated description of phenomena occurring at the nanoscale that involve locally bilayered structures. Similarities and differences are underlined and critically compared in terms of biomimetic fidelity and instrumental accessibility to structural and dynamical parameters, focusing on some recent reports that either explicitly address this comparison or introducing some studies that separately investigate the same process in SLB and lipid vesicles. Despite the structural similarity on the nanoscale, the different topology implies radically different characterization techniques that have evolved in sectorial and separated approaches. The quest for increasing levels of compositional complexity for bilayered systems should not result in a loss of structural and dynamical control: this is the central challenge of future research in this area, where the integrated approach highlighted in this contribution would enable improved levels of understanding., (© The Owner Societies 2011)

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipids bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper describes the anchoring of novel cholesterol-modified DNA-18mers in supported lipid bilayers (SLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: we compared the behavior of two anchoring functionalities differing in the number of cholesterol units and in the length of a spacer group. Quartz Crystal Microbalance with impedance monitoring (QCM-Z) measurements showed that both oligonucleotides insert into the bilayer membrane through cholesterol anchoring; however, dramatic differences, in terms of surface organization and thickness, are found as the number of anchoring units increases. In the case of multiple cholesterol units, a peculiar three-regimes concentration dependence was revealed and correlated to the effective size of the adsorbing units. Interestingly, for high oligonucleotide concentration, the adsorption process was rationalized in terms of a compaction model of amphiphilic DNA molecules. QCM-Z temperature cycles of the SLB-anchored double strands provided clear evidence for reversible hybridization at the bilayer interface.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipid bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper compares the behavior of two cholesteryl modified oligonucleotides, differing in the architecture and hydrophobicity of the lipophilic moiety, in the self-aggregation, hybridization, and insertion in phospholipid vesicle membranes. We have focused our attention on a singly substituted derivative (SC-ON(1)) and a multicholesterol (MC-ON(1)) derivative, where the cholesteryl units are inserted at the desired positions along a noncoupling T-sequence. The self-aggregation properties in solution are also explicitly taken into account and evaluated as competitive with respect to the adsorption at fluid or solid interfaces and to hybridization with the complementary ON(2) sequence. By exploring a wide range of ON derivative concentrations, different peculiar scenarios emerge for different hydrophobicity of the amphiphilic DNA guest molecules on the vesicles, in terms of distribution and conformation of the single strand and consequent coupling properties with the complementary strand in solution.

We focused our studies on the fabrication of nano-organized systems incorporating redox mediators for NADH. We fabricated gold-modified electrodes of phospholipid layers containing a redox mediator for NADH by means of the Langmuir-Blodgett (LB) technique. We chose tetramethylbenzidine (TMB) which is known to decrease the very large overvoltage involved in the direct oxidation of NADH. The electrochemical characterisation of the films allowed to determine the presence and the electrochemical activity of the mediator in the LB layers. The response of the sensing layer in solutions containing NADH was investigated by means of cyclic voltammetry and fluorescence spectroscopy. We observed that NADH has a strong affinity with the film, and it interacts with the TMB-containing phospholipid layers. In fact, we found that TMB molecules mediate the NADH oxidation reaction, even if the surrounding matrix relatively hinders their mobility.