Quantitative Attribution of the Protective Effects of Aminosterols against Protein Aggregates to Their Chemical Structures and Ability to Modulate Biological Membranes.

Natural aminosterols are promising drug candidates against neurodegenerative diseases, like Alzheimer and Parkinson, and one relevant protective mechanism occurs via their binding to biological membranes and displacement or binding inhibition of amyloidogenic proteins and their cytotoxic oligomers. We compared three chemically different aminosterols, finding that they exhibited different (i) binding affinities, (ii) charge neutralizations, (iii) mechanical reinforcements, and (iv) key lipid redistributions within membranes of reconstituted liposomes. They also had different potencies (EC50) in protecting cultured cell membranes against amyloid-ß oligomers. A global fitting analysis led to an analytical equation describing quantitatively the protective effects of aminosterols as a function of their concentration and relevant membrane effects. The analysis correlates aminosterol-mediated protection with well-defined chemical moieties, including the polyamine group inducing a partial membrane-neutralizing effect (79 ± 7%) and the cholestane-like tail causing lipid redistribution and bilayer mechanical resistance (21 ± 7%), linking quantitatively their chemistry to their protective effects on biological membranes.

Insights in Cell Biomechanics through Atomic Force Microscopy.

We review the advances obtained by using Atomic Force Microscopy (AFM)-based approaches in the field of cell/tissue mechanics and adhesion, comparing the solutions proposed and critically discussing them. AFM offers a wide range of detectable forces with a high force sensitivity, thus allowing a broad class of biological issues to be addressed. Furthermore, it allows for the accurate control of the probe position during the experiments, providing spatially resolved mechanical maps of the biological samples with subcellular resolution. Nowadays, mechanobiology is recognized as a subject of great relevance in biotechnological and biomedical fields. Focusing on the past decade, we discuss the intriguing issues of cellular mechanosensing, i.e., how cells sense and adapt to their mechanical environment. Next, we examine the relationship between cell mechanical properties and pathological states, focusing on cancer and neurodegenerative diseases. We show how AFM has contributed to the characterization of pathological mechanisms and discuss its role in the development of a new class of diagnostic tools that consider cell mechanics as new tumor biomarkers. Finally, we describe the unique ability of AFM to study cell adhesion, working quantitatively and at the single-cell level. Again, we relate cell adhesion experiments to the study of mechanisms directly or secondarily involved in pathologies.

Cholesterol Hinders the Passive Uptake of Amphiphilic Nanoparticles into Fluid Lipid Membranes.

Plasma membranes represent pharmacokinetic barriers for the passive transport of site-specific drugs within cells. When engineered nanoparticles (NPs) are considered as transmembrane drug carriers, the plasma membrane composition can affect passive NP internalization in many ways. Among these, cholesterol-regulated membrane fluidity is probably one of the most biologically relevant. Herein, we consider small (2-5 nm in core diameter) amphiphilic gold NPs capable of spontaneously and nondisruptively entering the lipid bilayer of plasma membranes. We study their incorporation into model 1,2-dioleoyl-sn-glycero-3-phosphocholine membranes with increasing cholesterol content. We combine dissipative quartz crystal microbalance experiments, atomic force microscopy, and molecular dynamics simulations to show that membrane cholesterol, at biologically relevant concentrations, hinders the molecular mechanism for passive NP penetration within fluid bilayers, resulting in a dramatic reduction in the amount of NP incorporated.

Making biological membrane resistant to the toxicity of misfolded protein oligomers: a lesson from trodusquemine.

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.

Amphiphilic gold nanoparticles perturb phase separation in multidomain lipid membranes.

Amphiphilic gold nanoparticles with diameters in the 2-4 nm range are promising as theranostic agents thanks to their spontaneous translocation through cell membranes. This study addresses the effects that these nanoparticles may have on a distinct feature of plasma membranes: lipid lateral phase separation. Atomic force microscopy, quartz crystal microbalance, and molecular dynamics are combined to study the interaction between model neuronal membranes, which spontaneously form ordered and disordered lipid domains, and amphiphilic gold nanoparticles having negatively charged surface functionalization. Nanoparticles are found to interact with the bilayer and form bilayer-embedded ordered aggregates. Nanoparticles also suppress lipid phase separation, in a concentration-dependent fashion. A general, yet simple thermodynamic model is developed to show that the change of lipid-lipid enthalpy is the dominant driving force towards the nanoparticle-induced destabilization of phase separation.