Building a biomimetic membrane for neutron reflectivity investigation : complexity, asymmetry and contrast

The preparation and investigation of model membranes is deserving growing interest both for the physics of complex systems, and for biology. The need of simplified models should preserve mimicking the qualifying characteristics of biological membranes, and keep non-invasive and detailed description. As a main feature, biological membranes are non-homogeneous in the disposition of components, both in the lateral and in the transverse direction. We prepared asymmetric supported membranes containing GM1 ganglioside in biomimetic proportion according to different protocols. Then, we studied their internal structure by neutron reflectometry, providing few-Angstrom sensitivity in the cross direction meanwhile avoiding radiation damage. This technique can also be profitably applied to study interactions at the membrane surface. The best protocol has proven to be the Langmuir-Blodgett/Langmuir-Schaefer depositions. Notably, also the simpler and most accessible protocol of vesicle fusion was found to be suitable for straightforward and good quality deposition of compositionally asymmetric membranes

Decoration of nanovesicles with pH (low) insertion peptide (pHLIP) for targeted delivery

Acidity at surface of cancer cells is a hallmark of tumor microenvironments, which does not depend on tumor perfusion, thus it may serve as a general biomarker for targeting tumor cells. We used the pH (low) insertion peptide (pHLIP) for decoration of liposomes and niosomes. pHLIP senses pH at the surface of cancer cells and inserts into the membrane of targeted cells, and brings nanomaterial to close proximity of cellular membrane. DMPC liposomes and Tween 20 or Span 20 niosomes with and without pHLIP in their coating were fully characterized in order to obtain fundamental understanding on nanocarrier features and facilitate the rational design of acidity sensitive nanovectors. The samples stability over time and in presence of serum was demonstrated. The size, ζ-potential, and morphology of nanovectors, as well as their ability to entrap a hydrophilic probe and modulate its release were investigated. pHLIP decorated vesicles could be useful to obtain a prolonged (modified) release of biological active substances for targeting tumors and other acidic diseased tissues

Polystyrene perturbs the structure, dynamics, and mechanical properties of DPPC membranes: An experimental and computational study

Synthetic plastic oligomers can interact with the cells of living organisms by different ways. They can be intentionally administered to the human body as part of nanosized biomedical devices. They can be inhaled by exposed workers, during the production of multicomponent, polymer-based nanocomposites. They can leak out of food packaging. Most importantly, they can result from the degradation of plastic waste, and enter the food chain. A physicochemical characterization of the effects of synthetic polymers on the structure and dynamics of cell components is still lacking. Here, we combine a wide spectrum of experimental techniques (calorimetry, x-ray, and neutron scattering) with atomistic Molecular Dynamics simulations to study the interactions between short chains of polystyrene (25 monomers) and model lipid membranes (DPPC, in both gel and fluid phase). We find that doping doses of polystyrene oligomers alter the thermal properties of DPPC, stabilizing the fluid lipid phase. They perturb the membrane structure and dynamics, in a concentration-dependent fashion. Eventually, they modify the mechanical properties of DPPC, reducing its bending modulus in the fluid phase. Our results call for a systematic, interdisciplinary assessment of the mechanisms of interaction of synthetic, everyday use polymers with cell membranes

Pathogenic Aβ A2V versus protective Aβ A2T mutation : early stage aggregation and membrane interaction

We investigated the effects of punctual A-to-V and A-to-T mutations in the amyloid precursor protein APP, corresponding to position 2 of A\u3b21\u201342. Those mutations had opposite effects on the onset and progression of Alzheimer disease, the former inducing early AD pathology and the latter protecting against the onset of the disease. We applied Static and Dynamic Light Scattering and Circular Dichroism, to study the different mutants in the early stages of the aggregation process, essential for the disease. Comparative results showed that the aggregation pathways differ in the kinetics and extent of the process, in the size of the aggregates and in the evolution of the secondary structure, resulting in fibrils of different morphology, as seen by AFM. Mutated peptides had comparable toxic effects on N2a cells. Moreover, as assessed by X-ray scattering, all of them displayed disordering effects on the internal structure of mixed phospholipids-gangliosides model membranes

Reflectivity from floating bilayers: can we keep the structural asymmetry?

To assess the structure of complex biomembranes, the use of asymmetric model systems is rare, due to the difficulty of realizing artificial membranes with desired heterogeneous composition and applicable for single membrane structural investigation. We developed an experimental model with a single macroscopic bilayer floating on top of another adhering to a silicon flat surface, prepared by Langmuir-Blodgett Langmuir-Schaefer technique, then investigated by neutron reflectivity. On the way to more complex systems, containing lipids of different nature, we tested whether a simple imposed asymmetry is kept in time and whether it can stand some standard experimental protocols commonly employed in treating model membranes. We focused on cholesterol, a basic component with a transverse distribution that is not symmetric in biomembranes, and may assume specific location in functional domains. So we forced different asymmetries in the "adhering + floating" bilayers system composed of phospholipids and cholesterol in bio-similar mole ratios. The neutron reflection accessible length-scale and its sensitivity, enhanced by the possibility to play with deuteration, allowed assessing the cross profile of the membrane and revealing that lipid redistribution can occur

Inflammatory role of extracellular sphingolipids in Cystic Fibrosis

Ceramide is emerging as one of the players of inflammation in lung diseases. However, data on its inflammatory role in Cystic Fibrosis (CF) as part of the extracellular machinery driven by lung mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are missing. We obtained an in vitro model of CF-MSC by treating control human lung MSCs with a specific CFTR inhibitor. We characterized EVs populations derived from MSCs (ctr EVs) and CF-MSCs (CF-EVs) and analyzed their sphingolipid profile by LC-MS/MS. To evaluate their immunomodulatory function, we treated an in vitro human model of CF, with both EVs populations. Our data show that the two EVs populations differ for the average size, amount, and rate of uptake. CF-EVs display higher ceramide and dihydroceramide accumulation as compared to control EVs, suggesting the involvement of the de novo biosynthesis pathway in the parental CF-MSCs. Higher sphingomyelinase activity in CF-MSCs, driven by inflammation-induced ceramide accumulation, sustains the exocytosis of vesicles that export new formed pro-inflammatory ceramide. Our results suggest that CFTR dysfunction associates with an enhanced sphingolipid metabolism leading to the release of EVs that export the excess of pro-inflammatory Cer to the recipient cells, thus contributing to maintain the unresolved inflammatory status of CF

The nasal delivery of nanoencapsulated statins – An approach for brain delivery

© 2016 Clementino et al. Purpose: Along with their cholesterol-lowering effect, statins have shown a wide range of pleiotropic effects potentially beneficial to neurodegenerative diseases. However, such effects are extremely elusive via the conventional oral administration. The purpose of the present study was to prepare and characterize the physicochemical properties and the in vivo biodistribution of simvastatin-loaded lecithin/chitosan nanoparticles (SVT-LCNs) suitable for nasal administration in view of an improved delivery of the statins to the brain. Materials and methods: Chitosan, lecithin, and different oil excipients were used to prepare nanocapsules loaded with simvastatin. Particle size distribution, surface charge, structure, simvastatin loading and release, and interaction with mucus of nanoparticles were determined. The nanoparticle nasal toxicity was evaluated in vitro using RPMI 2651 nasal cell lines. Finally, in vivo biodistribution was assessed by gamma scintigraphy via Tc99m labeling of the particles. Results: Among the different types of nanoparticles produced, the SVT-LCN_MaiLab showed the most ideal physicochemical characteristics, with small diameter (200 nm), positive surface charge (+48 mV) and high encapsulation efficiency (EE; 98%). Size distribution was further confirmed by nanoparticle tracking analysis and electron microscopy. The particles showed a relatively fast release of simvastatin in vitro (35.6%±4.2% in 6 hours) in simulated nasal fluid. Blank nanoparticles did not show cytotoxicity, evidencing that the formulation is safe for nasal administration, while cytotoxicity of simvastatin-loaded nanoparticles (IC50) was found to be three times lower than the drug solution (9.92 vs 3.50 μM). In rats, a significantly higher radioactivity was evidenced in the brain after nasal delivery of simvastatin-loaded nanoparticles in comparison to the administration of a similar dose of simvastatin suspension. Conclusion: The SVT-LCNs developed presented some of the most desirable characteristics for mucosal delivery, that is, small particle size, positive surface charge, long-term stability, high EE, and mucoadhesion. In addition, they displayed two exciting features: First was their biodegradability by enzymes present in the mucus layer, such as lysozyme. This indicates a new Trojan-horse strategy which may enhance drug release in the proximity of the nasal mucosa. Second was their ability to enhance the nose-to-brain transport as evidenced by preliminary gamma scintigraphy studies

Membrane restructuring following in situ sialidase digestion of gangliosides : complex model bilayers by synchrotron radiation reflectivity

Synchrotron radiation reflectometry was used to access the transverse structure of model membranes under the action of the human sialidase NEU2, down to the \uc5ngstr\uf6m length scale. Model membranes were designed to mimic the lipid composition of so-called Glycosphingolipids Enriched Microdomains (GEMs), which are membrane platforms specifically enriched in cholesterol and sphingolipids, and where also typical signalling molecules are hosted. Gangliosides, glycosphingolipids containing one or more sialic acid residues, are asymmetrically embedded in GEMs, in the outer membrane leaflet where gangliosides are claimed to interact directly with growth-factor receptors, modulating their activation and then the downstream intracellular signalling pathways. Thus, membrane dynamics and signalling could be strongly influenced by the activity of enzymes regulating the membrane ganglioside composition, including sialidases. Our results, concerning the structure of single membranes undergoing in-situ enzymatic digestion, show that the outcome of the sialidase action is not limited to the emergence of lower-sialylated ganglioside species. In fact, membrane reshaping occurs, involving a novel arrangement of the headgroups on its surface. Thus, sialidase activity reveals to be a potential tool to control dynamically the structural properties of the membrane external leaflet of living cells, influencing both the morphology of the close environment and the extent of interaction among active molecules belonging to signalling platforms

Amyloidβ Peptides in interaction with raft-mime model membranes : a neutron reflectivity insight

The role of first-stage \u3b2-amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between \u3b2-amyloid (A\u3b2) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of A\u3b2(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the A\u3b2(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the A\u3b2(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller A\u3b2(1-6) fragment, the N-terminal portion of A\u3b2, was also used. A\u3b2 N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment

The A2V mutation as a new tool for hindering Aβ aggregation: A neutron and x-ray diffraction study

We have described a novel C-to-T mutation in the APP gene that corresponds to an alanine to valine substitution at position 673 in APP (A673V), or position 2 of the amyloid-\uce\ub2 (A\uce\ub2) sequence. This mutation is associated with the early onset of AD-type dementia in homozygous individuals, whereas it has a protective effect in the heterozygous state. Correspondingly, we observed differences in the aggregation properties of the wild-type and mutated A\uce\ub2 peptides and their mixture. We have carried out neutron diffraction (ND) and x-ray diffraction (XRD) experiments on magnetically-oriented fibers of A\uce\ub21-28WT and its variant A\uce\ub21-28A2V. The orientation propensity was higher for A\uce\ub21-28A2V suggesting that it promotes the formation of fibrillar assemblies. The diffraction patterns by A\uce\ub21-28WT and A\uce\ub21-28A2V assemblies differed in shape and position of the equatorial reflections, suggesting that the two peptides adopt distinct lateral packing of the diffracting units. The diffraction patterns from a mixture of the two peptides differed from those of the single components, indicating the presence of structural interference during assembly and orientation. The lowest orientation propensity was observed for a mixture of A\uce\ub21-28WT and a short N-terminal fragment, A\uce\ub21-6A2V, which supports a role of A\uce\ub2's N-terminal domain in amyloid fibril formation