Search

Rhamnolipids (RLs) are among the most important biosurfactants produced by microorganisms, and have been widely investigated because of their multiple biological activities. Their action appears to depend on their structural interference with lipid membranes, therefore several studies have been performed to investigate this aspect. We studied by X-ray scattering, neutron reflectometry and molecular dynamic simulations the insertion of dirhamnolipid (diRL), the most abundant RL, in model cellular membranes made of phospholipids and glycosphingolipids. In our model systems the affinity of diRL to the membrane is highly promoted by the presence of the glycosphingolipids and molecular dynamics simulations unveil that this evidence is related to sugar-sugar attractive interactions at the membrane surface. Our results improve the understanding of the plethora of activities associated with RLs, also opening new perspectives in their selective use for pharmaceutical and cosmetics formulations. Additionally, they shed light on the still debated role of carbohydrate-carbohydrate interactions as driving force for molecular contacts at membrane surface.

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-kappa B (siNF kappa B) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air-liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NF kappa B gene silencing was achieved in LPS-stimulated 16HBE14o-cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

Phenylketonuria (PKU) is a metabolic disorder connected to an excess of phenylalanine (Phe) in the blood and tissues, with neurological consequences. The disease's molecular bases seem to be related to the accumulation of Phe at the cell membrane surface. Radiological outcomes in the brain demonstrate decreased water diffusivity in white matter, involving axon dysmyelination of not yet understood origin. We used a biophysical approach and model membranes to extend our knowledge of Phe–membrane interaction by clarifying Phe's propensity to affect membrane structure and dynamics based on lipid composition, with emphasis on modulating cholesterol and glycolipid components to mimic raft domains and myelin sheath membranes. Phe showed affinity for the investigated membrane mimics, mainly affecting the Phe-facing membrane leaflet. The surfaces of our neuronal membrane raft mimics were strong anchoring sites for Phe, showing rigidifying effects. From a therapeutic perspective, we further investigated the role of doxycycline, known to disturb Phe packing, unveiling its action as a competitor in Phe interactions with the membrane, suggesting its potential for treatment in the early stages of PKU. Our results suggest how Phe accumulation in extracellular fluids can impede normal growth of myelin sheaths by interfering with membrane slipping and by remodulating free water and myelin-associated water contents.

Extracellular vesicles (EVs) are a potent intercellular communication system. Such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite their extreme biological relevance, their mechanisms of interaction with the membranes of recipient cells are still hotly debated. Here, we propose a multiscale investigation based on atomic force microscopy, small angle X-ray scattering, small angle neutron scattering and neutron reflectometry to reveal structure-function correlations of purified EVs in interaction with model membrane systems of variable complex compositions and to spot the role of different membrane phases on the vesicle internalization routes. Our analysis reveals strong interactions of EVs with the model membranes and preferentially with the borders of protruding phase domains. Moreover, we found that upon vesicle breaking on the model membrane surface, the biomolecules carried by/on EVs diffuse with different kinetics rates, in a process distinct from simple fusion. The biophysical platform proposed here has clear implications on the modulation of EV internalization routes by targeting specific domains at the plasma cell membrane and, as a consequence, on EV-based therapies.

Extracellular vesicles (EVs)-mediated communication relies not only on the delivery of complex molecular cargoes as lipids, proteins, genetic material, and metabolites to their target cells but also on the modification of the cell surface local properties induced by the eventual fusion of EVs’ membranes with the cells’ plasma membrane. Here we applied scanning calorimetry to study the phase transition of single phospholipid (DMPC) monolamellar vesicles, investigating the thermodynamical effects caused by the fusion of doping amounts of mesenchymal stem cells-derived EVs. Specifically, we studied EVs-induced consequences on the lipids distributed in the differently curved membrane leaflets, having different density and order. The effect of EV components was found to be not homogeneous in the two leaflets, the inner (more disordered one) being mainly affected. Fusion resulted in phospholipid membrane flattening associated with lipid ordering, while the transition cooperativity, linked to membrane domains’ coexistence during the transition process, was decreased. Our results open new horizons for the investigation of the peculiar effects of EVs of different origins on target cell membrane properties and functionality.

Protein uptake at the interface of a millimeter-sized air bubble in water is investigated by a recently developed differential interferometric technique. The technique allows the study of capillary waves with amplitudes around 10

Extracellular vesicles (EVs) represent a potent intercellular communication system. Within a lipid bilayer such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite of their extreme biological relevance, the small size (30 to a few hundred nanometers in diameter) of EVs still poses a great challenge for their isolation, quantification and biophysical/biochemical characterization, therefore the complex network of EVs and cells as well as their interaction remains to be further revealed. Here we propose a multiscale platform based on Atomic Force Microscopy, Small Angle X-ray Scattering, Small Angle Neutron Scattering and Neutron Reflectometry to reveal structure-function correlations of purified EVs through the analysis of their interaction with model membrane systems, in form of both supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. The analysis reveals a strong interaction of EVs with the model membranes and preferentially with liquid ordered raft-like lipid domains, and opens the way to understand uptake mechanisms in different vesicle to cell membrane relative compositions.

We present a study on biosurfactant uptake at the interface of a millimeter-sized drop of squalene in water, by a recently developed differential interferometric technique. The technique allows detecting capillary waves, with amplitudes of the order of 10−9 m, excited on the surface of the drop by an electric field in the order of 5 V/cm. By studying the resonant surface modes of the drop it is possible to assess the interfacial properties as a function of the surfactant concentration in the water bulk. The technique allows to follow the adsorption process at extremely low surfactant concentration, not accessible by other methods, investigating the gas state in the π-A diagrams of 2D amphiphilic monolayers.

Synchrotron radiation reflectometry was used to access the transverse structure of model membranes under the action of the human sialidase NEU2, down to the Ångström 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.

C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-beta-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections.

Scattering techniques are applied to studying the structural features of ganglioside aggregates in solution. Here it is described how different probing radiations allow to access different structural and dynamical parameters on different lengthscales. Besides a brief but comprehensive description of the scattering measurements, several practical suggestions are given concerning the experiments and the data analysis.

We investigated the insertion of small potassium (K+) channel proteins (Kcv(MA-1D) and Kcv(NTS)) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Scattering techniques are applied to studying the structural features of ganglioside aggregates in solution. Here it is described how different probing radiations allow to access different structural and dynamical parameters on different lengthscales. Besides a brief but comprehensive description of the scattering measurements, several practical suggestions are given concerning the experiments and the data analysis.

Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system. Background: Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system. Methods: A panel of DPPC/PLGA hNPs was prepared by emulsion/solvent diffusion and fully characterized. A combination of model siRNAs against the sodium transepithelial channel (ENaC) was entrapped in optimized hNPs comprising or not poly(ethylenimine) (PEI) as third component. siRNA-loaded hNPs were characterized for encapsulation efficiency, release kinetics, aerodynamic properties, and stability in artificial mucus (AM). The fate and cytotoxicity of hNPs upon aerosolization on a triple cell co-culture model (TCCC) mimicking human epithelial airway barrier were assessed. Finally, the effect of siRNA-loaded hNPs on ENaC protein expression at 72 hours was evaluated in A549 cells. Results: Optimized muco-inert hNPs encapsulating model siRNA with high efficiency were produced. The developed hNPs displayed a hydrodynamic diameter of approximate to 150nm, a low polydispersity index, a negative potential close to -25mV, and a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI. siRNA formulations showed optimal in vitro aerosol performance after delivery with a vibrating mesh nebulizer. Furthermore, small-angle X-ray scattering analyses highlighted an excellent stability upon incubation with AM, confirming the potential of hNPs for direct aerosolization on mucus-lined airways. Studies in TCCC confirmed that fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute proinflammatory effect. Finally, a prolonged inhibition of ENaC protein expression was observed in A549 cells upon treatment with siRNA-loaded hNPs. Conclusions: Results demonstrate the great potential of hNPs as carriers for pulmonary delivery of siRNA, prompting toward investigation of their therapeutic effectiveness in severe lung diseases.

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 Aβ1–42. 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.

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.

In the present study, we explore the effect of concentration on micelles made by different gangliosides, which are ionic biological glycolipids bearing multisugar headgroups with huge steric hindrance. Moreover, strong preferential interactions exist among like-conformer headgroups that can keep the ganglioside micelles in a trapped configuration. We extend the well-known ionic-amphiphiles paradigm, where local condensation and micelle crowding are matched by forming larger aggregates at increasing concentration. In fact, we force the balance between interparticle and intraparticle interactions while allowing for like conformers to modulate rebalancing. In the vast experimental framework, obtained by Small Angle X-ray scattering (SAXS) experiments, a theoretical model, accounting for a collective conformational transition of the bulky headgroups, is developed and successfully tested. It allows us to shed some light on the nature and coupling of the intermolecular forces involved in the interactions among glycolipid micelles. Energy minimization leads to complex behavior of the aggregation number on increasing concentration, fully consistent with the experimental landscape. From a biological perspective, this result could be reflected in the properties of ganglioside-enriched rafts on cell membranes, with a nonlinear structural response to approaching bodies such as charged proteins.

We revisit the structural investigation we performed over the years on gangliosides, biological amphiphiles typically found in the cell membranes of the nervous system of mammalians. Their molecular features, a large and charged saccharidic headgroup connected to a sticky and extended ceramide double tail, strongly dictate their aggregation properties and place ganglioside aggregates at the borderline between the curved world and the flatland. All along we found that unexpected interesting behaviours were induced by the hierarchical propagation of such extreme monomer properties, from the aggregate scale to the mesoscopic phases. In fact, even small changes in the monomer geometry or hindrance result in dramatic aggregate reshaping, due to collective amplification. Surface packing optimization requires preferential mutual orientation of headgroups, giving rise to trapped solid-disordered configurations. The interplay between interparticle and intraparticle interactions gives rise to unexpected behaviours and counterintuitive phase's landscape. In situ modification of monomer properties, operated by enzymatic digestion of aggregated ganglioside headgroups, either causes collective rearrangement or is overwhelmed by collective trapping, depending on their surface density. This aspect is interesting as gangliosides are not evenly distributed in cell membranes, but only in the outer leaflet, where they p]articipate in rafts, functional microdomains enriched in special lipids including cholesterol. We recently found that ganglioside GM1 forces a preferential distribution of cholesterol, constituting a collective structural pair across the membrane. In summary, ganglioside assemblies, through cooperativity, reach a structural complexity comparable or even bigger and more adaptive than that of a protein.

Neutron scattering techniques have been employed to investigate 1,2-dimyristoyl-sn -glycero-3-phosphocholine (DMPC) membranes in the form of multilamellar vesicles (MLVs) and deposited, stacked multilamellar-bilayers (MLBs), covering transitions from the gel to the liquid phase. Neutron diffraction was used to characterise the samples in terms of transition temperatures, whereas elastic incoherent neutron scattering (EINS) demonstrates that the dynamics on the sub-macromolecular length-scale and pico- to nano-second time-scale are correlated with the structural transitions through a discontinuity in the observed elastic intensities and the derived mean square displacements. Molecular dynamics simulations have been performed in parallel focussing on the length-, time- and temperature-scales of the neutron experiments. They correctly reproduce the structural features of the main gel-liquid phase transition. Particular emphasis is placed on the dynamical amplitudes derived from experiment and simulations. Two methods are used to analyse the experimental data and mean square displacements. They agree within a factor of 2 irrespective of the probed time-scale, i.e. the instrument utilized. Mean square displacements computed from simulations show a comparable level of agreement with the experimental values, albeit, the best match with the two methods varies for the two instruments. Consequently, experiments and simulations together give a consistent picture of the structural and dynamical aspects of the main lipid transition and provide a basis for future, theoretical modelling of dynamics and phase behaviour in membranes. The need for more detailed analytical models is pointed out by the remaining variation of the dynamical amplitudes derived in two different ways from experiments on the one hand and simulations on the other.

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single “supported + floating” bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir–Blodgett Langmuir–Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.

It is well known that the curvature of ganglioside-containing nanoparticles strongly depends on their headgroup structure, as determined in aggregates with 'stationary' composition, that is, when the system finds its optimal structure at the moment of lipid dissolution in aqueous solution. In the present work, we directly followed the structural change in model aggregates, induced by on-line molecular modification of already-packed gangliosides, namely the one brought about by a sialidase, acting on the ganglioside GD1a and leading to the lower-curvature-aggregating GM1. We applied small-angle X-ray and neutron scattering techniques to follow the time evolution of the aggregate structure. We found that, while chemically undergoing the enzymatic action in both cases, the aggregated structure could be either very stable, in single component systems, or structurally responsive, in mixed model systems. Moreover, while in progress, the sialidase-ganglioside interaction seems to define a time lag where the system is structurally off the smooth route between the initial and the final states. We hypothesize that, in this time lag, the local structure could be very sensitive to the environment and eventually readdressed to a specific final structural fate.

Solid Lipid Nanoparticles (SLNs) composed of biodegradable physiological lipids have been widely proposed as efficient drug delivery systems, also for ophthalmic administration. Recently, chitosan-associated-SLNs have been developed to further improve the residence time of these colloidal systems in the precorneal area by means of mucoadhesive interaction. In the present study, a one-step preparation protocol was used aiming both at scale-up ease and at stronger coupling between chitosan and SLNs. The resulting particles were chitosan associated-SLNs (CS-SLNs). These nanoparticles were characterized, as compared to both the chitosan-free and the usual chitosan-coated ones, by applying a multi-technique approach: light, neutron and X-ray scattering, Zeta-potential, AFM, calorimetry. It was assessed that, while keeping the features of nano-size and surface-charge required for an efficient vector, these new nanoparticles display a strong and intimate interaction between chitosan and SLNs, far more settled than the usual simple coverage. Moreover, this one-step preparation method allows to obtain a strong and intimate interaction between chitosan and SLNs, firmer than the usual simple coating. This confers to the CS-SLNs an improved mucoadhesion, opening the way for a high-performing ophthalmic formulation.

Background Gangliosides are biological glycolipids participating in rafts, structural and functional domains of cell membranes. Their headgroups are able to assume different conformations when packed on the surface of an aggregate, more lying or standing. Switching between different conformations is possible, and is a collective event. Switching can be induced, in model systems, by concentration or temperature increase, then possibly involving ganglioside–water interaction. In the present paper, the effect of GM1 ganglioside headgroup conformation on the water structuring and interactions is addressed. Methods Depolarized Rayleigh Scattering, Raman Scattering, Quasielastic Neutron Scattering and NMR measurements were performed on GM1 ganglioside solutions, focusing on solvent properties. Results All used techniques agree in evidencing differences in the structure and dynamics of solvent water on different time-and-length scales in the presence of either GM1 headgroup conformations. Conclusions In general, all results indicate that both the structural properties of solvent water and its interactions with the sugar headgroups of GM1 respond to surface remodelling. The extent of this modification is much higher than expected and, interestingly, ganglioside headgroups seem to turn from cosmotropes to chaotropes upon collective rearrangement from the standing- to the lying-conformation. Significance In a biological perspective, water structure modulation could be one of the physico-chemical elements contributing to the raft strategy, both for rafts formation and persistence and for their functional aspects. In particular, the interaction with approaching bodies could be favoured or inhibited or triggered by complex-sugar-sequence conformational switch. This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazu and Dr. Federica Migliardo.

The use of nanocarriers, which respond to different stimuli controlling their physicochemical properties and biological responsivness, shows a growing interest in pharmaceutical science. The stimuli are activated by targeting tissues and biological compartments, e.g., pH modification, temperature, redox condition, enzymatic activity, or can be physically applied, e.g., a magnetic field and ultrasound. pH modification represents the easiest method of passive targeting, which is actually used to accumulate nanocarriers in cells and tissues. The aim of this paper was to physicochemically characterize pH-sensitive niosomes using different experimental conditions and demonstrate the effect of surfactant composition on the supramolecular structure of niosomes. In this attempt, niosomes, made from commercial (Tween21) and synthetic surfactants (Tween20 derivatives), were physicochemically characterized by using different techniques, e.g., transmission electron microscopy, Raman spectroscopy, and small-angle X-ray scattering. The changes of niosome structure at different pHs depend on surfactants, which can affect the supramolecular structure of colloidal nanocarriers and their potential use both in vitro and in vivo. At pH 7.4, the shape and structure of niosomes have been maintained; however, niosomes show some differences in terms of bilayer thicknesses, water penetration, membrane coupling, and cholesterol dispersion. The acid pH (5.5) can increase the bilayer fluidity, and affect the cholesterol depletion. In fact, Tween21 niosomes form large vesicles with lower curvature radius at acid pH; while Tween20-derivative niosomes increase the intrachain mobility within a more interchain correlated membrane. These results demonstrate that the use of multiple physicochemical procedures provides more information about supramolecular structures of niosomes and improves the opportunity to deeply investigate the effect of stimuli responsiveness on the niosome structure.

The local structure of a GM3 ganglioside bilayer, whose wide-angle X-ray spectrum is reconstructed from molecular dynamics simulations, is found to compare quantitatively well with the experimental one. By separating inter- and intramolecular contributions, correlations between distinct head groups are shown to contribute in a substantial way to the total scattering intensity. This finding supports the hypothesis of a strong local head group order as recently formulated on the basis of calorimetry and X-ray experimental data.

The interest in the glyco-world is widespread and covers quite a number of different areas. We restrict ourselves to underline some features concerning interactions and organization in the hydrophilic region of glycolipid aggregates that, besides the biological or technological aspects, constitute an experimental and theoretical challenge, as they invest some aspects of colloidal self-assembly that keep on being not completely understood.

We present results of molecular dynamics simulations of fully hydrated dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers in the disordered liquid crystalline phase (Lalpha) and compare them to wide-angle X-ray scattering experiments. Though we find a generally good agreement between the simulated and experimental spectra, there are some deviations whose origin has been investigated by a reparametrization of the aliphatic chains' force field. A detailed analysis of the various contribution to the X-ray spectra shows that a non-negligible contribution to the total scattered intensity comes from the headgroups and the head-tail cross correlation.

We report experimental and theoretical results on the concentration dependence of the micellar size of GM1 and GM1acetyl gangliosides, five-sugar-headed anionic glycolipids. Contrary to one of the mainstays of colloid science, that the aggregation number of amphiphile aggregates grows with concentration, an anomalous region is found at intermediate concentrations, where a sharp decrease of the aggregation number occurs. Experiments were performed by small-angle X-ray and neutron scattering (SAXS and SANS). Two models are discussed, reproducing the observed behavior of either GM1acetyl or GM1. The first one is a conventional picture of interacting micelles where a reduction in the molecular surface area, leading to an increase of the aggregate dimension, is paid to reduce intermicellar interactions: it foresees a monotonous increase of the aggregation number with concentration. The second one accounts for a conformational bistability of the bulky headgroups of GM1, modifying the amphiphilic molecular surface area and protrusion from the aggregate surface, and contributing to the inter- and intramicellar interaction balance. Energy minimization leads to a complex behavior of the aggregation number, which is consistent with the anomalous behavior of GM1.

A new paradigm for the aetiopathology of phenylketonuria suggests the presence of amyloid-like assemblies in the brains of transgenic mouse models and patients with phenylketonuria, possibly shedding light on the selective cognitive deficit associated with this disease. Paralleling the amyloidogenic route that identifies different stages of peptide aggregation, corresponding to different levels of toxicity, we experimentally address for the first time, the physico-chemical properties of phenylalanine aggregates via Small Angle, Wide Angle X-ray Scattering and Atomic Force Microscopy. Results are consistent with the presence of well-structured, aligned fibres generated by milliMolar concentrations of phenylalanine. Moreover, the amyloid-modulating doxycycline agent affects the local structure of phenylalanine aggregates, preventing the formation of well-ordered crystalline structures. Phenylalanine assemblies prove toxic in vitro to immortalized cell lines and primary neuronal cells. Furthermore, these assemblies also cause dendritic sprouting alterations and synaptic protein impairment in neurons. Doxycycline counteracts these toxic effects, suggesting an approach for the development of future innovative non-dietary preventive therapies.

The fate of lipid-based nanovectors, used in genetic targeting inside cells, depends on their behavior in biological media. In fact, during both in vitro and in vivo transfection, nanovectors come in contact with proteins that compete for their surface and build the protein corona, their true biological identity while engaging the cell membrane. Nonetheless, after cell internalization, the efficacy of transfection may depend also on structural modifications that occurred under the protein cover, following interaction with biological fluids. Here, based on previous in vivo experiments, two widely used lipid mixtures, namely DOTAP/DOPC and DC-Chol/DOPE, were identified as paradigms to investigate the impact of the inner structure of nanovectors on the transfection efficiency, all being proficiently internalized. The evolution of the inner structure of cationic lipoplexes and nanoparticles based on such lipid mixtures, following interaction with human plasma, could be unraveled. Particles were investigated in high dilution, approaching the biosimilar conditions. Data have demonstrated that the modulation of their inner structure depends on their lipid composition and the plasma concentration, still preserving the genetic payload. Interestingly, protein contact induces a variety of inner structures with different perviousness, including reshaping into cubic phases of different porosity, sometimes observed upon interaction between carrier-lipids and cell-lipids. Cubic reshaping is of biological relevance, as lipid cubic phases have been recently associated to both fusogenicity and to the readiness in releasing the payload to the final target via endosomal escape.

We have carried out density and DSC measurements on large unilamellar vesicles (LUVs) of different phosphocholines in the temperature range corresponding to the anomalous swelling regime of multibilayer systems, adjacent to the chain melting transition region. Experiments were performed for DPPC, DMPC and DC 13 PC, as a function of aggregate size. The steep drop in density occurring around the main transition gets broader as the vesicle radius decreases and a two-step process is clearly observed for DMPC and DC 13 PC vesicles smaller than 200 nm. No similar effect is observed for DPPC vesicles, whatever the aggregate dimension. DSC measurements corroborate density results, showing a splitting of the main peak that becomes more evident as the chain length decreases and as the curvature of the aggregate increases.

We report on wide angle X-ray scattering (WAXS) and density experiments performed on the ganglioside GM3 in water solution. As compared to what usually found in lipid assemblies, GM3 bilayers show a degree of cooperativity feebler in the region of the lipid chains and stronger in the region of the headgroups. The collected WAXS spectra indicate the existence of a strong degree of order on the molecular scale, which we propose to be due to a highly constrained and symmetric disposition of the trisaccharide charged headgroups of GM3 at the bilayer surface. It is the first time that such an order in the headgroup region is seen in dispersed lipid assemblies, in normal pressure conditions. Experiments were carried out with no added salt, in the semidilute range of concentration, 5–15% bw, and in the range of temperature from 15 to 60 °C. Parallel SAXS experiments indicate that on the scale of the bilayer cross section the same structure is preserved from the very dilute (0.1%) to the semidilute range of concentration.

Gangliosides are double-tailed biological amphiphiles naturally abundant in the nervous system. We present a quasielastic neutron scattering study on the dynamics of ganglioside molecules in 15% concentration micellar solution. The scattering contribution due to gangliosides has been analysed in terms of a simple model of confined diffusion within a sphere with rigid walls.

A concise discussion of the role of different geometrical conformational states in the process of self-assembling of gangliosides is given. The report focuses on the effects of the geometrical variations occurring in the head group region of gangliosides as reflected on the geometrical properties of the whole assembly. Collective phenomena happening at the water interfacial region are found to be coupled to the phase transition of the lipid moiety, that is, to the well-known order-disorder conformational transition involving the hydrophobic tails. The possible biological relevance of the head group bistability is envisaged.

The conformations and dynamics of gangliosides GM1, GM2, 6′-GM2 and GM4 have been studied by computational means, and the results compared to NMR data. Unconstrained conformational searches were run using the AMBER* force field augmented by MNDO derived parameters for the Neu5Ac anomeric torsion, the GB/SA water solvation model, and the MC/EM alogorithm; extended (10–12[emsp4 ]ns) dynamic simulations in GB/SA water were performed with the MC/SD protocol, and the stored structures were minimized. The overall mobility of the Neu5Acα2,3Gal linkage and the position of its minimum energy conformation have been shown to depend mainly on the presence or the absence of a GalNAc residue at the adjacent position. The best quantitative agreement with the available NOE data was achieved after minimization of the structures stored during the MC/SD dynamic runs. The latter protocol appears to reproduce satisfactorily the available experimental data, and can be used with confidence to build three-dimensional models of ganglioside headgroups.

The conformational properties of GM2, GalNacβ-4(Neu5Acα-3) Galβ-4Glcβ-1Cer have been compared to those of 6′-GM2, in which the linkage between the GalNAc and Gal was altered from GalNacβ-4Galβ- to GalNacβ-6Galβ-, and to those of GD1a, Neu5Acα-3Galβ-3GalNAcβ-4(Neu5Acα-3)Galβ-4Glcβ-1Cer, and GalNAc-GD1a. Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6′-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions. The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Acα-3Galβ-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two ‘core’ GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1--4(NeuAcalpha2--3)Galbeta1--4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1--6(NeuAcalpha2--3)Galbeta1--4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1--4Gal linkage, 6'-GM2 has GalNAcbeta1--6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1--4Galbeta1--4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.

The paper describes the pseudosugar 2 [Galβ1−3GalNAcβ1−4(NeuAcα2−3)DCCHD], a high affinity binder of cholera toxin (CT). The molecule was designed using molecular modeling techniques to mimic the natural CT membrane receptor, ganglioside GM1. The central residue of GM1, a 3,4-disubstituted galactose unit, was recognized as the ganglioside scaffold element and substituted with a conformationally locked cyclohexanediol (DCCHD). DCCHD was synthesized in enantiopure form using enantioselective Diels Alder methodology and regioselectively α-sialylation at the equatorial position. Glycosylation with a Galβ(1−3)GalNAc donor completed the synthesis of 2. The solution structure of 2 and its binding ability to CT were found to be analogous to those of the GM1 oligosaccharide.

膜ガングリオシドの特性の研究に適した水環境中での界面モデルが開発されつつある。このモデルは、高分子量のガングリオシドミセルとともに、ガングリオシドとホスファチジルコリンの低分子量混合ミセルや、中間分子量のセラミド修飾ガングリオシドミセルから成る。これらの界面モデルに挿入したガングリオ系列ガングリオシドのコンフォメーション的および立体的特性に関する実験情報について論じ、マルチラメラ凝集モデルを用いて得られた情報と比較、総括する。結果から、鎖のコンフォメーション特性は、シアル酸とN-アセチルガラクトサミンユニットとの残基間相互作用によって決定され、全体のオリゴ糖構造や、セラミドの構造、可能なリン脂質極性基やCaイオンとの相互作用、高いガングリオシド含量の環境、膜コレステロールの存在や温度変化に無関係であることが示された。さらにオリゴ糖鎖は限定された平均的コンフォメーションおよび運動の程度で、二重膜表面を越えて巧みに伸長していると思われ、これは調べた全てのガングリオシドに共通している。

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. © 2013 American Chemical Society.

The conformational properties of the oligosaccharide chain of GM1 ganglioside containing N-glycolyl-neuraminic acid, beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal- (1-4)1-4)-beta-Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core -beta- GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal- were found to fix the relative orientation of the three saccharides, while the glycosidic linkage of the terminal beta-Gal- was found to be quite mobile as the beta-Gal-(1-3)-beta-GalNAc- disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containing N-acetyl-neuraminic acid and neuraminic acid [1].

A ganglioside preparation containing two structurally related minor gangliosides (Gg 1 + 2) was isolated from bovine brain ganglioside mixture and characterized. Treatment of 50 g ganglioside mixture with Clostridium perfrigens sialidase, followed by chromatography on DEAE-Sepharose and silica gel columns, yielded 20 mg Gg 1 + 2. By chemical analyses, 1H- and 13C-NMR spectroscopy, enzymic hydrolyses using human beta-hexosaminidase A and clostridial sialidase, and TLC overlay with the conjugated cholera toxin B subunit, the two novel gangliosides Gg 1 and Gg 2 were identified to be: Gg 1, GalNAc-GD1a(Neu5Ac/Neu5Gc), beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta- Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal-(1-4)-be ta- Glc-(1-1)-Cer; Gg 2, GalNAc-GD1a(Neu5Gc/Neu5Ac), beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]- beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal-(1- 4)-beta- Glc-(1-1)-Cer. These two gangliosides contain the identical pentasaccharide backbone except that the substitution of the two sialic acids, Neu5Ac and Neu5Gc, are in the reversed position of the external and the internal Gal residues. Our analyses showed that the content of Gg 1 and Gg 2 were approximately 0.12% and 0.08%, respectively, of the total brain ganglioside mixture.

GD1a ganglioside containing an acetyl group as acyl moiety, GD1a(acetyl), was synthesized from natural GD1a. The aggregative properties in aqueous solution of GD1a(acetyl) have been studied by static and dynamic laser light-scattering measurements. GD1a(acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular mass of 33 A and 96 kDa, respectively. Vibrio cholerae sialidase showed a very high activity on the micelles of GD1a(acetyl), compared to GD1a. This has been explained as a consequence of the high surface curvature of the the small micelles. High resolution proton NMR spectra were recorded from micelles of GD1a(acetyl) in deuterated water. The low overall correlation time of the GD1a(acetyl) micelles was calculated to be about 2 x 10(-8)s, a value one order of magnitude lower than that determined for natural GD1a.

We followed the process of enzymatic digestion of ganglioside GD1a, operated by sialidase on aggregated micelles. The product is the ganglioside GM1, lacking the external sialic acid. The structural aspects and the kinetics connected to the process occurring on a fragmented-condensed substrate, the ganglioside micelles, are investigated by small angle X-ray scattering (SAXS). Observed at short times, the kinetics of the reaction shows a transient step-like decay, while it tends to a smooth Michaelis-Menten kinetics in the late stages. We propose a model, based on the fragmented-condensed nature of the substrate, that well reproduces the experimental observation without invoking any feedback mechanism in the reaction, usually required for an oscillatory behavior. The model predicts an initial regime dominated by the strict enzyme-substrate interaction, with a step-like appearance.

It is well known that the curvature of ganglioside-containing nanoparticles strongly depends on their headgroup structure, as determined in aggregates with 'stationary' composition, that is, when the system finds its optimal structure at the moment of lipid dissolution in aqueous solution. In the present work, we directly followed the structural change in model aggregates, induced by on-line molecular modification of already-packed gangliosides, namely the one brought about by a sialidase, acting on the ganglioside GD1a and leading to the lower-curvature-aggregating GM1. We applied small-angle X-ray and neutron scattering techniques to follow the time evolution of the aggregate structure. We found that, while chemically undergoing the enzymatic action in both cases, the aggregated structure could be either very stable, in single component systems, or structurally responsive, in mixed model systems. Moreover, while in progress, the sialidase-ganglioside interaction seems to define a time lag where the system is structurally off the smooth route between the initial and the final states. We hypothesize that, in this time lag, the local structure could be very sensitive to the environment and eventually readdressed to a specific final structural fate.

We developed a simple model to investigate the effect of lipid clustering on the local interlayer distance in a cluster of interacting lamellae. The model, based on nonequilibrium thermodynamics and linear stability theories, explores the early stages of the lamella-lamella phase separation process where the lateral diffusion is much faster than the interlamellar lipid exchange. Results indicate, in the early stages, the presence of locally distorted regions with a higher concentration of one lipid component and an anomalous repeat distance. Experimental cases are presented, consisting of multilamellar-oriented depositions of phospholipids containing minority amounts of ganglioside or sphingomyelin under a low-hydration condition. The minority components are known to form domains within the phospholipid bilayer matrix. The low water content inhibits the lipid exchange among nearby lamellae and strengthens lamella-lamella interaction, allowing for a straightforward comparison with the model. Small-angle and wide-angle neutron diffraction experiments were performed in order to detect interlayer distances and local chain order, respectively. Lamellar stacking splitting has been observed for the ganglioside-containing lamellae, induced by in-phase lipid clustering. In excess water and after long equilibration times, these local structures may further evolve, leading to coexisting lamellar phases with different lipid compositions and interlayer distances.

Colloidal aggregates of amphiphilic molecules provide an example of self-confined liquid crystal, being finite both in extension, nanometric scale, and in dimension, nearly-two dimensional. In fact, although disperse on the macroscopic scale, amphiphiles demix in dilute aqueous solution on the microscopic scale due to their bifunctional nature and form condensed assemblies with a complex internal structure, dictated by the compromise between hydrophobic and hydrophilic requirements. On raising the concentration, these amphiphile-aggregate containing solutions start to develop organised structures on the mesoscale, evidencing a long-range crystalline order, but still preserving the ability to rearrange on the nanoscale giving rise to rich phase diagrams. We present here some peculiar and unexpected effects observed for aggregates of gangliosides, glycosidic amphiphiles with both an extended hydrophobic portion, a double-tailed ceramide, and a bulky saccharidic headgroup, displaying large conformational ada...

The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems. Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity. Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug loading efficiency. © 2008 American Chemical Society.