Influence of adhesion-promoting glycolipids on the structure and stability of solid-supported lipid double-bilayers.

Glycolipids have a considerable influence on the interaction between adjacent biomembranes and can promote membrane adhesion trough favorable sugar-sugar "bonds" even at low glycolipid fractions. Here, in order to obtain structural insights into this phenomenon, we utilize neutron reflectometry in combination with a floating lipid bilayer architecture that brings two glycolipid-loaded lipid bilayers to close proximity. We find that selected glycolipids with di-, or oligosaccharide headgroups affect the inter-bilayer water layer thickness and appear to contribute to the stability of the double-bilayer architecture by promoting adhesion of adjacent bilayers even against induced electrostatic repulsion. However, we do not observe any redistribution of glycolipids that would maximize the density of sugar-sugar contacts. Our results point towards possible strategies for the investigation of interactions between cell surfaces involving specific protein-protein, lipid-lipid, or protein-lipid binding.

Modulation of plant plasma membrane structure by exogenous fatty acid hydroperoxide is a potential perception mechanism for their eliciting activity.

Oxylipins are lipid-derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone-dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this study, the potential of 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid (13-HPOD) and 13(S)-hydroperoxy-(9Z, 11E, 15Z)-octadecatrienoic acid (13-HPOT), as plant defence elicitors and the underlying mechanism of action is investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity-related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. The relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.

PEGylated mucus-penetrating nanocrystals for lung delivery of a new FtsZ inhibitor against Burkholderia cenocepacia infection.

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-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-ß-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.

Aß Beyond the AD Pathology: Exploring the Structural Response of Membranes Exposed to Nascent Aß Peptide.

The physiological and pathological roles of nascent amyloid beta (Aß) monomers are still debated in the literature. Their involvement in the pathological route of Alzheimer's Disease (AD) is currently considered to be the most relevant, triggered by their aggregation into structured oligomers, a toxic species. Recently, it has been suggested that nascent Aß, out of the amyloidogenic pathway, plays a physiological and protective role, especially in the brain. In this emerging perspective, the study presented in this paper investigated whether the organization of model membranes is affected by contact with Aß in the nascent state, as monomers. The outcome is that, notably, the rules of engagement and the resulting structural outcome are dictated by the composition and properties of the membrane, rather than by the Aß variant. Interestingly, Aß monomers are observed to favor the tightening of adjacent complex membranes, thereby affecting a basic structural event for cell-cell adhesion and cell motility.

Protein Adsorption at the Air-Water Interface by a Charge Sensing Interferometric Technique.

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 10-9 m, excited at the surface of the bubble by an electric field of intensity on the order of 10 V/cm. When one studies the resonant modes of the bubble (radial and shape modes), it is possible to assess variations of interfacial properties and, in particular, of the net surface charge as a function of bulk protein concentration. Sensing the interfacial charge, the technique enables us to follow the absorption process in conditions of low concentrations, not easily assessable by other methods. We focus on bovine serum albumin (BSA) and lysozyme as representatives of typical globular proteins. To provide comprehensive insight into the novelty of the technique, we also investigated the equilibrium adsorption of sodium dodecyl sulfate (SDS) ionic surfactant for bulk concentrations at hundreds of times lower than the Critical Micelle Concentration (CMC). Results unveil how the absorption of charged molecules affects the amplitudes of the bubble resonant modes even before affecting the frequencies in a transition-like fashion. Different adsorption models are proposed and developed. They are validated against the experimental findings by comparing frequency and amplitude data. By measuring the charging rate of the bubble interface, we have followed the absorption kinetics of BSA and lysozyme recognizing a slow, energy barrier limited phenomena with characteristic times in agreement with data in the literature. The evaluation of the surface excess concentration (Γ) of BSA and SDS at equilibrium is obtained by monitoring charge uptake. At the investigated low bulk concentrations, reliable comparisons with literature data from equilibrium surface tension isotherm models are reported.

Mucin Thin Layers: A Model for Mucus-Covered Tissues.

The fate of macromolecules of biological or pharmacological interest that enter the mucus barrier is a current field of investigation. Studies of the interaction between the main constituent of mucus, mucins, and molecules involved in topical transmucoidal drug or gene delivery is a prerequisite for nanomedicine design. We studied the interaction of mucin with the bio-inspired arginine-derived amphoteric polymer d,l-ARGO7 by applying complementary techniques. Small angle X-ray scattering in bulk unveiled the formation of hundreds of nanometer-sized clusters, phase separated from the mucin mesh. Quartz microbalance with dissipation and neutron reflectometry measurements on thin mucin layers deposited on silica supports highlighted the occurrence of polymer interaction with mucin on the molecular scale. Rinsing procedures on both experimental set ups showed that interaction induces alteration of the deposited hydrogel. We succeeded in building up a new significant model for epithelial tissues covered by mucus, obtaining the deposition of a mucin layer 20 Å thick on the top of a glycolipid enriched phospholipid single membrane, suitable to be investigated by neutron reflectometry. The model is applicable to unveil the cross structural details of mucus-covered epithelia in interaction with macromolecules within the Å discreteness.

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 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.

pH-sensitive niosomes: Effects on cytotoxicity and on inflammation and pain in murine models.

pH-sensitive nonionic surfactant vesicles (niosomes) by polysorbate-20 (Tween-20) or polysorbate-20 derivatized by glycine (added as pH sensitive agent), were developed to deliver Ibuprofen (IBU) and Lidocaine (LID). For the physical-chemical characterization of vesicles (mean size, size distribution, zeta potential, vesicle morphology, bilayer properties and stability) dynamic light scattering (DLS), small angle X-ray scattering and fluorescence studies were performed. Potential cytotoxicity was evaluated on immortalized human keratinocyte cells (HaCaT) and on immortalized mouse fibroblasts Balb/3T3. In vivo antinociceptive activity (formalin test) and anti-inflammatory activity tests (paw edema induced by zymosan) in murine models were performed on drug-loaded niosomes. pH-sensitive niosomes were stable in the presence of 0 and 10% fetal bovine serum, non-cytotoxic and able to modify IBU or LID pharmacological activity in vivo. The synthesis of stimuli responsive surfactant, as an alternative to add pH-sensitive molecules to niosomes, could represent a promising delivery strategy for anesthetic and anti-inflammatory drugs.

Niosomes as Drug Nanovectors: Multiscale pH-Dependent Structural Response.

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.

Optimizing the crowding strategy: sugar-based ionic micelles in the dilute-to-condensed regime.

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.

Integrated Strategies for a Holistic View of Extracellular Vesicles.

Extracellular vesicles (EVs) are receiving increasing attention for their role in spreading both beneficial and harmful information during cell-cell communication. The complexity and heterogeneity of the origin of EVs make integrated molecular, structural, and functional studies extremely challenging but necessary at the same time. In fact, a comprehensive interdisciplinary approach is needed to correlate the features of EVs, target cells/organs, and the pathophysiological outcomes exerted by the EVs' actions. Based on these premises, after introducing a brief state-of-the-art outline on the current analytical approaches exploited to characterize EVs, this review aims to highlight the effectiveness of those studies where authors put in correlation the diverse EV data collected from different points of view. Although these examples are still just a few, they still represent an excellent starting point to be taken as a reference in the perspective for improving the correlation among EV-related clinical aspects. Of course, to fully reach this goal, several points need to be further improved and developed. Undoubtedly, new avenues in diagnostic, prognostic, and therapeutic applications by EVs will be initiated by integrative strategies, combining biophysical approaches, high-throughput omics technologies, and computational models.

Calorimetry of extracellular vesicles fusion to single phospholipid membrane.

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.

Carbohydrate-carbohydrate interaction drives the preferential insertion of dirhamnolipid into glycosphingolipid enriched membranes.

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.

Dysmyelination and glycolipid interference caused by phenylalanine in phenylketonuria.

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.

Hybrid Lipid/Polymer Nanoparticles to Tackle the Cystic Fibrosis Mucus Barrier in siRNA Delivery to the Lungs: Does PEGylation Make the Difference?

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-κB (siNFκB) 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 NFκB 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.

Nanoscale structural response of ganglioside-containing aggregates to the interaction with sialidase.

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.

Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes.

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.

Correction: Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes.

Correction for 'Structural insights into fusion mechanisms of small extracellular vesicles with model plasma membranes' by Fabio Perissinotto et al., Nanoscale, 2021, 13, 5224-5233, DOI: .

Iron-mediated interaction of alpha synuclein with lipid raft model membranes.

The aberrant misfolding and aggregation of alpha synuclein (αS) into toxic oligomeric species is one of the key features associated with the pathogenesis of Parkinson's disease (PD). It involves different biochemical and biophysical factors as plasma membrane binding and interaction with heavy metal ions. In the present work, atomic force microscopy (AFM) is combined with Fourier Transform Infrared Spectroscopy (FTIR) measurements to investigate the interaction of wild-type (WT) and A53T mutated alpha synuclein with artificial lipid bilayers mimicking lipid raft (LR) domains, before and after ferrous cations (Fe2+) treatment. In the absence of iron, protein monomers produce a thinning of the membrane, targeting the non-raft phase of the bilayer preferentially. On the contrary, iron actively promotes the formation of globular protein aggregates, resembling oligomers, targeted to LR domains. In both aggregation states, monomer and oligomer, the mutated A53T protein exhibits a greater and faster membrane-interaction. These results underlie a new mechanism of membrane-protein interaction in PD. The targeting of Fe2+-promoted αS oligomers to LRs might be functional for the disease and be helpful for the development of new therapeutic strategies.

Attractive Interaction between Fully Charged Lipid Bilayers in a Strongly Confined Geometry.

We investigate the interaction between highly charged lipid bilayers in the presence of monovalent counterions. Neutron and X-ray reflectivity experiments show that the water layer between like-charged bilayers is thinner than for zwitterionic lipids, demonstrating the existence of counterintuitive electrostatic attractive interaction between them. Such attraction can be explained by taking into account the correlations between counterions within the Strong Coupling limit, which falls beyond the classical Poisson-Boltzmann theory of electrostatics. Our results show the limit of the Strong Coupling continuous theory in a highly confined geometry and are in agreement with a decrease in the water dielectric constant due to a surface charge-induced orientation of water molecules.