Importance of the Electrostatic Correlations in Surface Tension of Hydrated Reline Deep Eutectic Solvent from Combined Experiments and Molecular Dynamics Simulations.

In this study, the surface tension and the structure of hydrated reline are investigated by using diverse methods. Initially, the surface tension displays a nonlinear pattern as water content increases, decreasing until reaching 45 wt %, then gradually matching that of pure water. This fluctuation is associated with strong electrostatic correlations present in pure reline, which decrease as more water is added. Changes in surface tension reflect a shift from charge layering in pure reline to an increased interfacial hydrogen bonding as the water content rises. This shift causes the segregation of urea molecules into the bulk phase and a gradual anchoring of water molecules to the air-reline interface. An interesting observation is the antisurfactant effect, where heightened interfacial anchoring results in an unexpected increase in real contribution of surface tension. This, along with weakened electrostatic correlations beyond 45 wt % due to reinforced interfacial hydrogen bonding, contributes to the complex behavior of surface tension observed in this study.

Acidic Osteoid Templates the Plywood Structure of Bone Tissue.

Bone is created by osteoblasts that secrete osteoid after which an ordered texture emerges, followed by mineralization. Plywood geometries are a hallmark of many trabecular and cortical bones, yet the origin of this texturing in vivo has never been shown. Nevertheless, extensive in vitro work revealed how plywood textures of fibrils can emerge from acidic molecular cholesteric collagen mesophases. This study demonstrates in sheep, which is the preferred model for skeletal orthopaedic research, that the deeper non-fibrillar osteoid is organized in a liquid-crystal cholesteric geometry. This basophilic domain, rich in acidic glycosaminoglycans, exhibits low pH which presumably fosters mesoscale collagen molecule ordering in vivo. The results suggest that the collagen fibril motif of twisted plywood matures slowly through self-assembly thermodynamically driven processes as proposed by the Bouligand theory of biological analogues of liquid crystals. Understanding the steps of collagen patterning in osteoid-maturation processes may shed new light on bone pathologies that emerge from collagen physico-chemical maturation imbalances.

Engineering of brick and staple components for ordered assembly of synthetic repeat proteins.

Synthetic ɑRep repeat proteins are engineered as Brick and Staple protein pairs that together self-assemble into helical filaments. In most cases, the filaments spontaneously form supercrystals. Here, we describe an expanded series of ɑRep Bricks designed to stabilize the interaction between consecutive Bricks, to control the length of the assembled multimers, or to alter the spatial distribution of the Staple on the filaments. The effects of these Brick modifications on the assembly, on the final filament structure and on the crystal symmetry are analyzed by biochemical methods, electron microscopy and small angle X-ray scattering. We further extend the concept of Brick/Staple protein origami by designing a new type of "Janus"-like Brick protein that is equally assembled by orthogonal staples binding its inner or outer surfaces and thus ending inside or outside the filaments. The relative roles of longitudinal and lateral associations in the assembly process are discussed. This set of results demonstrates important proofs-of-principle for engineering these remarkably versatile proteins toward nanometer-to-micron scale constructions.

Solubilization of free ß-sitosterol in milk sphingomyelin and polar lipid vesicles as carriers: Structural characterization of the membranes and sphingosome morphology.

High consumption of plant sterols reduces the risk of cardiovascular diseases in humans and provides health benefits. Increasing the amount of plant sterols in the diet is therefore necessary to reach the recommended daily dietary intake. However, food supplementation with free plant sterols is challenging because of their low solubility in fats and water. The objectives of this study were to investigate the capacity of milk-sphingomyelin (milk-SM) and milk polar lipids to solubilise ß-sitosterol molecules in bilayer membranes organised as vesicles called sphingosomes. The thermal and structural properties of milk-SM containing bilayers composed of various amounts of ß-sitosterol were examined by differential scanning calorimetry (DSC) and temperature-controlled X-ray diffraction (XRD), the molecular interactions were studied using the Langmuir film technique, the morphologies of sphingosomes and ß-sitosterol crystals were observed by microscopy. We showed that the milk-SM bilayers devoid of ß-sitosterol exhibited a gel to fluid Lα phase transition for Tm = 34.5 °C and formed facetted spherical sphingosomes below Tm. The solubilisation of ß-sitosterol within milk-SM bilayers induced a liquid-ordered Lo phaseabove 25 %mol (1.7 %wt) ß-sitosterol and a softening of the membranes leading to the formation of elongated sphingosomes. Attractive molecular interactions revealed a condensing effect of ß-sitosterol on milk-SM Langmuir monolayers. Above 40 %mol (25.7 %wt) ß-sitosterol, partitioning occured with the formation of ß-sitosterol microcrystals in the aqueous phase. Similar results were obtained with the solubilization of ß-sitosterol within milk polar lipid vesicles. For the first time, this study highlighted the efficient solubilization of free ß-sitosterol within milk-SM based vesicles, which opens new market opportunities for the formulation of functional foods enriched in non-crystalline free plant sterols.

Design, synthesis, and characterization of protein origami based on self-assembly of a brick and staple artificial protein pair.

A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.

Flexible and Transparent Luminescent Cellulose-Transition Metal Cluster Composites.

Red-NIR luminescent polymers are principally obtained from petroleum-based derivatives in which emitters, usually a critical raw material such as rare-earth or platinum group metal ions, are embedded. Considering the strong ecological impact of their synthesis and the major risk of fossil fuel energy shortage, there is an urgent need to find alternatives. We describe a luminescent nanocomposite based on red-NIR phosphorescent molybdenum nanoclusters, namely Cs2Mo6I8(OCOC2F5)6, embedded in an eco-friendly cellulose biopolymer matrix that is obtained by a simple solvent casting technique. While homogeneity is kept up to 20 wt% of cluster complex doping, annealing hybrids leads to a large increase of their emission efficiency, as demonstrated by quantum yield measurements.

Metallogels: a novel approach for the nanostructuration of single-chain magnets.

In this study we demonstrate that single-chain magnets (SCMs) can be assembled in gel phase and transferred intact on surface. We take advantage of a family of SCMs based on TbIII ions and nitronyl-nitroxides radicals functionalized with short alkyl chains known to form crystalline supramolecular nanotubes interacting with heptane acting as crystallizing solvent. When the radicals are functionalized with long aliphatic chains a robust gel is formed with similar structural and functional properties respect to its crystalline parent. Indeed, a small-angle X-ray scattering (SAXS) study unambiguously demonstrates that the gel is made of supramolecular nanotubes: the high stability of the gel allows the determination from SAXS data of precise nanotube metrics such as diameter, helical pitch and monoclinic cell of the folded 2D crystal lattice along the tube direction. Additionally, static and dynamic magnetic investigations show the persistence of the SCM behavior in the metallogel. Last, on-surface gelation provides thick films as well as sub-monolayer deposits of supramolecular nanotubes on surface as evidenced by atomic force microscopy (AFM) observations. This paves the road toward magnetic materials and devices made of SCMs profiting of their isolation on surface as individual chains.

Milk sphingosomes as lipid carriers for tocopherols in aqueous foods: Thermotropic phase behaviour and morphology.

Foods containing polyunsaturated lipids are prone to oxidation. Designing food-grade hydrocolloidal encapsulation systems able to load lipophilic antioxidant molecules, such as tocopherols (vitamin E), is necessary to prevent oxidation and its deleterous consequences. In this study, we hypothesised that α-tocopherol molecules could incorporate in a host membrane composed of milk sphingomyelin (milk-SM) and performed a multi-scale biophysical study. The thermal properties of milk-SM bilayers with various molar proportions of α-tocopherol were characterised by differential scanning calorimetry (DSC), their structural properties were examined by X-ray diffraction (XRD). The miscibility between milk-SM and α-tocopherol was investigated in mixed Langmuir monolayers. The morphology of milk-SM sphingosomes was observed by confocal laser scanning microscopy (CLSM). We found that molecules of α-tocopherol inserted into the milk-SM bilayers and induced a physical desorganisation in the membrane packing, both in the ordered and fluid states. In the presence of α-tocopherol, the bilayers were no longer in a gel phase below the phase transition temperature Tm, but in the liquid ordered Lo phase. Furthermore, the sphingosomes formed elongated structures in presence of α-tocopherol as a result of membrane softening and changes in the bilayer curvature associated to membrane fusion. The findings of this work contribute in a better understanding of the capacity of milk-SM bilayers to incorporate guest molecules. Milk-SM sphingosomes loaded with tocopherols could be used to prevent oxidation in aqueous foods containing polyunsaturated lipids such as oil-in-water emulsions.

Adjusting the water-sensitivity of sugar/boronate-based organogels.

During the investigation of the water-sensitivity of (arylboronate alkylglucoside)-based organogels, we evaluated a series of twelve potential organogelators. They were synthesised in a single step from the corresponding arylboronic acids and alkylglucosides. Eight of them showed organogelation abilities in three solvents (toluene, cyclohexane, and ethyl myristate). Conformational minimisations of the potential organogelators permitted a clear relationship between the arylboronate orientation and the gelation effectiveness to be established. These gels were characterised by rheometry and SEM which revealed a gel-state originating from the self-assembly of the organogelators into long entangled fibres. SAXS confirmed the mode of packing in a hexagonal phase. Gels in toluene were found to be water-sensitive both after addition of a small amount of water and immersion into water. This study demonstrated that the main parameter impacting the water-sensitivity was the length of the alkyl chain at the anomeric position of the glucoside unit, much more than the functionalisation of an arylboronate moiety.

Encapsulation of Luminescent Gold Nanoclusters into Synthetic Vesicles.

Gold nanoclusters (Au NCs) are attractive luminescent nanoprobes for biomedical applications. In vivo biosensing and bioimaging requires the delivery of the Au NCs into subcellular compartments. In this view, we explore here the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Different methods were investigated to prepare vesicles containing Au NCs in their lumen. The efficiency of the process was correlated to the structural and morphological aspect of the Au NCs' encapsulating vesicles thanks to complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Furthermore, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.

Luminescent Gold Nanoclusters Interacting with Synthetic and Biological Vesicles.

According to their high electron density and ultrasmall size, gold nanoclusters (AuNCs) have unique luminescence and photoelectrochemical properties that make them very attractive for various biomedical fields. These applications require a clear understanding of their interaction with biological membranes. Here we demonstrate the ability of the AuNCs as markers for lipidic bilayer structures such as synthetic liposomes and biological extracellular vesicles (EVs). The AuNCs can selectively interact with liposomes or EVs through an attractive electrostatic interaction as demonstrated by zetametry and fluorescence microscopy. According to the ratio of nanoclusters to vesicles, the lipidic membranes can be fluorescently labeled without altering their thickness until charge reversion, the AuNCs being located at the level of the phosphate headgroups. In presence of an excess of AuNCs, the vesicles tend to adhere and aggregate. The strong adsorption of AuNCs results in the formation of a lamellar phase as demonstrated by cryo-transmission electron microscopy and small-angle X-ray scattering techniques.

Interfacial organization and phase behavior of mixed galactolipid-DPPC-phytosterol assemblies at the air-water interface and in hydrated mesophases.

The structural behavior of model assemblies composed of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the two main galactolipids found in plants, was investigated at the air/water interface and in aqueous dispersion. To approach the composition of the natural photosynthetic membranes, tunable Langmuir model membrane of galactolipids (GL) were used, and were complexified to form either heterogenous binary or ternary assemblies of GL, phospholipids (PL), and phytosterols (pS). The impact of pS, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or both on the structural properties of GL membrane was studied. The nature of the interactions between the different molecules was investigated using biophysical characterizations (ellipsometry, tensiometry, atomic force microscopy). In addition, the phase behavior was determined by SAXS analysis on the model assemblies in aqueous dispersions. Results revealed the good interfacial stability of these specific plant membrane lipids. The morphology of the GL film was characteristic of a fluid phase, with an interfacial roughness induced by the intercalation of monogalactosyl and digalactosyl polar heads of MGDG and DGDG, respectively. A phase heterogeneity in the monolayer was induced by the addition of DPPC and/or pS, which resulted in the modification of galactolipid organization and headgroup interactions. These structural changes were confirmed by SAXS analysis, showing more favorable interactions between MGDG and DPPC than between DGDG and DPPC in aqueous dispersion. This phenomenon was exacerbated in the presence of pS.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Molecular Engineering onto RuII Bis(1,2-diphenylphosphinoethane) Synthon: Toward an Original Organometallic Gelator.

In this article, we report the successful molecular engineering of Ru bis-acetylides that led for the first time to a gelator and more specifically in aromatic solvents. By means of a nonlinear ligand and an extended aromatic platform, the bulky Ru bis-acetylides were able to self-assemble into lamellar structures as evidenced by scanning electron microscopy (SEM) in benzene, toluene, and o- and m-xylene, which in turn induced gelation of the solution with a critical gelation concentration of 30 mg/mL. Nuclear magnetic resonance (NMR), variable temperature (VT)-NMR, and Fourier transform infrared (FT-IR) spectroscopies evidenced that hydrogen bonds are mainly responsible for the self-organization. VT-NMR and small-angle X-ray scattering (SAXS) have also suggested that the pro-ligand and the complex stack in different ways.

In vitro solubilization of fat-soluble vitamins in structurally defined mixed intestinal assemblies.

The structures of fed state intestinal assemblies containing bile components, dietary fat, and fat-soluble vitamins are not well known, although they are involved in lipid transport. In this study, several methods were used to investigate structural transitions upon various dietary lipids or various fat-soluble vitamins incorporation in bile intestinal assemblies. In particular, DLS and turbidimetry were used to study transition points as a function of component concentration, and cryo-TEM and SAXS were used to resolve assembly structures at microscopic and supramolecular scales, respectively. Results showed that increasing the concentration of dietary lipids in bile assembly induced a transition from core-shell micelles to unilamellar vesicles (except with caprylate lipids, always yielding micelles). In these specific assemblies, increasing the concentration of a fat-soluble vitamin either induced a systematic structural transition, defining a solubilization capacity (α-tocopherol or phylloquinone), or induced a structural transition only in micelles (retinol), or did not induce any structural transition up to very high concentrations (cholecalciferol). Using SAXS data, ideal molecular organizations are proposed for assemblies in the absence or presence of α-tocopherol.

Loading of lutein in egg-sphingomyelin vesicles as lipid carriers: Thermotropic phase behaviour, structure of sphingosome membranes and lutein crystals.

Lutein is a xanthophyll carotenoid provided exclusively by the diet, that has protective functions and beneficial effects on human health. Supplementation in lutein is necessary to reach the recommended daily dietary intake. However, the introduction of lutein into foods and beverages is a real challenge since this lipophilic nutrient has a poor aqueous solubility and a low bioavailability. In this study, we investigated the capacity of egg-sphingomyelin (ESM) vesicles called sphingosomes to solubilise lutein into the bilayers. The thermal and structural properties of ESM bilayers were examined in presence of various amounts of lutein by differential scanning calorimetry (DSC) and temperature-controlled X-ray diffraction (XRD), the structures of sphingosomes and lutein crystals were observed by microscopic techniques. ESM bilayers were in the fluid Lα phase above the phase transition temperature Tm = 39.6 °C and in the lamellar ripple Pß' phase below Tm where ESM sphingosomes exhibited ondulations and were facetted. Lutein molecules were successfully incorporated into the ESM bilayers where they induced a structural disorganisation. For ESM/lutein 90/10 %mol (91.8/8.2 %wt; 89 mg lutein / g ESM), lutein partitioning occured with the formation of lutein crystals in the aqueous phase together with lutein-loaded ESM vesicles. This study highlighted the capacity of new lipid carriers such as egg-sphingosomes to solubilise lutein and opens perspectives for the formulation of effective lutein-fortified functionnal foods and beverages providing health benefits.

Force-Field Simulations of a Hydrated Lanreotide-Based Derivative: Hydration, Dynamics, and Numerical Evidence of Self-Assembly in Dimers.

Recently, self-organization of the cyclic octapeptide lanreotide and lanreotide-based derivatives in a nanotube to from a dimer structure has been experimentally evidenced. While the nature of the interactions between both monomers has been strongly investigated no molecular details of the hydration of the monomer and the formation of the dimer have been provided. Using molecular dynamics simulations, this work focuses on the structure, hydration, and dynamics of water and an analog of lanreotide. To do so, several models of monomers based on different schemes of partial charges and electrostatic interaction calculations are considered. By comparison with the experiments, we show that the model based on the combination of the AMBER force-field, CHELPG charge calculation, Ewald sum is the most relevant. Additionally, by mapping the interfacial hydration of the lanreotide monomer we evidence a heterogeneous surface in terms of hydrophilicity involving heterogeneous hydration. Furthermore, we show a slowdown in the translational dynamics of water molecules located close to the lanreotide surface. We also provide the molecular details of the self-assembly in the dimer in terms of structure, hydration, and energy.

Boron Effect on Sugar-Based Organogelators.

The reaction of several alkylglucosides with phenyl boronic acid permitted easy access to a series of alkylglucoside phenyl boronate derivatives. This type of compound has structures similar to those of known benzylidene glucoside organogelators except for the presence of a boronate function in place of the acetal one. Low to very low concentrations of these amphiphilic molecules produced gelation of several organic solvents. The rheological properties of the corresponding soft materials characterized them as elastic solids. They were further characterized by SEM to obtain more information on their morphologies and by SAXS to determine the type of self-assembly involved within the gels. The sensitivity of the boronate function towards hydrolysis was also investigated. We demonstrated that a small amount of water (5 % v/v) was sufficient to disrupt the organogels leading to the original alkylglucoside and phenyl boronic acid; an important difference with the stable benzylidene-based organogelators. Such water-sensitive boronated organogelators could be suitable substances for the preparation of smart soft material for topical drug delivery.

Origin of transparency in scattering biomimetic collagen materials.

Living tissues, heterogeneous at the microscale, usually scatter light. Strong scattering is responsible for the whiteness of bones, teeth, and brain and is known to limit severely the performances of biomedical optical imaging. Transparency is also found within collagen-based extracellular tissues such as decalcified ivory, fish scales, or cornea. However, its physical origin is still poorly understood. Here, we unveil the presence of a gap of transparency in scattering fibrillar collagen matrices within a narrow range of concentration in the phase diagram. This precholesteric phase presents a three-dimensional (3D) orientational order biomimetic of that in natural tissues. By quantitatively studying the relation between the 3D fibrillar network and the optical and mechanical properties of the macroscopic matrices, we show that transparency results from structural partial order inhibiting light scattering, while preserving mechanical stability, stiffness, and nonlinearity. The striking similarities between synthetic and natural materials provide insights for better understanding the occurring transparency.

Hybrid gold nanoparticle-quantum dot self-assembled nanostructures driven by complementary artificial proteins.

Hybrid nanostructures are constructed by the direct coupling of fluorescent quantum dots and plasmonic gold nanoparticles. Self-assembly is directed by the strong affinity between two artificial α-repeat proteins that are introduced in the capping layers of the nanoparticles at a controlled surface density. The proteins have been engineered to exhibit a high mutual affinity, corresponding to a dissociation constant in the nanomolar range, towards the protein-functionalized quantum dots and gold nanoparticles. Protein-mediated self-assembly is evidenced by surface plasmon resonance and gel electrophoresis. The size and the structure of colloidal superstructures of complementary nanoparticles are analyzed by transmission electron microscopy and small angle X-ray scattering. The size of the superstructures is determined by the number of proteins per nanoparticle. The well-defined geometry of the rigid protein complex sets a highly uniform interparticle distance of 8 nm that affects the emission properties of the quantum dots in the hybrid ensembles. Our results open the route to the design of hybrid emitter-plasmon colloidal assemblies with controlled near-field coupling and better optical response.