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.

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.

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.

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.

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.

Organic Nanoscrolls from Electrostatic Interactions between Peptides and Lipids: Assembly Steps and Structure.

An important aspect of cells is their shape flexibility that gives them motion but also a high adaptation versatility to their environment. This shape versatility is mediated by different types of protein-membrane interactions among which electrostatic plays an important role. In the present work we examined the interaction between a small dicationic peptide, that possesses self-assembly properties, and lipid model membranes. The peptide, lanreotide, spontaneously forms nanotubes in water that have a strictly uniform diameter. In the current work, we show that the interaction between the cationic peptide and negatively charged bilayers of lipids induces the formation of myelin sheath-like structures that we call nanoscrolls. By deciphering the different steps of formation and the molecular structure of the self-assembly, we show how electrostatics modify the spontaneous peptide and lipid way of packing.

Molecular and Material Engineering of Photocathodes Derivatized with Polyoxometalate-Supported {Mo3S4} HER Catalysts.

Molecular engineering of efficient HER catalysts is an attractive approach for controlling the spatial environment of specific building units selected for their intrinsic functionality required within the multistep HER process. As the {Mo3S4} core derived as various coordination complexes has been identified as one as the most promising MoSx-based HER electrocatalysts, we demonstrate that the covalent association between the {Mo3S4} core and the redox-active macrocyclic {P8W48} polyoxometalate (POM) produces a striking synergistic effect featured by high HER performance. Various experiments carried out in homogeneous conditions showed that this synergistic effect arises from the direct connection between the {Mo3S4} cluster and the toroidal {P8W48} units featured by a stoichiometry that can be tuned from two to four {Mo3S4} cores per {P8W48} unit. In addition, we report that this effect is preserved within heterogeneous photoelectrochemical devices where the {Mo3S4}-{P8W48} (thio-POM) assembly was used as cocatalyst (cocat) onto a microstructured p-type silicon. Using a drop-casting procedure to immobilize cocat onto the silicon interface led to high initial HER performance under simulated sunlight, achieving a photocurrent density of 10 mA cm-2 at +0.13 V vs RHE. Furthermore, electrostatic incorporation of the thio-POM anion cocat into a poly(3,4-ethylenedioxythiophene) (PEDOT) film is demonstrated to be efficient and straightforward to durably retain the cocat at the interface of a micropyramidal silicon (SimPy) photocathode. The thio-POM/PEDOT-modified photocathode is able to produce H2 under 1 Sun illumination at a rate of ca. 100 µmol cm-2 h-1 at 0 V vs RHE, highlighting the excellent performance of this photoelectrochemical system.

Casein interaction with lipid membranes: Are the phase state or charge density of the phospholipids affecting protein adsorption?

Casein micelles are ~200 nm electronegative particles that constitute 80 wt% of the milk proteins. During synthesis in the lactating mammary cells, caseins are thought to interact in the form of ~20 nm assemblies, directly with the biological membranes of the endoplasmic reticulum and/or the Golgi apparatus. However, conditions that drive this interaction are not yet known. Atomic force microscopy imaging and force spectroscopy were used to directly observe the adsorption of casein particles on supported phospholipid bilayers with controlled compositions to vary their phase state and surface charge density, as verified by X-ray diffraction and zetametry. At pH 6.7, the casein particles adsorbed onto bilayer phases with zwitterionic and liquid-disordered phospholipid molecules, but not on phases with anionic or ordered phospholipids. Furthermore, the presence of adsorbed caseins altered the stability of the yet exposed bilayer. Considering their respective compositions and symmetry/asymmetry, these results cast light on the possible interactions of casein assemblies with the organelles' membranes of the lactating mammary cells.

Polyoxothiometalate-Derivatized Silicon Photocathodes for Sunlight-Driven Hydrogen Evolution Reaction.

Silicon photocathodes coated with drop-casted {Mo3S4}-based polyoxothiometalate assemblies are demonstrated to be effective for sunlight-driven hydrogen evolution reaction (HER) in acid conditions. These photocathodes are catalytically more efficient than that coated with the parent thiomolybdate incorporating an organic ligand, as supported by a higher onset potential and a lower overvoltage at 10 mA cm-2. At pH 7.3, the trend is inversed and the beneficial effect of the polyoxometalate for the HER is not observed. Moreover, the polyoxothiometalate-modified photocathode is found to be also more stable under acid conditions and can be operated at the light-limited catalytic current for more than 40 h. Furthermore, X-ray photoelectron spectroscopy and atomic force microscopy measurements indicate that the cathodic polarization of both photocathodes leads to the release of a large amount of the deposited material into the electrolyte solution concomitantly with the formation of mixed valence species {Mo(IV)3-x Mo(III) x O4-n S n }(4-x)+ resulting from the replacement of S2- sulfido ligands in the cluster by oxo O2- groups; these combined effects are shown to be beneficial for the photoelectrocatalysis.

Mechanical Properties of Membranes Composed of Gel-Phase or Fluid-Phase Phospholipids Probed on Liposomes by Atomic Force Spectroscopy.

In many liposome applications, the nanomechanical properties of the membrane envelope are essential to ensure, e.g., physical stability, protection, or penetration into tissues. Of all factors, the lipid composition and its phase behavior are susceptible to tune the mechanical properties of membranes. To investigate this, small unilamellar vesicles (SUV; diameter < 200 nm), referred to as liposomes, were produced using either unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in aqueous buffer at pH 6.7. The respective melting temperatures of these phospholipids were -20 and 41 °C. X-ray diffraction analysis confirmed that at 20 °C DOPC was in the fluid phase and DPPC was in the gel phase. After adsorption of the liposomes onto flat silicon substrates, atomic force microscopy (AFM) was used to image and probe the mechanical properties of the liposome membrane. The resulting force-distance curves were treated using an analytical model based on the shell theory to yield the Young's modulus (E) and the bending rigidity (kC) of the curved membranes. The mechanical investigation showed that DPPC membranes were much stiffer (E = 116 ± 45 MPa) than those of DOPC (E = 13 ± 9 MPa) at 20 °C. The study demonstrates that the employed methodology allows discrimination of the respective properties of gel- or fluid-phase membranes when in the shape of liposomes. It opens perspectives to map the mechanical properties of liposomes containing both fluid and gel phases or of biological systems.

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins.

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended ß-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials.

Effects of a novel archaeal tetraether-based colipid on the in vivo gene transfer activity of two cationic amphiphiles.

Gene therapy for treating inherited diseases like cystic fibrosis might be achieved using multimodular nonviral lipid-based systems. To date, most optimizations have concerned cationic lipids rather than colipids. In this study, an original archaeal tetraether derivative was used as a colipid in combination with one or the other of two monocationic amphiphiles. The liposomes obtained, termed archaeosomes, were characterized regarding lipid self-assembling properties, macroscopic/microscopic structures, DNA condensation/neutralization/relaxation abilities, and colloidal stability in the presence of serum. In addition, gene transfer experiments were conducted in mice with lipid/DNA complexes being administered via systemic or local delivery routes. Altogether, the results showed that the tetraether colipid can provide complexes with different in vivo transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the administration route. This original colipid appears thus as an innovative modular platform endowed with properties possibly beneficial for fine-tuning of in vivo lipofection and other biomedical applications.

Thermotropic luminescent clustomesogen showing a nematic phase: a combination of experimental and molecular simulation studies.

Octahedral Mo6 nanoclusters are functionalized with two organic ligands containing cyanobiphenyl (CB) units, giving luminescent hybrid liquid crystals (LC). Although the mesogenic density around the bulky inorganic core is constant, the two hybrids show different LC properties. Interestingly, one of them shows a nematic phase, which is particularly rare for this kind of supermolecular system. This surprising result is explained by using large-scale molecular dynamic simulations.

Directing peptide crystallization through curvature control of nanotubes.

In the absence of efficient crystallization methods, the molecular structures of fibrous assemblies have so far remained rather elusive. In this paper, we present a rational method to crystallize the lanreotide octapeptide by modification of a residue involved in a close contact. Indeed, we show that it is possible to modify the curvature of the lanreotide nanotubes and hence their diameter. This fine tuning leads to crystallization because the radius of curvature of the initially bidimensional peptide wall can be increased up to a point where the wall is essentially flat and a crystal is allowed to grow along a third dimension. By comparing X-ray diffraction data and Fourier transform Raman spectra, we show that the nanotubes and the crystals share similar cell parameters and molecular conformations, proving that there is indeed a structural continuum between these two morphologies. These results illustrate a novel approach to crystallization and represent the first step towards the acquisition of an Å-resolution structure of the lanreotide nanotubes ß-sheet assembly.

Experimental observation of double-walled peptide nanotubes and monodispersity modeling of the number of walls.

Self-assembled nanoarchitectures based on biological molecules are attractive because of the simplicity and versatility of the building blocks. However, size control is still a challenge. This control is only possible when a given system is deeply understood. Such is the case with the lanreotide acetate, an octapeptide salt that spontaneously forms monodisperse nanotubes when dissolved into pure water. Following a structural approach, we have in the past demonstrated the possibility to tune the diameter of these nanotubes while keeping a strict monodispersity, either by chemical modification of one precise amino acid on the peptide sequence or by changing the size of the counterions. On the basis of these previous studies, we replaced monovalent counterions by divalent ones to vary the number of walls. Indeed, in the present work, we show that lanreotide associated with a divalent counterion forms double-walled nanotubes while keeping the average diameter constant. However, the strict monodispersity of the number of walls was unexpected. We propose that the divalent counterions create an adhesion force that can drive the wall packing. This adhesion force is counterbalanced by a mechanical one that is related to the stiffness of the peptide wall. By taking into account these two opposite forces, we have built a general model that fully explains why the lanreotide nanotubes formed with divalent counterions possess two walls and not more.

Electroless patterned assembly of metal nanoparticles on hydrogen-terminated silicon surfaces for applications in photoelectrocatalysis.

The deposition of gold and platinum nanoparticles (NPs) on hydrogen-terminated Si(100) (Si(100)-H) surfaces has been performed by galvanic displacement using fluoride-free sub-millimolar metallic salt solutions. The scanning electron microscopy (SEM) images showed the formation of oblate hemispherical NPs, with an average diameter of ca. 40 nm and an average height of 20 ± 10 and 10 ± 5 nm for Au and Pt, respectively. Furthermore, the calculated number density was (6.0 ± 0.8) × 10(9) Au NPs cm(-2) and (6.6 ± 1.3) × 10(9) Pt NPs cm(-2) with a larger size distribution measured for Au NPs. The Au 4f and Pt 4f X-ray photoelectron spectra of the metallized surfaces were characterized by a principal component corresponding to either the metallic gold or platinum. However, two other components located at higher binding energies were also visible and ascribed to gold or platinum silicides. Using this fluoride-free deposition process and a "reagentless" UV photolithography technique, we have also demonstrated that it was possible to prepare metallic NP micropatterns. Following this approach, single metal (Au) and two metals (Au and Pt) patterns have been produced and characterized by energy-dispersive X-ray spectroscopy (EDS) which revealed the presence of the expected metal(s). Such metallic NP micropatterned surfaces were used as photocathodes for H(2) evolution from water as a proof-of-concept experiment. These electrodes exhibited much higher electrocatalytic performance than that of nonmetallized Si(100)-H, both in the absence of light and under illumination. The overpotential for hydrogen evolution was significantly decreased by ca. 450 mV with respect to Si(100)-H (measured for a current density of 0.1 mA cm(-2)) under identical illumination conditions.

Stereochemical effect revealed in self-assemblies based on archaeal lipid analogues bearing a central five-membered carbocycle: a SAXS study.

The relative stereochemistry (cis or trans) of a 1,3-disubstituted cyclopentane unit in the middle of tetraether archaeal bipolar lipid analogues was found to have a dramatic influence on their supramolecular self-assembly properties. SAXS studies of two synthetic diastereomeric archaeal lipids bearing two lactosyl polar head groups at opposite ends revealed different lyotropic behaviors. The cis isomer led to L(c)-L(α)-Q(II) transitions whereas the trans isomer retained an L(α) phase from 20 to 100 °C. These main differences originate from the conformational equilibrium (pseudorotation) of 1,3-disubstituted cyclopentanes. Indeed, this pseudorotation exhibits quite similar orientations of the two substituents in a trans isomer whereas several orientations of the two alkyl chains are expected in a cis-1,3-dialkyl cyclopentane, thus authorizing more conformational flexibility in the lipid packing.