Insights into gliadin supramolecular organization at digestive pH 3.0.

Alpha-gliadin is a highly immunogenic protein from wheat, which is associated with many human diseases, like celiac disease and non-celiac gluten sensitivity. Because of that, gliadin solution is subject to intense biomedical research. However, the physicochemical nature of the employed gliadin solution at physiological pH is not understood. Herein, we present a supramolecular evaluation of the alpha-gliadin protein in water at pH 3.0 by dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and small-angle-.X-ray scattering (SAXS). We report that at 0.5 wt% concentration (0.1 mg/ml), gliadin is already a colloidal polydisperse system with an average hydrodynamic radius of 30 ±â€¯10 nm. By cryo-TEM, we detected mainly large clusters. However, it was possible to visualise for the first time prolate oligomers of around 68 nm and 103 nm, minor and major axis, respectively. SAXS experiments support the existence of prolate/rod-like structures. At 1.5 wt% concentration gliadin dimers, small oligomers and large clusters coexist. The radius of gyration (Rg1) of gliadin dimer is 5.72 ±â€¯0.23 nm with a dimer cross-section (Rc) of 1.63 nm, and an average length of around 19 nm, this suggests that gliadin dimers are formed longitudinally. Finally, our alpha-gliadin 3D model, obtained by ab initio prediction and analysed by molecular dynamics (MD), predicts that two surfaces prone to aggregation are exposed to the solvent, at the C-terminus. We hypothesise that this region may be involved in the dimerisation process of alpha-gliadin.

Polyaspartic acid facilitates oxolation within iron(iii) oxide pre-nucleation clusters and drives the formation of organic-inorganic composites.

The interplay between polymers and inorganic minerals during the formation of solids is crucial for biomineralization and bio-inspired materials, and advanced material properties can be achieved with organic-inorganic composites. By studying the reaction mechanisms, basic questions on organic-inorganic interactions and their role during material formation can be answered, enabling more target-oriented strategies in future synthetic approaches. Here, we present a comprehensive study on the hydrolysis of iron(iii) in the presence of polyaspartic acid. For the basic investigation of the formation mechanism, a titration assay was used, complemented by microscopic techniques. The polymer is shown to promote precipitation in partly hydrolyzed reaction solutions at the very early stages of the reaction by facilitating iron(iii) hydrolysis. In unhydrolyzed solutions, no significant interactions between the polymer and the inorganic solutes can be observed. We demonstrate that the hydrolysis promotion by the polymer can be understood by facilitating oxolation in olation iron(iii) pre-nucleation clusters. We propose that the adsorption of olation pre-nucleation clusters on the polymer chains and the resulting loss in dynamics and increased proximity of the reactants is the key to this effect. The resulting composite material obtained from the hydrolysis in the presence of the polymer was investigated with additional analytical techniques, namely, scanning and transmission electron microscopies, light microscopy, atomic force microscopy, zeta potential measurements, dynamic light scattering, and thermogravimetric analyses. It consists of elastic, polydisperse nanospheres, ca. 50-200 nm in diameter, and aggregates thereof, exhibiting a high polymer and water content.

Neutrophilic granulocytes - promiscuous accelerators of atherosclerosis.

Neutrophils, as part of the innate immune system, are classically described to be main actors during the onset of inflammation enforcing rapid neutralisation and clearance of pathogens. Besides their well-studied role in acute inflammatory processes, recent advances strongly indicate a so far underappreciated importance of neutrophils in initiation and development of atherosclerosis. This review focuses on current findings on the role of neutrophils in atherosclerosis. As pro-inflammatory mechanisms of neutrophils have primarily been studied in the microvascular environment; we here aim at translating these into the context of macrovascular inflammation in atherosclerosis. Since much of the pro-inflammatory activities of neutrophils stem from instructing neighbouring cell types, we highlight the promiscuous interplay between neutrophils and platelets, monocytes, T lymphocytes, and dendritic cells and its possible relevance to atherosclerosis.

CXCL4L1 inhibits angiogenesis and induces undirected endothelial cell migration without affecting endothelial cell proliferation and monocyte recruitment.

BACKGROUND AND OBJECTIVES: The non-allelic variant of CXCL4/PF4, CXCL4L1/PF4alt, differs from CXCL4 in three amino acids of the C-terminal α-helix and has been characterized as a potent anti-angiogenic regulator. Although CXCL4 structurally belongs to the chemokine family, it does not behave like a 'classical' chemokine, lacking significant chemotactic properties. Specific hallmarks are its angiostatic, anti-proliferative activities, and proinflammatory functions, which can be conferred by heteromer-formation with CCL5/RANTES enhancing monocyte recruitment. METHODS AND RESULTS: Here we show that tube formation of endothelial cells was inhibited by CXCL4L1 and CXCL4, while only CXCL4L1 triggered chemokinesis of endothelial cells. The chemotactic response towards VEGF and bFGF was attenuated by both variants and CXCL4L1-induced chemokinesis was blocked by bFGF or VEGF. Endothelial cell proliferation was inhibited by CXCL4 (IC(50) 6.9 µg mL(-1)) but not by CXCL4L1, while both chemokines bound directly to VEGF and bFGF. Moreover, CXCL4 enhanced CCL5-induced monocyte arrest in flow adhesion experiments and monocyte recruitment into the mouse peritoneal cavity in vivo, whereas CXCL4L1 had no effect. CXCL4L1 revealed lower affinity to CCL5 than CXCL4, as quantified by isothermal fluorescence titration. As evidenced by the reduction of the activated partial thromboplastin time, CXCL4L1 showed a tendency towards less heparin-neutralizing activity than CXCL4 (IC(50) 2.45 vs 0.98 µg mL(-1)). CONCLUSIONS: CXCL4L1 may act angiostatically by causing random endothelial cell locomotion, disturbing directed migration towards angiogenic chemokines, serving as a homeostatic chemokine with a moderate structural distinction yet different functional profile from CXCL4.

Colloidal dispersions for the delivery of acyclovir: a comparative study.

This paper describes a comparative study on the performances of ethosomes and solid lipid nanoparticle as delivery systems for acyclovir. Ethosomes were spontaneously produced by dissolution of phosphatidylcholine and acyclovir in ethanol followed by addition of an aqueous buffer while solid lipid nanoparticle were produced by homogenization and ultrasonication. Both colloidal systems were morphologically characterized by cryo-transmission electron microscopy. The encapsulation efficiency was 94.2±2.8% for ethosomes and 53.2±0.2% for solid lipid nanoparticle. Concerning Z potential, both formulations are close to neutrality. The diffusion coefficients of the drug from ethosomes and solid lipid nanoparticle, determined by a Franz cell method, were 9.4 and 1.2-fold lower as compared to the free acyclovir in solution, thus evidencing the ability of both colloidal systems in enhancing the diffusion of the drug. The antiviral activity against HSV-1 of both systems was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that no significant differences in the antiviral activity were observed by acyclovir in the free or loaded forms. Taken together these results, colloidal systems could be interesting to mediate the penetration of acyclovir within Vero cells.

Ethosomes for the delivery of anti-HSV-1 molecules: preparation, characterization and in vitro activity.

This paper describes the production, characterization and in vitro activity of ethosomes containing two molecules with antiviral activity, such as acyclovir (ACY) and N1-beta-D-ribofuranosyl-pyrazole [3,4d]pyridazin-7(6p-chlorine-phenyl)-one nucleoside (N1CP). Ethosomes were prepared and morphologically characterized by Cryo-TEM. The encapsulation efficiency was 92.3 +/- 2.5% for ACY and 94.2 +/- 2.8% for N1CP. The release of the drug from vesicles, determined by a Franz cell method, indicated that both drugs were released in a controlled manner. In order to possibly guarantee the stability during long-term storage ethosome suspensions was freeze-dried. It was found that the freeze-dried ethosomes' cakes were compact, glassy characterized by low density and quick re-hydration. However, the storage time slightly influences the percentage of drug encapsulation within ethosomes showing a drug leakage after re-hydration around 10%. The antiviral activity against HSV-1 of both drugs was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that ethosomes allowed a reduction of the ED50 of N1CP evidencing an increase of its antiviral activity. However, ACY remains more active than N1CP. No differences are appreciable between drug-containing ethosomes before and after freeze-drying. Taken together these results, ethosomal formulation could be possibly proposed as mean for topical administration of anti-herpetic molecules.

The solubilisation of a water insoluble functionalized silicone oil in an aqueous surfactant solution: a novel mechanism for the solubilisation process.

The solubilisation of the functionalized silicone oil aminoethyl aminopropyl methylpolysiloxane WR 1300 has been investigated in aqueous solutions of the surfactant pentaethoxy-iso-tridecanol (iC(13)E(5)). The silicone oil consists of a linear backbone with an average of 200 dimethylsiloxane units and 2-3 functionalized side groups. In various applications the compound is considered a silicone oil. The surfactant iC(13)E(5) forms a L(alpha)-phase in water, and with decane a microemulsion can be obtained. The solubilisation experiments started with lamellar iC(13)E(5) phases. With proper amounts of WR 1300 transparent, optically isotropic but highly viscous single phases are formed. These phases cannot be considered true microemulsions with an oil core and a surrounding surfactant layer. Cryo-TEM micrographs show micelles with irregular shapes and about 25 nm diameter. Their size is independent of the oil/surfactant ratio. It can be explained on the basis of a model where the amino groups of the oil are all found at the surface of the micelles, limiting the radius of the aggregates to the largest length of the polydimethylsiloxane backbone between two such groups or between one end of the oil and one functionalized group. The micellar structures contain only a few siloxane molecules exposing their hydrophilic groups to the water. The functionalized silicone oil molecules can thus be considered as surfactant molecules with a few polar groups and a large hydrophobic chain from dimethyl siloxane.

Combining FIB milling and conventional Argon ion milling techniques to prepare high-quality site-specific TEM samples for quantitative EELS analysis of oxygen in molten iron.

This paper reports a procedure to combine the focused ion beam micro-sampling method with conventional Ar-milling to prepare high-quality site-specific transmission electron microscopy cross-section samples. The advantage is to enable chemical and structural evaluations of oxygen dissolved in a molten iron sample to be made after quenching and recovery from high-pressure experiments in a laser-heated diamond anvil cell. The evaluations were performed by using electron energy-loss spectroscopy and high-resolution transmission electron microscopy. The high signal to noise ratios of electron energy-loss spectroscopy core-loss spectra from the transmission electron microscopy thin foil, re-thinned down to 40 nm in thickness by conventional Argon ion milling, provided us with oxygen quantitative analyses of the quenched molten iron phase. In addition, we could obtain lattice-fringe images using high-resolution transmission electron microscopy. The electron energy-loss spectroscopy analysis of oxygen in Fe(0.94)O has been carried out with a relative accuracy of 2%, using an analytical procedure proposed for foils thinner than 80 nm. Oxygen K-edge energy-loss near-edge structure also allows us to identify the specific phase that results from quenching and its electronic structure by the technique of fingerprinting of the spectrum with reference spectra in the Fe-O system.

Quantitative analysis of polymer colloids by cryo-transmission electron microscopy.

The structure of colloidal latex particles in dilute suspension at room temperature is investigated by cryogenic transmission electron microscopy (cryo-TEM). Two types of particles are analyzed: (i) core particles made of polystyrene with a thin layer of poly(N-isopropylacrylamide) (PNIPAM) and (ii) core-shell particles consisting of core particles onto which a network of cross-linked PNIPAM is affixed. Both systems are also studied by small-angle X-ray scattering (SAXS). The radial density profile of both types of particles have been derived from the cryo-TEM micrographs by image processing and compared to the results obtained by SAXS. Full agreement is found for the core particles. There is a discrepancy between the two methods in case of the core-shell particles. The discrepancy is due to the buckling of the network affixed to the surface. The buckling is clearly visible in the cryo-TEM pictures. The overall dimensions derived from cryo-TEM agree well with the hydrodynamic radius of the particles. The comparison of these data with the analysis by SAXS shows that SAXS is only sensitive to the average radial structure as expected. All data show that cryo-TEM micrographs can be evaluated to yield quantitative information about the structure of colloidal particles.

Shear stresses of colloidal dispersions at the glass transition in equilibrium and in flow.

We consider a model dense colloidal dispersion at the glass transition, and investigate the connection between equilibrium stress fluctuations, seen in linear shear moduli, and the shear stresses under strong flow conditions far from equilibrium, viz., flow curves for finite shear rates. To this purpose, thermosensitive core-shell particles consisting of a polystyrene core and a cross-linked poly(N-isopropylacrylamide) shell were synthesized. Data over an extended range in shear rates and frequencies are compared to theoretical results from integrations through transients and mode coupling approaches. The connection between nonlinear rheology and glass transition is clarified. While the theoretical models semiquantitatively fit the data taken in fluid states and the predominant elastic response of glass, a yet unaccounted dissipative mechanism is identified in glassy states.

Blastulae aggregates: new intermediate structures in the micelle-to-vesicle transition of catanionic systems.

A new type of intermediate structure was found in the salt-induced micelle-to-vesicle transition in a catanionic system composed of sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) in aqueous solution with an excess of anionic surfactant. The appearance of symmetrically shaped hollow structures, which we named blastulae vesicles, is presented.

Specific alkali cation effects in the transition from micelles to vesicles through salt addition.

The transition of ionic micelles to vesicles with added salts is explored in this paper. The catanionic surfactant solution was comprised of sodium dodecylsulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) with an excess of SDS. The micellar size increased with concentration for all salts. No anion specificity was found, probably because of the excess of SDS. However, when the cation of the added salt was varied, large differences were observed in the hydrodynamic radii of the aggregates. A classification of the cations according to their ability to increase the measured hydrodynamic radii follows a Hofmeister series. The change in aggregate size can be explained by modified counterion binding and dehydration of the surfactant headgroups.

Influence of composition and preparation parameters on the properties of aqueous monoolein dispersions.

Colloidal cubic phase particles formed in the monoolein/poloxamer/water system are being investigated as potential drug carriers for, e.g., intravenous administration. Preparation methods must, however, still be further developed to reliably yield monoolein dispersions with cubic particles in a size range acceptable for i.v. administration and adequate long-term stability. In this context, the influence of different composition and preparation parameters on the properties of monoolein dispersions prepared by high-pressure homogenization was studied. High pressure homogenization of coarse poloxamer 407-stabilized monoolein/water mixtures leads to dispersions with a large fraction of micrometer-sized particles at low poloxamer concentrations. Higher poloxamer concentrations lead to lower mean particle sizes but the fraction of cubic particles becomes smaller and vesicular particles are observed instead. A study of the characteristics of a dispersion with a standard composition indicated that the homogenization temperature has a much stronger influence on the dispersion properties than the homogenization pressure or the type of homogenizer used. Temperatures around 40-60 degrees C lead to the most favorable dispersion properties. The high temperature sensitivity of the preparation process appears to be at least partly correlated with the phase behavior of the dispersed particles determined by temperature-dependent X-ray diffraction.

Nanosystems for skin hydration: a comparative study.

The present investigation describes a comparative study for the design of innovative topical formulation for skin hydration. In particular, different colloidal forms based on lipidic components have been produced and characterized. Morphology and dimensional distribution have been investigated by means of electron microscopy and photon correlation spectroscopy. Nanoparticulate systems characterized by different morphology and dimensions depending on production procedures have been obtained, namely cubosomes, nanovesicles, solid lipid nanoparticles and liposomes. Hydration power has been studied by means of a corneometer, measuring the skin electrical capacitance before and after the application of opportunely viscosized nanoparticulate systems. It has been demonstrated that nanovesicle gel displayed a pronounced hydration power with respect to the other nanostructured forms, its hydration effect on skin was 3.5-fold higher, with respect to the untreated area, after 5 min from the application and 1.5-fold higher after 2 h.

Thermosensitive core-shell particles as model systems for studying the flow behavior of concentrated colloidal dispersions.

We report on a comprehensive investigation of the flow behavior of colloidal thermosensitive core-shell particles at high densities. The particles consist of a solid core of poly(styrene) onto which a network of cross-linked poly(N-isopropylacrylamide) is affixed. Immersed in water the shell of these particles will swell if the temperature is low. Raising the temperature above 32 degrees C leads to a volume transition within this shell which leads to a marked shrinking of the shell. The particles have well-defined core-shell structure and a narrow size distribution. The remaining electrostatic interactions due to a small number of charges affixed to the core particles can be screened by adding 0.05M KCl to the suspensions. Below the lower critical solution temperature at 32 degrees C the particles are purely repulsive. Above this transition, a thermoreversible coagulation takes place. Lowering the temperature again leads to full dissociation of the aggregates formed by this process. The particles crystallize for effective volume fractions between 0.48 and 0.55. The crystallites can be molten by shear in order to reach a fluid sample again. The reduced shear stress measured in this metastable disordered state was found to be a unique function of the shear rate and the effective volume fraction. These reduced flow curves thus obtained can be described quantitatively by the theory of Fuchs and Cates [Phys. Rev. Lett. 89, 248304 (2002)] which is based on the mode-coupling theory of the glass transition.

Crystallization behavior of supercooled smectic cholesteryl myristate nanoparticles containing phospholipids as stabilizers.

Supercooled smectic nanoparticles based on physiological cholesterol esters are under investigation as a potential novel carrier system for lipophilic drugs. The present study investigates the very complex crystallization behavior of such nanoparticles stabilized with the aid of phospholipids. Phospholipid and phospholipid/bile salt stabilized cholesteryl myristate dispersions were prepared by high-pressure melt homogenization and characterized by particle size measurements, differential scanning calorimetry, X-ray diffraction and electron microscopy. To obtain fractions with very small smectic nanoparticles, selected dispersions were ultracentrifuged. A mixture of cholesteryl myristate and the phospholipid used for the stabilization of the dispersions was also investigated by light microscopy. The nanoparticles usually display a bimodal crystallization event which depends on the thermal treatment and cannot be attributed to crystalline polymorphism. The ratio of the particle fractions crystallizing in the two successive steps strongly depends on the particle size of the dispersions. The presence of larger particles leads to an increased fraction crystallizing at higher temperature and a higher recrystallization tendency upon storage. The observed peculiarities of the crystallization behavior seem to be mainly caused by the presence of particles with different shapes (cylindrical and spherical) as observed in electron microscopy. Alterations in the composition of the nanoparticles may also play a role.

Supercooled smectic nanoparticles: a potential novel carrier system for poorly water soluble drugs.

PURPOSE: The possibility of preparing nanoparticles in the supercooled thermotropic liquid crystalline state from cholesterol esters with saturated acyl chains as well as the incorporation of model drugs into the dispersions was investigated using cholesteryl myristate (CM) as a model cholesterol ester. METHODS: Nanoparticles were prepared by high-pressure melt homogenization or solvent evaporation using phospholipids, phospholipid/ bile salt, or polyvinyl alcohol as emulsifiers. The physicochemical state and phase behavior of the particles was characterized by particle size measurements (photon correlation spectroscopy, laser diffraction with polarization intensity differential scattering), differential scanning calorimetry, X-ray diffraction, and electron and polarizing light microscopy. The viscosity of the isotropic and liquid crystalline phases of CM in the bulk was investigated in dependence on temperature and shear rate by rotational viscometry. RESULTS: CM nanoparticies can be obtained in the smectic phase and retained in this state for at least 12 months when stored at 230C in optimized systems. The recrystallization tendency of CM in the dispersions strongly depends on the stabilizer system and the particle size. Stable drug-loaded smectic nanoparticles were obtained after incorporation of 10% (related to CM) ibuprofen, miconazole, etomidate, and 1% progesterone. CONCLUSIONS: Due to their liquid crystalline state, colloidal smectic nanoparticles offer interesting possibilities as carrier system for lipophilic drugs. CM nanoparticles are suitable model systems for studying the crystallization behavior and investigating the influence of various parameters for the development of smectic nanoparticles resistant against recrystallization upon storage.