Idit Amar-Yuli

Disassembly and reassembly of amyloid fibrils in water-ethanol mixtures.

This work presents the structural analysis of amyloid-like β-lactoglobulin fibrils incubated in ethanol-water mixtures after their formation in water. We observe for the first time the disassembly of semiflexible heat-denatured β-lactoglobulin fibrils and reassembly into highly flexible wormlike fibrils in ethanol-water solutions. Tapping mode atomic force microscopy is performed to follow structural changes. Our results show that in addition to their growth in length, there is a continuous nucleation process of new wormlike objects with time at the expense of the original β-lactoglobulin fibrils. The persistence length of wormlike fibrils (29.43 nm in the presence of 50% ethanol), indicative of their degree of flexibility, differs by 2 orders of magnitude from that of untreated β-lactoglobulin fibrils (2368.75 nm in pure water). Interestingly, wormlike fibrils do not exhibit a multiple strands nature like the pristine fibrils, as revealed by the lower maximum height and the lack of clear height periodicity along their contour length profile. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates that the set of polypeptides obtained by ethanol degradation differs in some fractions from that present in pristine β-lactoglobulin fibrils. ATR-FTIR (attenuated total reflectance-Fourier transform infrared) spectroscopy also supports a different composition of the secondary structure of wormlike fibrils with a decreased amount of α-helix and increased random coils and turns content. These findings can contribute to deciphering the molecular mechanisms of protein aggregation into amyloid fibrils and their disassembly as well as enabling tailor-made production of protein fibrils.

Low viscosity reversed hexagonal mesophases induced by hydrophilic additives.

This study reports on the formation of a low viscosity H(II) mesophase at room temperature upon addition of Transcutol (diethylene glycol mono ethyl ether) or ethanol to the ternary mixture of GMO (glycerol monooleate)/TAG (tricaprylin)/water. The microstructure and bulk properties were characterized in comparison with those of the low viscosity HII mesophase formed in the ternary GMO/TAG/water mixture at elevated temperatures (35-40 degrees C). We characterized the role of Transcutol or ethanol as inducers of disorder and surfactant mobility. The techniques used were rheology, differential scanning calorimetry (DSC), wide- and small-angle X-ray scattering (WAXS and SAXS, respectively), NMR (self-diffusion and (2)H NMR), and Fourier transform infrared (FTIR) spectroscopies. The incorporation of either Transcutol or ethanol induced the formation of less ordered HII mesophases with smaller domain sizes and lattice parameters at room temperature (up to 30 degrees C), similar to those found for the GMO/TAG/water mixture at more elevated temperatures (35-40 degrees C). On the basis of our measurements, we suggest that Transcutol or ethanol causes dehydration of the GMO headgroups and enhances the mobility of the GMO chains. As a result, these two small molecules, which compete for water with the GMO polar headgroups, may increase the curvature of the cylindrical micelles and also perhaps reduce their length. This results in the formation of fluid H(II) structures at room temperature (up to 30 degrees C). It is possible that these phases are a prelude to the H(II)-L(2) transformation, which takes place above 35 degrees C.

Solubilization of Nutraceuticals into Reverse Hexagonal Mesophases

The solubilization of four bioactive molecules with different polarities, in three reverse hexagonal (HII) systems has been investigated. The three HII systems were a typical reverse hexagonal composed of glycerol monooleate (GMO)/tricaprylin/water and two fluid hexagonal systems containing either 2.75 wt % Transcutol or ethanol as a fourth component. The phase behavior of the liquid crystalline phases in the presence of ascorbic acid, ascorbyl palmitate, D-alpha-tocopherol and D-alpha-tocopherol acetate were determined by small-angle X-ray scattering (SAXS) and optical microscopy. Differential scanning calorimetry (DSC) and Fourier-transform infrared (FT-IR) techniques were utilized to follow modifications in the thermal behavior and in the vibrations of different functional groups upon solubilizing the bioactive molecules. The nature of each guest molecule (in both geometry and polarity) together with the different HII structures (typical and fluids) determined the corresponding phase behavior, swelling or structural transformations and its location in the HII structures. Ascorbic acid was found to act as a chaotropic guest molecule, localized in the water-rich core and at the interface. The AP was also a chaotropic guest molecule with its head located in the vicinity of the GMO headgroup while its tail embedded close to the surfactant tail. D-alpha-tocopherol and D-alpha-tocopherol acetate were incorporated between the GMO tails; however, the D-alpha-tocopherol was located closer to the interface. Once Transcutol or ethanol was present and upon guest molecule incorporation, partial migration was detected.

Transitions and loci of solubilization of nutraceuticals in U-type nonionic microemulsions studied by self-diffusion NMR

U-type microemulsions based on five food-grade ingredients, water, R(+)-limonene, ethanol, propylene glycol, (or glycerol) and ethoxylated sorbitan esters (Tween 60 or Tween 80) were studied. The U-type phase diagram is characterized by a unique composition, fully dilutable with the aqueous phase that inverts progressively from an L2 phase to an L1 phase via a bicontinuous structure without phase separation. The ‘oil concentrate’ (surfactant, oil, alcohol) is loaded with water-insoluble solubilizates (phytosterols, lutein and lycopene) a few times more than the solubility capacity of the oil phase (without the surfactant). The maximum solubilization capacity (μ-value) was obtained for phytosterols and the minimum solubilization capacity was for lycopene. All solubilization values decrease with aqueous phase dilution. If solubilization is calculated on the basis of the oil content (α-value) or the oil + surfactant (γ-value) it is obvious that the interface plays a key role in the solubilization.The lipophilic solubilizates (by SD-NMR) are tightly packed and well accommodated at interfaces that convex (hydrophobic-in-nature) toward the water (water-in-oil microemulsions). Solubilization at the bicontinuous interface is lower and the solubilizates are loose packed. Once phase inversion occurs, and the interface becomes more hydrophilic and transforms into oil-in-water microemulsion, the solubilization becomes minimal, and most of the solubilizate desorbs. Phytosterols and lycopene induce the transition from W/O to bicontinuous and it occurs at lower water content (ca. 25 wt% aqueous phase in the presence of solubilizate vs. 35 wt% in its absence). The transition from bicontinuous to O/W is mostly unaffected (or undetected) since the interface flattens out and the solubilizate does not affect the curvature much. Lutein displays different behavior, the transition, from bicontinuous phase to O/W, occurs at higher water contents because its adsorption and packing are significantly tighter. Solubilization capacities of each of the nutraceuticals were determined for all dilution compositions.

Tuning in-meso-Crystallized Lysozyme Polymorphism by Lyotropic Liquid Crystal Symmetry

Lipid-based lyotropic liquid crystals (LLCs) show great potential for applications in fields as diverse as food technology, cosmetics, pharmaceutics, or structural biology. Recently, these systems have provided a viable alternative to the difficult process of membrane protein crystallization, owing to their similarities with cell membranes. Nonetheless, the process of in-meso crystallization of proteins still remains poorly understood. In this study, we demonstrate that in-meso crystal morphologies of lysozyme (LSZ), a model hydrophilic protein, can be controlled by both the composition and symmetry of the mesophase, inferring a possible general influence of the LLC space group on the protein crystal polymorphism. Lysozyme was crystallized in-meso from three common LLC phases (lamellar, inverse hexagonal, and inverse bicontinuous cubic) composed of monolinolein and water. Different mixing ratios of mesophase to crystallization buffer were used in order to tune crystallization both in the bulk mesophase and in excess water conditions. Two distinct mechanisms of crystallization were shown to take place depending on available water in the mesophases. In the bulk mesophases, protein nuclei form and grow within structural defects of the mesophase and partially dehydrate the system inducing order-to-order transitions of the liquid crystalline phase toward stable symmetries in conditions of lower hydration. The formed protein crystals eventually macrophase separate from the mesophase allowing the system to reach its final symmetry. On the other hand, when excess water is available, protein molecules diffuse from the water channels into the excess water, where the crystallization process can take place freely, and with little to no effect on the structure and symmetry of the lyotropic liquid crystals.

Controlled embedment and release of DNA from lipidic reverse columnar hexagonal mesophases

DNA–lipid interactions have important implications for biological functions, gene therapy and biotechnology. In the present work, we exploit hydrogen bonding and ionic interactions between lipids and DNA to control the entrapment, the binding and the release properties of DNA confined within the water channels of reverse hexagonal columnar phases. Two lipid formulations were considered, consisting of glycerol monooleate/tricaprylin and glycerol monooleate/oleyl amine/tricaprylin, yielding the nonionic and cationic-based systems, respectively. In the presence of water, or water–DNA dilute solutions, both formulations led to the formation of reverse hexagonal columnar mesophases. To study the confinement of DNA in the reverse hexagonal mesophases, and to understand its interactions with the nonionic and cationic lipid formulations, we relied on small-angle X-ray scattering (SAXS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The release of the DNA from these hosting systems in excess water was monitored by UV spectrophotometry and single molecule atomic force microscopy (AFM). In the case of the nonionic columnar system, DNA confined within the water cylinders, was stabilized by hydrogen bonding with the lipid polar heads, as revealed by the dehydration of the glycerol monooleate polar headgroups and a decrease in the water channel diameter. The diffusion of DNA out of the mesophase water channels was found to occur in three steps correlated with the different contour lengths of the DNA fragments generated enzymatically from the same pristine DNA macromolecule. In contrast, the presence of a low dose of cationic surfactants in the formulation enabled strong electrostatic interactions with the DNA molecules, swelling the water cylinders and entirely suppressing the release of DNA. These results show that lipidic mesophases constitute an appealing, fully biocompatible carrier, allowing a fine control on the encapsulation and delivery of DNA in excess water environment.

Templating effects of lyotropic liquid crystals in the encapsulation of amyloid fibrils and their stimuli-responsive magnetic behavior

The relationship between the symmetry of inverted lyotropic liquid crystals (LLC), used as hosting complex fluid, and amyloid fibrils, confined within the LLC matrix, was studied with the aim of exploiting the LLC water reservoirs for encapsulation of large protein aggregates. We used β-lactoglobulin (βlg) fibrils as a model system for high aspect ratio amyloid fibrils and encapsulated them into three different types of LLC mesophases composed of glycerol monolinoleate, with or without linoleic acid and water, yielding respectively lamellar, inverse bicontinuous cubic and inverse columnar hexagonal symmetries. The impact of fibrils confinement within the LLC on their secondary structure and spatial organization was studied by combining small angle X-ray scattering (SAXS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and atomic force microscopy (AFM) techniques. FTIR indicated that the βlg fibrils were incorporated within the aqueous layers of the lamellar phase while in the cubic and hexagonal structures they were mostly located at the lipid–water interface, along the channels. Furthermore, each mesophase affected the assembly of the amyloid fibers in accordance with the nature of the space group of the LLC structure considered. The two-dimensional order parameter of the fibrils, which was calculated on the basis of AFM images, revealed mainly random orientation distribution of the fibrils when these were confined within the lamellar and cubic phases, which can be understood by the orientation degeneracy of the fibrils in a 2D confinement and by the isotropic nature of the cubic phases. On the contrary, the fibers exhibited nearly perfect orientation when confined within the columnar hexagonal phase as a consequence of the unidirectional orientation of the LLC and the high aspect ratio of the fibrils. These trends were further exploited to induce orientation of the LLC by decorating the encapsulated amyloid fibrils with magnetic nanoparticles capable to respond to an external magnetic field stimulus: a coupling of the orientation of the nanoparticle-decorated amyloid fibrils and that of the LLC mesophase was demonstrated by small angle neutron scattering under the application of a constant magnetic field.

Crystallization of cyclosporin A in lyotropic reverse hexagonal liquid crystals

In this communication we demonstrate for the first time that lyotropic reverse hexagonal mesophase can serve as a crystallization medium for a hydrophobic peptide, resulting in high quality single crystals suitable for crystallographic analysis. The obtained crystals of cyclosporin A enabled excellent X-ray data collection, diffracting to a resolution limit of 1.0 A. Current findings open a new perspective in the field of crystallization of biological macromolecules in lyotropic mesophases.

Coenzyme Q10: functional benefits, dietary uptake and delivery mechanisms.

Abstract: Coenzyme Q10, present in all cells and membranes, is essential for electron transport in the mitochondrial respiratory chain, antioxidant defence, and other functions of great importance for cellular metabolism. The essential advantages of CoQ10, combined with its poor and slow absorption, generated the need to develop new technologies for delivery. Therefore, much effort has been expended to design delivery vehicles that can improve its aqueous solubility and overall bioavailability for oral and topical administration. This chapter summarizes the findings available today concerning CoQ10 chemical characteristics, human functionalities, its impact on human diseases, and new techniques developed to enhance its delivery.