Transitions induced by solubilized fat into reverse hexagonal mesophases

Lyotropic liquid crystals of glycerol monooleate (GMO) and water binary mixtures have been extensively studied and their resemblance to human membranes has intrigued many scientists. Biological systems as well as food mixtures are composed of lipids and fat components including triacylglycerols (TAGs, triglycerides) that can affect the nature of the assembly of the mesophase. The present study examines the effect of TAGs of different chain lengths (C(2)-C(18)) at various water/GMO compositions, on phase transitions from lamellar or cubic to reverse hexagonal (L(alpha)-H(II) and Q-H(II)). The ability of the triglycerides to promote the formation of an H(II) mesophase is chain length-dependent. It was found that TAG molecules with very short acyl chains (triacetin) can hydrate the head groups of the lipid and do not affect the critical packing parameter (CPP) of the amphiphile; therefore, they do not affect the self-assembly of the GMO in water, and the mesophase remains lamellar or cubic. However, TAGs with medium chain fatty acids will solvate the tails of the lipid, and will affect the CPP of the GMO, and transform the lamellar or cubic phases into hexagonal mesophase. TAGs with long chain fatty acids are very bulky, not very miscible with the GMO, and therefore, kinetically are very slow to solvate the lipid tails of the amphiphile and are difficult to accommodate into the lipophilic parts of the GMO. Their effect on the transitions from a lamellar or cubic phase to hexagonal is detected only after months of equilibration. In order to enhance the effect of the TAG on the phase transitions in the GMO/triglyceride/water systems, temperature and electrolytes effects were examined. In the presence of short and medium chain triglycerides, increasing temperature caused a transition from lamellar or hexagonal to L(2) phase (highest CPP value). However, in the presence of long chain TAGs, increasing temperature to ca. 40 degrees C caused a formation of H(II) mesophase. In addition, it was found that in tricaprylin/GMO/water systems, the increase in temperature caused a decrease in the lattice parameter. The effect of NaCl on the H(II) mesophase revealed interesting results. At low concentration of tricaprylin (5 wt%), the addition of only 0.1 wt% of NaCl was sufficient to cause the formation of well-defined H(II) mesophase, while further addition of electrolyte increased the hexagonal lattice parameters. At higher TAGs concentrations (10 wt%), addition of electrolyte resulted in the formation of H(II) with modifications of the lattice parameter. All the examined effects were more pronounced with increasing water content.     

Bayesian inference for the power law process

The power law process has been used to model reliability growth, software reliability and the failure times of repairable systems. This article reviews and further develops Bayesian inference for such a process. The Bayesian approach provides a unified methodology for dealing with both time and failure truncated data. As well as looking at the posterior densities of the parameters of the power law process, inference for the expected number of failures and the probability of no failures in some given time interval is discussed. Aspects of the prediction problem are examined. The results are illustrated with two data examples.     

Solubilization of food bioactives within lyotropic liquid crystalline mesophases

Liquid crystals are widely utilized as model systems to mimic biological processes where the phase behavior of lipids plays a mediating role. In various foods and pharmaceutical and biotechnical applications, the liquid crystalline phases formed by surfactants in an aqueous medium represent useful host systems for drugs, amino acids, peptides, proteins and vitamins.Various biologically active food additives are soluble in neither aqueous nor oil phase and require environmental protection against hydrolysis or oxidation. Lyotropic liquid crystals meet these requirements mainly due to their high solubilization capacities for hydrophilic, lipophilic and amphiphilic guest molecules. Moreover, recent studies demonstrated controlled and/or sustained release of solubilized molecules from different liquid crystalline matrices.This paper surveys the solubilization of hydrophilic, lipophilic and amphiphilic guest molecules for food applications and illustrates the corresponding structural transformations. Recent developments in liquid crystal characterization methods are discussed.     

Determining the binding and intracellular transporting abilities of a host-[3]rotaxane

The cellular permeability of compounds can be enhanced in the presence of a host-[2]rotaxane (HR). The effective concentration of an HR is limited by the stoichiometry of the complex formation of the HR and the delivered compound. We speculate that a complex forms between the HR and a guest during membrane passage. To further explore the relationship between guest binding and guest delivery and to obtain more efficient delivery devices, we present, in this report, the first example of a cyclophane-[3]rotaxane (Cy3R), which has two wheels and a cyclophane as a blocking group. The properties of Cy3R were compared to a new cyclophane-[2]rotaxane (Cy2R) that has the same cyclophane pocket as Cy3R but only a single wheel. The second wheel of Cy3R can form additional noncovalent bonds, e.g., salt bridges, cation-pi interactions or aromatic-aromatic interactions, with appropriately functionalized guests. We show by flow cytometric analysis that Cy3R transfers Fl-AVWAL (76%) and to a lesser degree Fl-QEAVD (26%) into live cells. The level of Fl-peptide within a cell is concentration dependent and largely temperature and ATP independent, suggesting that a Cy3R.Fl-peptide complex passes through the cellular membrane without requiring active cell-mediated processes. Cy2R, on the other hand, forms weaker complexes and requires a higher concentration to transfer materials into cells. These results demonstrate that the addition of a second wheel on a rotaxane can improve guest binding in various solvents and hence delivery through cellular membranes.     

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.     

The role of glycerol and phosphatidylcholine in solubilizing and enhancing insulin stability in reverse hexagonal mesophases

The potential of reverse hexagonal mesophases based on monoolein (GMO) and glycerol (as cosolvent) to facilitate the solubilization of proteins, such as insulin was explored. H(II) mesophases composed of GMO/decane/water were compared to GMO/decane/glycerol/water and GMO/phosphatidylcholine (PC)/decane/glycerol/water systems. The stability of insulin was tested, applying external physical modifications such as low pH and heat treatment (up to 70°C), in which insulin is known to form ordered amyloid-like aggregates (that are associated with several neurodegenerative diseases) with a characteristic cross β-pleated sheet structure. The impact of insulin confinement within these carriers on its stability, unfolding, and aggregation pathways was studied by combining SAXS, FTIR, and AFM techniques. These techniques provided a better insight into the molecular level of the "component interplay" in solubilizing and stabilizing insulin and its conformational modifications that dictate its final aggregate morphology. PC enlarged the water channels while glycerol shrank them, yet both facilitated insulin solubilization within the channels. The presence of glycerol within the mesophase water channels led to the formation of stronger hydrogen bonds with the hosting medium that enhanced the thermal stability of the protein and remarkably affected the unfolding process even after heat treatment (at 70°C for 60 min).     

Determining the intracellular transport mechanism of a cleft-[2]rotaxane

Rotaxanes are a class of interlocked compounds that have been extensively investigated for their potential utility as switches or sensors. We recently demonstrated that rotaxanes have further application as agents that transport material into cells. This novel finding prompted our investigation into the mechanism by which rotaxanes are involved in transmembrane transport. Two-dimensional NMR analysis showed that a cleft-containing rotaxane exists in two dominant conformations ("closed" and "open"). To determine the importance of conformational flexibility on the ability of the rotaxanes to bind guests and transport material into cells, the rotaxane was chemically modified to lock it in the closed conformation. Charged guests interact less favorably with the locked rotaxane, as compared to the unmodified rotaxane, both in an aqueous solution and in DMSO. In a chloroform solution, both rotaxanes bind the guests with similar affinities. The locked rotaxane exhibited a reduced capacity to transport a fluoresceinated peptide into cells, whereas the unmodified rotaxane efficiently delivers the peptide. Flow cytometry experiments demonstrated that a high percentage of the cells contained the delivered peptide (89-98%), the level of delivery is concentration dependent, and the rotaxanes and peptide have low toxicity. Cellular uptake of the peptide was largely temperature and ATP independent, suggesting that the rotaxane-peptide complex passes through the cellular membrane without requiring active cell-mediated processes. The results show that the sliding motion of the wheel is necessary for the delivery of materials into cells and can enhance the association of guests. These studies demonstrate the potential for rotaxanes as a new class of mechanical devices that deliver a variety of therapeutic agents into targeted cell populations.     

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.     

Investigation of the intracellular delivery of fluoresceinated peptides by a host-[2]rotaxane

The development of methods to transport peptides into cells via a passive mechanism would greatly aid in the development of therapeutic agents. We recently demonstrated that an impermeable fluoresceinated pentapeptide enters the cytoplasm and nucleus of COS 7 cells in the presence of a host-[2]rotaxane by a mechanism that does not depend on an active cell-mediated process. In this report, we further investigate the ability of the host-[2]rotaxane to deliver peptides possessing a wide range of polarities (negatively charged, positively charged, polar, and apolar side chains) into live cells. Only in the presence of the host-[2]rotaxane were the Fl-peptides taken up by COS 7 and ES2 cells. Flow cytometry experiments demonstrated that the level of delivery is largely temperature and adenosine 5'-triphosphate (ATP) independent, and the membranes remain intact. Although the level of transport does depend upon the nature of the side chains, it does not correlate with calculated LogD values, indicating that an additional interaction with the host-[2]rotaxane is modifying the permeability properties of the peptide. The amount of Fl-peptides transported from an aqueous phase into a chloroform phase in the presence of the host-[2]rotaxane correlates with the intensity of cellular fluorescence. Extraction and U-tube studies show that the Fl-peptide can be released from its complex with the host-[2]rotaxane into an aqueous phase, and the host-[2]rotaxane can transport a greater than a stoichiometric amount of an Fl-peptide through a CHCl3 layer. These studies demonstrate the utility of the host-[2]rotaxane in delivering peptides of all polarities across a cell membrane.     

Hexosome and hexagonal phases mediated by hydration and polymeric stabilizer

In this research, we studied the factors that control formation of GMO/tricaprylin/water hexosomes and affect their inner structure. As a stabilizer of the soft particles dispersed in the aqueous phase, we used the hydrophilic nonionic triblock polymer Pluronic 127. We demonstrate how properties of the hexosomes, such as size, structure, and stability, can be tuned by their internal composition, polymer concentration, and processing conditions. The morphology and inner structure of the hexosomes were characterized by small-angle X-ray scattering, cryo-transmission electron microscope, and dynamic light scattering. The physical stability (to creaming, aggregation, and coalescence) of the hexosomes was further examined by the LUMiFuge technique. Two competing processes are presumed to take place during the formation of hexosomes: penetration of water from the continuous phase during dispersion, resulting in enhanced hydration of the head groups, and incorporation of the polymer chains into the hexosome structure while providing a stabilizing surface coating for the dispersed particles. Hydration is an essential stage in lyotropic liquid crystal (LLC) formation. The polymer, on the other hand, dehydrates the lipid heads, thereby introducing disorder into the LLC and reducing the domain size. Yet, a critical minimum polymer concentration is necessary in order to form stable nanosized hexosomes. These competing effects require the attention of those preparing hexosomes. The competition between these two processes can be controlled. At relatively high polymer concentrations (1-1.6 wt % of the total formulation of the soft particles), the hydration process seems to occur more rapidly than polymer adsorption. As a result, smaller and more stable soft particles with high symmetry were formed. On the other hand, when the polymer concentration is fixed at lower levels (<1.0 wt %), the homogenization process encourages only partial polymer adsorption during the dispersion process. This adsorption is insufficient; hence, maximum hydration of the surfactant head group is reached prior to obtaining full adsorption, resulting in the formation of less ordered hexosomes of larger size and lower stability.