Temperature effect on the bilayer stacking in multilamellar lipid vesicles.

The small-angle region of the Bragg diffraction of MLV samples is a simple and powerful tool for the study of mesoscopic lipid structures either alone or in interaction with molecules of biological interest. It is also a helpful tool to obtain the much needed thermotropic phase diagrams of lipid mixtures. In the course of our work, we found that the analysis of the diffractograms obtained as a function of temperature is not as straightforward as we expected. When the aqueous medium is concentrated in univalent salts, the small-angle X-ray scattering, SAXS, develops several peaks that have been interpreted to result from regions with different thickness of the interbilayer region. However, a systematic study shows that nonzero ionic strength is by no means a necessary criterion for irregularity of bilayer stacking. We show that MLV in water are uniform and stable if made, kept, and measured at the same temperature. If not, the lamellar repeat distance is smaller than the equilibrated one, eventually developing transient multimodal SAXS diffractions. We present a detailed study of this phenomenon using SAXS and dynamic light scattering and conclude that the deviations from the equilibrium interbilayer distance is a consequence of geometric constraints created by the insufficient thermal expansion of the lipid bilayers.

Study of the miscibility of cholesteryl oleate in a matrix of ceramide, cholesterol and fatty acid.

Cholesteryl esters (CE) are not generally abundant but are ubiquitous in living organisms and have markedly different properties from cholesterol because of their acyl chain. The miscibility/immiscibility of CE with biological lipid structures is a key property for their functions. In this work we study the solubility of cholesteryl oleate (ChO) in a model of the stratum corneum lipid matrix composed of ceramide C16, cholesterol and palmitic acid in excess water. Experiments were done in conditions of fully ionized (pH=9.0) and fully neutralized fatty acid (pH=4.0), and differential scanning calorimetry of the ternary mixtures with added ChO at pH=9.0 clearly displayed a main transition with the same maximum temperature, peak shape, and enthalpy, suggesting that ChO was excluded from the remaining lipids. This technique is not conclusive at pH=4.0 because the transitions of the lipid matrix and ChO overlap. The insolubility of ChO at both pH values is supported by X-ray diffraction. Adding the ceramide:cholesterol:fatty acid lipid mixture to ChO did not change the X-ray pattern of the mixture nor that of the ChO. To supplement the above physical techniques, we applied (13)C MAS NMR spectroscopy with C-13 carbonyl-labeled ChO. A single (13)C carbonyl peak from the ChO at 171.5 ppm was observed, indicating exposure to only one environment. The chemical shift was identical to pure ChO below and above the temperature of isotropic liquid formation. Taken together, our results lead to the conclusion that the solubility of ChO is negligible in the ceramide:cholesterol:fatty acid lipid mixture.

The thermotropism and prototropism of ternary mixtures of ceramide C16, cholesterol and palmitic acid. An exploratory study.

Mixtures of ceramides with other lipids in the presence of water are key components of the structure of the lipid matrix of the stratum corneum and are involved in lateral phase separation processes occurring in lipid membranes. Besides their structural role, ceramides are functional for cell signaling and trafficking. We elected, as our object of study, a mixture of N-hexadecanoylceroyl-d-erythro-sphyngosine, C16-Cer, with cholesterol, Ch, in a molar proportion 54:46 in excess water to which palmitic acid, PA, is added in varying amounts. The chosen C16-Cer:Ch proportion replicates the relative abundance of ceramides and cholesterol found in the stratum corneum lipid matrix. For each lipidic composition, we identify the phases in equilibrium and study the thermotropism of the system, using differential scanning calorimetry and temperature-dependent small and wide-angle X-ray powder diffraction. Since the molecular aggregation of the system and its mesoscopic properties are affected by the degree of protonation of the PA, we explore mixtures with several PA contents at two extreme pH values, 9.0 and 4.0. A specific C16-Cer:Ch:PA composition forms at pH 9.0 a lamellar crystalline aggregate, to which we attribute the stoichiometry C16-Cer(5)Ch(4)PA(2), that melts at 88-90 °C to give a H(II) phase. For pH values at which there is partial or total protonation of PA another L(C) C16-Cer:Ch (2:3) stoichiometric aggregate is observed, identical to that previously reported for C16-Cer:Ch mixtures (Souza et al., 2009, J. Phys. Chem. B, 113, 1367-1375), coexisting with a lamellar fluid phase. For pH 4.0 and 7.0, the existing lamellar liquid crystalline converts into a isotropic fluid phase at high temperatures. It is also found that the miscibility of PA in the C16-Cer:Ch mixture at pH 4.0 does not exceed ca. 18 mol%, but for pH 9.0 no free PA is detected at least until 60 mol%.

Phase behavior of aqueous dispersions of mixtures of N-palmitoyl ceramide and cholesterol: a lipid system with ceramide-cholesterol crystalline lamellar phases.

Ceramides are particularly abundant in the stratum corneum lipid matrix, where they determine its unusual mesostructure, are involved in the lateral segregation of lipid domains in biological cell membranes, and are also known to act as signaling agents in cells. The importance attributed to ceramides in several biological processes has heightened in recent years, demanding a better understanding of their interaction with other membrane components, namely, cholesterol. Structural data concerning pure ceramides in water are relatively scarce, and this is even more the case for mixtures of ceramides with other lipids commonly associated with them in biological systems. We have derived the thermotropic binary phase diagram of mixtures of N-palmitoyl- D-erythro-sphingosine, C16:0-ceramide, and cholesterol in excess water, using differential scanning calorimetry and small- and wide-angle X-ray diffraction. These mixtures are self-organized in lamellar mesostructures that, between other particularities, show two ceramide to cholesterol crystalline phases with molar proportions that approach 2:3 and 1:3. The 2:3 phase crystallizes in a tetragonal arrangement with a lamellar repeat distance of 3.50 nm, which indicates an unusual lipid stacking, probably unilamellar. The uncommon mesostructures formed by ceramides with cholesterol should be considered in the rationalization of their singular structural role in biological systems.

On the self-aggregation and fluorescence quenching aptitude of surfactant ionic liquids.

The aggregation behavior in aqueous solution of a number of ionic liquids was investigated at ambient conditions by using three techniques: fluorescence, interfacial tension, and (1)H NMR spectroscopy. For the first time, the fluorescence quenching effect has been used for the determination of critical micelle concentrations. This study focuses on the following ionic liquids: [Cnmpy]Cl (1-alkyl-3-methylpyridinium chlorides) with different linear alkyl chain lengths (n=4, 10, 12, 14, 16, or 18), [C12mpip]Br (1-dodecyl-1-methylpiperidinium bromide), [C12mpy]Br (1-dodecyl-3-methylpyridinium bromide), and [C12mpyrr]Br (1-dodecyl-1-methylpyrrolidinium bromide). Both the influence of the alkyl side-chain length and the type of ring in the cation (head) on the CMC were investigated. A comparison of the self-aggregation behavior of ionic liquids based on 1-alkyl-3-methylpyridinium and 1-alkyl-3-methylpyridinium cations is provided. It was observed that 1-alkyl-3-methylpyridinium ionic liquids could be used as quenchers for some fluorescence probes (fluorophores). As a consequence, a simple and convenient method to probe early evidence of aggregate formation was established.

Encapsulation of adenoviral vectors into chitosan-bile salt microparticles for mucosal vaccination.

The objective of this study is the incorporation of adenoviral vectors into a microparticulate system adequate for mucosal delivery. Microencapsulation of the vectors was accomplished by ionotropic coacervation of chitosan, using bile salts as counter-anion. The process was optimized in order to promote high encapsulation efficiency, with a minimal loss of viral infectivity. The maintenance of sterility during all the encapsulation procedure was also taken into account. The principle relies on the simple addition of a solution containing adenoviral vectors to a solution of neutralized chitosan, under stirring. Some surfactants were added to the chitosan solution, to improve the efficiency of this process, such as Tween 80, and Pluronic F68 at 1% (w/v). Encapsulation efficiency higher than 84% was achieved with formulations containing sodium deoxycholate as counter-anion and Pluronic F68 as dispersant agent. The infectivity of the adenoviral vectors incorporated into microparticles was assessed by release assays in PBS and by direct inoculation in 293 and Caco-2 cells. The release in aqueous media was negligible but, when in contact with monolayers of the cells, an effective release of bioactive adenovirus was obtained. Our work shows that encapsulation in microparticles, not only appear to protect the adenovirus from the external medium, namely from low pH, but can also delay their release that is fully dependent on cell contact, an advantage for mucosal vaccination purposes. The formulations developed are able to maintain AdV infectivity and permit a delayed release of the bioactives that is promoted by digestion in situ of the microparticles by the cell monolayers. The onset of delivery is, that way, host-controlled. In view of these results, these formulations showed good properties for mucosal adenovirus delivery.

Kinetics of bimolecular reactions in model bilayers and biological membranes. A critical review.

The quantitative study of the probability of molecular encounters giving rise to a reaction in membranes is a challenging discipline. Model systems, model in the sense that they use model bilayers and model reactants, have been widely used for this purpose, but the methodologies employed for the analysis of the results obtained in experiments, and for experimental design, are so disparate that a concerned experimentalist has difficulty in deciding about the value of each approach. This review intends to examine the several approaches that can be found in the literature showing, when feasible, the weakness, strengths and limits of application of each of them. There is not, so far, a full experimental validation of the most promising theories for the analysis of reactions in two dimensions, what leaves open a large field for new research. The major challenge resides in the time range in which the processes take place, but the possibilities of the existing techniques for these studies are far from exhausted. We review also the attempts of several authors to quantitatively analyze the kinetics of reactions in biological membranes. Especially in this field, the recently developed microspectroscopies enclose a still unexplored potential.

Incorporation of a model protein into chitosan-bile salt microparticles.

In order to develop a mucosal delivery system based on biocompatible polymers, a new methodology for production of protein-loaded microparticles is developed. Chitosan anionic precipitation/coacervation is accomplished by the addition of sodium deoxycholate (DCA). These microparticles were prepared under mild conditions, where bovine serum albumin (BSA) and DCA were simply dipped into a chitosan solution under stirring. Platelet-like and/or spherical microparticles, having high protein loading efficiency and relatively low protein external exposure, are obtained. To achieve a better compaction of the microparticle matrix, block copolymers and other non-ionic surfactants are added to the formulation. BCA analysis and fluorescence quenching were used to assess the degree of protein exposure. BSA release profiles for chitosan-DCA formulations in PBS pH 7.4 and HCl 0.1 N revealed, in most cases, an initial burst release, but more than 55% of the BSA remains protected inside the microparticles. It is also observed that in acidic environment (HCl 0.1 N) the protein is better shielded from the environment. Some of the formulations show good properties for mucosal protein delivery, and one of those here developed is now being tested in vivo, for mucosal administration of an adenovirus vaccine.

Reappraisal of four different approaches for finding the mean reaction time in the multi-trap variant of the Adam-Delbrück problem.

Adam and Delbrück argued that the dimensionality of the diffusion space determines the average lifetime of a diffusing particle confined to a region with a central trap. Doubts have often been aired as to whether their calculation is relevant to real biological systems, where the number of traps is usually much larger than unity; or whether the rate enhancement is merely a manifestation of an increase in the concentration of the traps; or whether the diverse multi-trap versions of their expression for the mean lifetime in two dimensions are trustworthy. These issues are addressed, and the long-standing problem of finding the low-density limit of trapping time in two dimensions solved, by examining previous treatments of the problem, and by carrying out simulations of two-dimensional systems in which the particles undergo a Pearsonian random walk, and the traps are distributed randomly or on a square lattice. The mean lifetimes are found to be different in the two situations, and it is concluded that the neglect of this aspect lies at the root of the conflict between some of the existing expressions for the mean lifetime. Relations expressing the mean lifetime as a function of the concentration of the traps are presented together with a discussion of their applicability.

Partition of fish pheromones between water and aggregates of humic acids. Consequences for sexual signaling.

High-molecular-weight suspended organic matter of soil and aquatic origins competes with water for the dissolution of relatively water insoluble organic substances. The same happens with microalgae and other organisms present in natural waters. Several pheromones, which play a specific role in the reproductive cycle of fish, are secreted to the water and are generally, if not always, molecules with hydrophobic or amphiphilic characteristics. The natural tendency of these pheromones to dissolve in suspended or deposited organic matter may cause their signaling function to be adversely affected. In this work we study the partition constants between water and organic reservoirs in suspension, Kh, of two fish pheromones, 4-pregnene-17 alpha, 20beta-diol-3-one (17,20beta-P) and prostaglandin F2alpha (PGF2alpha), and also of 4-pregnene-11beta,21-diol-3,20-dione 21-sulfate (21-P-sulfate), used as a proxy for sulfated steroid pheromones. Two types of organic reservoirs are employed: aggregates of suspended humic substances and negatively charged phospholipid vesicles. We find that the three compounds have high affinities for both types of aggregates. However, 17,20beta-P, with pKh = 4.4 +/- 0.2, is the only one for which we may predict a significant decrease in availability in consideration of the normal content of dissolved organic matter in natural waters. Also to be considered is the fact that a relatively large amount of pheromones is retained and may be released at an inopportune moment. How significant these phenomena are in nature is not as yet clear, considering the variety of habitats in which fish spawn and the little that is known about the nature and mode of action of pheromones.

Solubility of amphiphiles in membranes: influence of phase properties and amphiphile head group.

The solubilities of two fluorescent lipid amphiphiles with comparable apolar structures and different polar head groups, NBD-hexadecylamine and RG-tetradecylamine (or -octadecylamine), were compared in lipid bilayers at a molar ratio of 1/50 at 23 degrees C. Bilayers examined were in the solid, liquid-disordered, or liquid-ordered phases. While NBD-hexadecylamine was soluble in all the examined bilayer membrane phases, RG-tetradecylamine was stably soluble only in the liquid-disordered phase. RG-tetradecylamine insolubility in solid and liquid-ordered phases manifests itself as an aggregation of the amphiphile over a period of several days and the kinetics of aggregation were studied. Solubility of these amphiphiles in the different phases examined seems to be related to the dipole moment of the amphiphile (in particular, of the polar fluorophore) and its orientation relative to the dipolar potential of the membrane. We propose that amphiphilic molecules inserted into membranes (including lipid-attached proteins) partition into different coexisting membrane phases based upon: (1) nature of the apolar structure (chain length, degree of saturation, and chain branching as has been proposed in the literature); (2) magnitude and orientation of the dipole moment of the polar portion of the molecules relative to the membrane dipolar potential; and (3) hydration forces that are a consequence of ordering of water dipoles at the membrane surface.