M. A. Kiselev

Properties of Ceramides and Their Impact on the Stratum Corneum Structure

The stratum corneum (SC) represents the outermost layer of the mammalian skin, exhibits the main skin barrier and plays an important role in the water penetration pathway through the SC. Knowing the structure and properties of the SC at the molecular level is essential for studying drug penetration through the SC and for the development of new dermal drug delivery systems. Therefore, research interest is focused on the SC lipid matrix and on water diffusion through it. Thus, the ultimate aim is to design a lipid mixture that mimics the barrier properties of the human SC to a high extent and that can substitute the SC in drug delivery systems. This review summarizes various studies performed on either isolated animal or human ceramide based SC model systems, coming to the result that using synthetic lipids with a well-defined architecture allows good extrapolation to the in vivo situation. This review is the continuation of part 1 that is focused on a detailed description of the thermotropic and/or lyotropic phase behaviour of single ceramide types obtained by various experimental techniques. The objective of part 2 is to reflect the numerous studies on SC lipid model systems, namely binary, ternary and multicomponent systems, during the last decade. In this context, neutron diffraction as a prospective tool for analyzing the internal membrane structure is addressed in particular. Based on these new insights, current SC models are presented, whose validations are still under discussion. A profound knowledge about SC lipid organization at the molecular level is still missing.

Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-structured omega-acylceramide [EOS] in a near natural ratio with neutron diffraction. In models proposed recently the presence of ceramide [EOS] and FFA are necessary for the formation of one of the two existent crystalline lamellar phases of the SC lipids, the long-periodicity phase as well as for the normal barrier function of the SC. The focus of this study was placed on the influence of the FFA BA on the membrane structure and its localization within the membrane based on the ceramides [EOS] and [AP]. The internal nanostructure of such membranes was obtained by Fourier synthesis from the experimental diffraction patterns. The resulting neutron scattering length density profiles showed that the exceptionally long ceramide [EOS] is arranged in a short-periodicity phase created by ceramide [AP] by spanning through the whole bilayer and extending even further into the adjacent bilayer. Specifically deuterated BA allowed us to determine the exact position of this FFA inside this SC lipid model membrane. Furthermore, hydration experiments showed that the presented SC mimic system shows an extremely small intermembrane hydration of approximately 1 A, consequently the headgroups of the neighboring leaflets are positioned close to each other.

Amphiphiles Self-Assembly: Basic Concepts and Future Perspectives of Supramolecular Approaches

Amphiphiles are synthetic or natural molecules with the ability to self-assemble into a wide variety of structures including micelles, vesicles, nanotubes, nanofibers, and lamellae. Self-assembly processes of amphiphiles have been widely used to mimic biological systems, such as assembly of lipids and proteins, while their integrated actions allow the performance of highly specific cellular functions which has paved a way for bottom-up bionanotechnology. While amphiphiles self-assembly has attracted considerable attention for decades due to their extensive applications in material science, drug and gene delivery, recent developments in nanoscience stimulated the combination of the simple approaches of amphiphile assembly with the advanced concept of supramolecular self-assembly for the development of more complex, hierarchical nanostructures. Introduction of stimulus responsive supramolecular amphiphile assembly-disassembly processes provides particularly novel approaches for impacting bionanotechnology applications. Leading examples of these novel self-assembly processes can be found, in fact, in biosystems where assemblies of different amphiphilic macrocomponents and their integrated actions allow the performance of highly specific biological functions. In this perspective, we summarize in this tutorial review the basic concept and recent research on self-assembly of traditional amphiphilic molecules (such as surfactants, amphiphile-like polymers, or lipids) and more recent concepts of supramolecular amphiphiles assembly which have become increasingly important in emerging nanotechnology.

Evidence of free fatty acid interdigitation in stratum corneum model membranes based on ceramide [AP] by deuterium labelling.

This research paper provides direct evidence concerning the localisation of free fatty acids in stratum corneum lipid model membranes. We employed partially deuterated free fatty acids to gain further information about the assembly of a stratum corneum lipid model membrane based on a ceramide of the phytosphingosine-type (ceramide [AP]) with particular respect to the position of the deuterated groups of the free fatty acids. The application of behenic-22,22,22-d(3)-acid and cerotic-12,12,13,13-d(4)-acid confirmed that the short-chain ceramide [AP] forces the longer-chained free fatty acids to incorporate into the bilayer created by ceramide [AP]. The ceramide [AP] molecules determine the structural assembly of this model membrane and obligate the long-chain free fatty acids to either arrange inside this formation or to separate as a fatty acid rich phase.

Does sucrose influence the properties of DMPC vesicles

Small-angle neutron and X-ray scattering, dynamic light scattering, X-ray diffraction coupled with differential scanning calorimetry, and Raman spectroscopy were applied to investigate unilamellar (ULVs) and multilamellar (MLVs) dimyristoylphosphatidylcholine (DMPC) vesicles in aqueous sucrose solutions with sucrose concentrations from 0 to 60% w/w. In case of ULVs, the addition of sucrose decreases the polydispersity of vesicle population. A minimum value of polydispersity was found at 20% sucrose. For sucrose concentration from 0 to 35% oligolamellar vesicles in the ULV population have a minimum presence. Vesicles with 5-10% sucrose exhibit the best stability in time. For the case of MLVs, sucrose influences the temperature of the phase transitions, but the internal membrane structure remains unchanged.

Conformation of Ceramide 6 Molecules and Chain-Flip Transitions in the Lipid Matrix of the Outermost Layer of Mammalian Skin, the Stratum Corneum

Neutron diffraction from oriented multilamellar model stratum corneum (SC) membranes provides information on the internal nanostructure and hydration of the lipid bilayer. The main distinguishing feature of model SC membranes based on ceramide 6 is the extremely small intermembrane space (1 Å). The role of the fully extended (FE) conformation of ceramide 6 molecules in the organization of the nanostructure of the lipid matrix is discussed. The FE conformation gives rise to extremely strong intermembrane attractions (armature reinforcement), which tighten the adjacent bilayers to form steric contacts. Chain-flip transitions in the conformation of ceramide molecules account for structural alterations in native and model SC membranes upon their hydration.

Localisation of partially deuterated cholesterol in quaternary SC lipid model membranes: a neutron diffraction study

This letter presents our first results in using the benefit of selective deuteration in neutron diffraction studies on stratum corneum (SC) lipid model systems. The SC represents the outermost layer of the mammalian skin and exhibits the main skin barrier. It is essential for studying drug penetration through the SC to know the internal structure and hydration behaviour on the molecular level. The SC intercellular matrix is mainly formed by ceramides (CER), cholesterol (CHOL) and long- chain free fatty acids (FFA). Among them, CHOL is the most abundant individual lipid, but a detailed knowledge about its localisation in the SC lipid matrix is still lacking. The structure of the quaternary SC lipid model membranes composed of either CER[AP]/CHOL-D6/palmitic acid (PA)/cholesterol sulphate (ChS) or CER[AP]/CHOL-D7/PA/ChS is characterized by neutron diffraction. Neutron diffraction patterns from the oriented samples are collected at the V1 diffractometer of the Hahn-Meitner-Institute, Berlin, measured at 32°C, 60% humidity and at different D2O contents. The neutron scattering length density profile in the direction normal to the surface is restored by Fourier synthesis from the experimental diffraction patterns. The analysis of scattering length density profile is a suitable tool for investigating the internal structure of the SC lipid model membranes. The major finding is the experimental proof of the CHOL localisation in SC model membrane by deuterium labelling at prominent positions in the CHOL molecules.

Definition of lipid membrane structural parameters from neutronographic experiments with the help of the strip function model

Neutron diffraction is an effective method for investigating model and biological membranes. Yet, to obtain accurate structural information it is necessary to use deuterium labels and much time is needed to acquire experimental data as there are a large number of diffraction reflections to register. This paper offers a way to define the hydrophobic boundary position in lipid membranes with high accuracy and for this purpose it is sufficient to take into consideration three structural factors. The method is based on modeling the density of the neutron diffraction amplitude rho(x) in the direction of the bilayer plane normal by means of a strip function, but it also takes into consideration the fact that the multiplication of the strip function amplitude rho i by the step width zi-zi-1 makes the sum of neutron scattering amplitudes of the atoms included in the step region. On the basis of the analysis of a large number of experimental data for different membranes, the effectiveness of this method in the determination of the position of hydrophilic/hydrophobic boundary is demonstrated, including the case of various rho(x) modifications in the region of polar heads and also the different phase states of membranes. However, it is shown in the present paper that the strip function model is not an adequate instrument for the determination of other structural parameters of membranes.

A detailed analysis of biodegradable nanospheres by different techniques—A combined approach to detect particle sizes and size distributions

Poly(d,l-lactide-co-glycolide) nanosupensions as intravenous nanosphere systems were produced by solvent deposition in aqueous Poloxamer 188 solutions. Light scattering techniques were applied to these colloidal systems to characterize particle sizes. Regularly shaped spherical particles were received as proved by freeze fracture replica and small-angle X-ray scattering (SAXS). SAXS was performed using intensive synchrotron radiation. Particle sizes were calculated from the small-angle part of scattering curve that were in good agreement with z-average values received from photon correlation spectroscopy (PCS). The flow field-flow fractionation (FlFFF) fractograms in combination with multi-angle light scattering (MALS) allowed an easy detection of maximum particle sizes what is most important for parenteral systems. Furthermore, high quality size distributions were received due to the separation step prior to size characterization. The calculated average size values exhibited a good correlation with z-averages determined by PCS. Only for suspensions of broader size distributions, higher deviations were observed. Comparing particle sizes with and without Poloxamer, differences in diameters resulted that were quantified. The additional Poloxamer shell was not able to be removed by an intensive washing during FlFFF focusing and separation. Especially FlFFF/MALS proved to be a valuable tool to characterize the pharmaceutical nanosuspensions in detail what is of great importance especially for controlled drug delivery systems.

Effect of anionic and cationic polyamidoamine (PAMAM) dendrimers on a model lipid membrane

In spite of the growing variety of biological applications of dendrimer-based nanocarriers, a major problem of their potential applications in bio-medicine is related to the disruption of lipid bilayers and the cytotoxicity caused by the aggregation processes involved onto cellular membranes. With the aim to study model dendrimer-biomembrane interaction, the self-assembly processes of a mixture of charged polyamidoamine (PAMAM) dendrimers and dipalmitoylphosphatidylcholine (DPPC) lipids were investigated by means of Zeta potential analysis, Raman and x-ray scattering. Zwitterionic DPPC liposomes showed substantially different behaviors during their interaction with negatively charged (generation G=2.5) sodium carboxylate terminated (COO- Na+) dendrimers or positively charged (generation G=3.0) amino terminated (-NH2) dendrimers. More specifically the obtained results evidence the sensitive interactions between dendrimer terminals and lipid molecules at the surface of the liposome, with an enhancement of the liposome surface zeta potential, as well as in the hydrophobic region of the bilayers, where dendrimer penetration produce a perturbation of the hydrophobic alkyl chains of the bilayers. Analysis of the SAXS structure factor with a suitable model for the inter-dendrimers electrostatic potential allows an estimation of an effective charge of 15 ǀeǀ for G=2.5 and 7.6 ǀeǀ for G=3.0 PAMAM dendrimers. Only a fraction (about 1/7) of this charge contributes to the linear increase of liposome zeta-potential with increasing PAMAM/DPPC molar fraction. The findings of our investigation may be applied to rationalize the effect of the nanoparticles electrostatic interaction in solution environments for the design of new drug carriers combining dendrimeric and liposomal technology.