Lipid Conformational Order Research Articles

Exploratory in vivo biophysical studies of stratum corneum lipid organization in human face and arm skin

The purpose of this in vivo exploratory study was to investigate human stratum corneum (SC) lipid conformational order and chain packing in healthy face (cheek) skin as a function of stratum corneum depth using a combination of tape-stripping and horizontal attenuated total reflection Fourier transform infrared (HATR-FTIR) spectroscopy. Equivalent data were also collected from volar forearm skin as we, and others, have previously characterized forearm SC lipid order as a function of depth, therefore these data served as a comparison site and an experimental internal standard for the previously unmeasured in vivo face skin data. An SC depth profile was achieved by using tape strips to sequentially remove “layers” of SC. Trans epidermal water loss (TEWL) measurements were recorded following each sequential tape strip. In vivo HATR-FTIR spectra were collected after each tape strip, providing a depth profile of spectral data through the SC of both faces and arms. Spectral data were analyzed at five discrete SC depths corresponding to baseline and SC depths at which the increase in TEWL was 25, 50, 75, and 100 percent (%) from the baseline measurement. Analysis of the SC in vivo HATR-FTIR spectroscopic data utilized mean spectra generated by averaging the spectra from all panelists (n = 10) at the five specific SC depths corresponding to when TEWL had increased by 25, 50, 75 and 100 % from baseline for each subject, respectively. While this is an exploratory study, and the data could be collated and processed in many ways, the average spectral data reveal clear trends in the face and arm SC. Increasing SC lipid order with depth is observed for face skin, as with arm skin, albeit with significant differences at all relative depths in the absolute lipid order between faces and arms. In vivo SC lipids are significantly more ordered at all depths in arms versus cheeks. The less ordered SC lipids of face skin can be in part attributed to the presence of a high amount of fluid sebaceous lipid species observed deep into the face SC. Interestingly, no evidence of orthorhombic packing is observed at any depth in face SC. This is consistent with the high degree of disorder indicated from the chain fluidity measurements. These in vivo HATR-FTIR depth studies of face skin highlight how different SC barrier organization is in the face compared to the more widely studied body sites such as arms and legs. Further studies of the SC lipid barrier in faces are needed to understand the significant differences in lipid organization and its implication for the penetration of drug and cosmetic actives through face skin.

Bipolar Imidazolium-Based Lipid Analogues for Artificial Archaeosomes.

Archaeal lipids have harvested biomedical and biotechnological interest because of their ability to form membranes with low permeability and enhanced temperature and pressure stability. Because of problems in isolating archaeal lipids, chemical synthesis appears to be a suitable means of producing model lipids that mimic the biological counterparts. Here, we introduce a new concept: we synthesized bipolar alkylated imidazolium salts of different chain lengths (BIm10-32) and studied their structure and lyotropic phase behavior. Furthermore, mixtures of the bolalipid analogues with phospholipid model biomembranes of diverse complexity were studied. DSC, fluorescence and FTIR spectroscopy, confocal fluorescence microscopy, DLS, SAXS, and TEM were used to reveal changes in lipid phase behavior, fluidity, the lipid's conformational order, and membrane morphology over a wide range of temperatures and for selected pressures. It could be shown that the long-chain BImN32 can form monolayer sheets. Integrated in phospholipid membranes, it reveals a fluidizing effect. Here, the two polar head groups, connected by a long alkyl chain, enable the integration into the bilayer. Interestingly, addition of BImN32 to fluid DPPC liposomes increased the lipid packing markedly, rendering the membrane system more stable at higher temperatures. The membrane system is also stable against compression as indicated by the high-pressure stability of the system, mimicking an archaeal lipid-like behavior. BImN32 incorporation into raft-like anionic model biomembranes led to marked changes in lateral membrane organization, topology, and fusogenicity of the membrane. Overall, it was found that long-chain imidazolium-based bolalipid analogues can help adjust membrane's biophysical properties, while the imidazolium headgroup provides the ability for crucial electrostatic interaction for vesicle fusion or selective interaction with membrane-related signaling molecules and polypeptides in a synthetically tractable manner. The results obtained may help to develop new approaches for rational design of extremophilic bolalipid-based liposomes for various applications, including delivery of drugs and vaccines.

Measurement of the biomechanical function and structure of ex vivo drying skin using raman spectral analysis and its modulation with emollient mixtures.

An important aspect of the biomechanical behaviour of the stratum corneum (SC) is the drying stresses that develop with water loss. These stresses act as a driving force for damage in the form of chapping and cracking. Betasitosterol is a plant sterol with a structure similar to cholesterol, a key component in the intercellular lipids of the outermost layer of human skin, the SC. Cholesterol plays an important role in stabilizing the SC lipid structure, and altered levels of cholesterol have been linked with SC barrier abnormalities. Betasitosterol is currently applied topically to skin for treatment of wounds and burns. However, it is unknown what effect betasitosterol has on the biomechanical barrier function of skin. Here, by analysing the drying stress profile of SC generated during a kinetics of dehydration, we show that betasitosterol, in combination with two emollient molecules, isocetyl stearoyl stearate (ISS) and glyceryl tri-2-ethylhexanoate (GTEH), causes a significant modulation of the drying stress behaviour of the SC by reducing both the maximal peak stress height and average plateau of the drying stress profile. Raman spectra analyses demonstrate that the combination of betasitosterol with the two emollients, ISS and GTEH, allows a high water retention capacity within the SC, while the lipid conformational order by increasing the amount of trans conformers. Our study highlights the advantage of combining a biomechanical approach together with Raman spectroscopy in engineering a suitable combination of molecules for alleviating dryness and dry skin damage.

Effect of mixed surfactants on stratum corneum: a drying stress and Raman spectroscopy study.

Stratum corneum (SC) lipids are known to play an important role in barrier properties of skin by maintaining the optimal hydration levels. The disruption of SC lipids by cleanser surfactants is believed to lead to dry skin damage which can be a precursor to other skin disorders. The purpose of this study is to investigate the effects of commonly used anionic and zwitterionic surfactants sodium lauryl ether sulphate (SLES) and cocoamidopropyl betaine (CAPB) on the generation of drying stresses in SC and the role played by lipids. Stratum corneum separated from pig skin was treated with various surfactants (SDS, SLES and CAPB) their mixtures and solvents. The tensile response to these treatments was measured by using a dynamic mechanical thermal analyzer. A Raman spectroscopy study of the treated samples was performed to investigate the effects of lipid modification (lipid chain conformational order and lipid removal) on stress generation in SC. The effects of commonly used anionic and zwitterionic surfactants on the generation of drying stresses in SC were studied. Although known to be milder in comparison with SDS, both SLES and CAPB generated high drying stresses individually. In mixtures, SLES-CAPB at 4:1 ratio leads to lower drying stress as compared to water alone. A Raman spectroscopic study of surfactant-treated SC shows changes in lipid chain conformational order as well as a decrease in lipid-protein ratio in SC. A chloroform-methanol 2:1 treatment leads to the highest drying stress as well delipidization of SC. The results show a correlation between generation of drying stress in SC and extent of lipid modification. We propose that the changes in lipid conformational order and removal of lipid components affect the stress relaxation properties of SC leading to high drying stresses.

Oleic Acid Disorders Stratum Corneum Lipids in Langmuir Monolayers

Oleic acid (OA) is well-known to affect the function of the skin barrier. In this study, the molecular interactions between OA and model stratum corneum (SC) lipids consisting of ceramide, cholesterol, and palmitic acid (PA) were investigated with Langmuir monolayer and associated techniques. Mixtures with different OA/SC lipid compositions were spread at the air/water interface, and the phase behavior was tracked with surface pressure-molecular area (π-A) isotherms. With increasing OA levels in the monolayer, the films became more fluid and more compressible. The thermodynamic parameters derived from π-A isotherms indicated that there are preferential interactions between OA and SC lipids and that films of their mixtures were thermodynamically stable. The domain structure and lipid conformational order of the monolayers were studied through Brewster angle microscopy (BAM) and infrared reflection absorption spectroscopy (IRRAS), respectively. Results indicate that lower concentrations of OA preferentially mix with and disorder the ceramide-enriched domains, followed by perturbation of the PA-enriched domains and disruption of SC lipid domain separation at higher OA levels.

In Vivo Barrier Challenge and Initial Recovery in Human Facial Skin

Many in vivo biophysical studies focus on parts of the body which are generally less exposed to the environment compared with facial skin. This study characterizes in vivo changes in the barrier function of cheek stratum corneum (SC) during controlled conditions of damage. The objective was to investigate structural and compositional changes in cheek SC in vivo using tape stripping and Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. In vivo ATR-FTIR spectra of the cheek were collected before and after tape stripping. Trans epidermal water loss (TEWL) measurements from the same site were conducted before and after stripping. Data were also collected 24 h after the last tape strip to investigate any initial recovery process. Sequential tape stripping of the cheek progressively increases TEWL to twice baseline values but some barrier recovery is evident 24 h later. ATR-FTIR spectra show increasing water content within the SC with each tape strip with concomitant increase in lipid conformational order. Several structural and compositional parameters have been analyzed for cheek SC and these have been correlated with barrier function. The depth dependent changes in face SC map those in body skin but over a much shorter distance.

Synthesis and properties of monofluorinated dimyristoylphosphatidylcholine derivatives: Potential fluorinated probes for the study of membrane topology

The synthesis of three monofluorinated dimyristoylphosphatidylcholine derivatives (F-DMPC), with the fluorine atom located on the acyl chain in position 2 of the glycerol (sn-2) is described. The synthetic strategy relies on the coupling of 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC) and three different fluorinated fatty acids. The latter were obtained from two different and complementary synthetic routes. Preliminary FTIR studies suggest that the presence of the fluorine atom does not significantly perturb the lipid conformational order and phase transition temperature and that these monofluorinated PC derivatives could be used as probes for the study of membrane topology, i.e. the location of drugs, peptides or proteins in membranes.

Role of Glycolipids in Lipid Rafts: A View through Atomistic Molecular Dynamics Simulations with Galactosylceramide

Even in small amounts, glycolipids are an integral part of lipid rafts and their cellular functions. Here we employ atomistic molecular dynamics simulations to consider galactosylceramide (GalCer), one of the common glycosphingolipids, and investigate its interactions with other raft components (cholesterol, POPC, sphingomyelin) as well as the role and the effects of GalCer on the physical properties of raft-like membranes. Our results for 5 mol % GalCer indicate that whereas the thickness of raft membranes is clearly increased by the addition of GalCer, the average area per lipid and lipid conformational order remain virtually unchanged. Notable changes are observed in lateral diffusion of the raft lipids. This is found to be associated with the interdigitation of GalCer. With cholesterol, GalCer is observed to interact specifically by shielding it from the water phase.

Stability of annexin V in ternary complexes with Ca2+ and anionic phospholipids: IR studies of monolayer and bulk phases.

Annexin V (AxV) is a member of a family of proteins that exhibit functionally relevant Ca2+-dependent binding to anionic phospholipid membranes. Protein structure and stability as a function of Ca2+ and phospholipids was studied by bulk phase infrared (IR) spectroscopy and by IR reflection-absorption spectroscopy (IRRAS) of monolayers in situ at the air/water (A/W) interface. Bulk phase experiments revealed that AxV undergoes an irreversible thermal denaturation at approximately 45-50 degreesC, as shown by the appearance of amide I bands at 1617 and 1682 cm-1. However, some native secondary structure is retained, even at 60 degreesC, consistent with a partially unfolded "molten globule" state. Formation of the Ca2+/phospholipid/protein ternary complex significantly protects the protein from thermal denaturation as compared to AxV alone, Ca2+/AxV, or lipid/AxV mixtures. Stabilization of AxV secondary structure by a DMPA monolayer in the presence of Ca2+ was also observed by IRRAS. Spectra of an adsorbed AxV film in the presence or absence of Ca2+ showed a 10 cm-1 shift in the amide I mode, corresponding to loss of ordered structure at the A/W interface. In both the bulk phase and IRRAS experiments, protection against H-->D exchange in AxV was enhanced only in the ternary complex. The combined data suggest that the secondary structure of AxV is strongly affected by the Ca2+/membrane component of the ternary complex whereas lipid conformational order is unchanged by protein.

Coupled External Reflectance FT-IR/Miniaturized Surface Film Apparatus for Biophysical Studies

An FT-IR spectrophotometer has been interfaced to a miniaturized surface film apparatus for external reflection studies of insoluble monolayers in situ at the air/water interface. Signal-to-noise ratios of 200:1 were routinely achieved for the CH2 stretching vibrations of phospholipids. We have monitored, using the acyl chain symmetric CH2 stretching frequency near 2850 cm−1 as a structural probe, lipid conformational order changes that occur during the surface pressure-induced two-dimensional phase transition in monolayers of 1,2-dipalmitoylphosphatidylserine. In addition, the small volume of the miniaturized film apparatus (30 mL) permitted replacement of H2O with D2O in the subphase. This capability, in turn, permits the acquisition of spectral data in the amide I region of proteins. We report the first external reflection FT-IR spectrum of an insoluble protein monolayer. The protein studied is pulmonary surfactant SP-C.