Article pubs.acs.org/Langmuir
Oleic Acid Disorders Stratum Corneum Lipids in Langmuir Monolayers Guangru Mao,*,† Dina VanWyck,† Xin Xiao,‡ M. Catherine Mack Correa,† Euen Gunn,† Carol R. Flach,‡ Richard Mendelsohn,‡ and Russel M. Walters† †
Johnson and Johnson Consumer Companies, Incorporated, 199 Grandview Road, Skillman, New Jersey 08558, United States Department of Chemistry, Rutgers University, 73 Warren Street, Newark, New Jersey 07102, United States
‡
ABSTRACT: 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.
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ment effect was demonstrated with in vitro,20 mouse model,21 and in vivo measurements.22 In the SC, OA predominantly interacts with lipids because it was observed to lower the SC lipid melting temperature but did not affect the keratin structure,20,23 although Langer et al. have recently suggested that OA increases corneocyte partitioning of the hydrophilic compound sulforhodamine B.24 The molecular mechanism of the permeation enhancement effect of OA on skin has long been debated. Francoeur et al.25 observed that acyl chain perdeuterated OA (OA-d) lowered the SC lipid phase transition temperature (Tm) in both isolated SC sheets and extracted SC lipid dispersions but did not change SC lipid acyl chain order at temperatures below Tm. They suggested that OA enhances skin permeation by forming a separate disordered OA phase among the SC lipids. However, Guy et al.26 reported that OA-d increased SC lipid disorder in vivo through attenuated total reflection infrared spectroscopy (ATR-IR) measurements and that the disordering effect correlates with depth into the SC. They proposed that OA enhances skin penetration through both disordering SC lipids and the formation of a separate OA-enriched domain. In a 2H nuclear magnetic resonance (NMR) study, Thewalt et al.27 found that OA did not change the Tm of model SC lipid dispersions; rather, it extracted some SC lipids and promoted phase separation. In the molecular dynamics simulations from
INTRODUCTION Stratum corneum (SC), the outermost layer of mammalian skin, serves as the main permeability barrier required for terrestrial life. It regulates the evaporation of water from skin and effectively hinders exogenous agents from entering.1 In the SC, anucleated keratin-filled corneocytes are dispersed in a lipid matrix, which is composed of cholesterol, long-chain free fatty acids, and several classes of ceramides. Its precise composition is under ongoing investigation.2−6 SC lipids form a highly ordered lamellar structure in the intercellular space7,8 with repeat distances of 6 and 13 nm, as measured with small-angle X-ray diffraction.9,10 It is generally accepted that SC lipids are packed in primarily orthorhombic, hexagonal, and liquid crystalline phases,11−14 consistent with the domain mosaic model of SC lipids.15 As the continuous phase in the SC, intercellular lipids are considered to be the major transport route through skin,16 so that barrier function is highly dependent upon the SC lipid structure.11,17 One feasible approach to improve impaired SC barrier function is to design topical products that can mimic or restore the proper SC lipid structure. Oleic acid (OA) is commonly used as an emollient and texture modifier in many skin-care products. It also constitutes the major fatty acid component of the triglycerides in several natural oils, such as olive oil and corn oil. The effects of natural oils on skin function have been linked to their fatty acid composition.18,19 The interaction between OA and SC lipids has also been studied from the perspective of drug delivery. OA is a well-known permeation enhancer. Its permeation enhance© 2013 American Chemical Society
Received: January 17, 2013 Revised: March 16, 2013 Published: March 21, 2013 4857
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Figure 1. (A) π−A isotherms and (B) monolayer compressibility modulus for SC lipids (black line), OA (red line), and their mixtures with OA mole percent at 10% (gray line), 20% (cyan line), 30% (magenta line), 40% (blue line), 60% (purple line), and 80% (green line).
Hoopes et al.,28 it is documented that OA increased diffusion of water and SC lipids in a bilayer system by reducing bilayer density and thickness. Overall, the interactions between OA and SC lipids are not thoroughly understood. In the current study, we investigate the effects of OA on model SC lipids composed of an equimolar ceramide, cholesterol, and palmitic acid (PA) mixture and, more specifically, the interactions of OA with different components of the SC lipids. The composition ratio between OA and SC lipids was either not controlled or examined at only a few values in the above-cited literature. We suspect that the interactions between OA and SC lipids vary with composition and the physical status of SC lipids. Thus, we took advantage of the ease of sample preparation in Langmuir monolayer experiments to study the interactions between OA and SC lipids over a wide range of composition and surface pressure values. The integration of several monolayer-oriented techniques offers a comprehensive approach to more fully understand this system from molecular-level interactions to microdomain structure to thermodynamics at the macroscale. The phase behavior of the mixed monolayers was monitored through classic monolayer π−A isotherms, while detailed interactions were studied at the molecular level with infrared reflection absorption spectroscopy (IRRAS). Monolayer morphology and domain structure were visualized directly through Brewster angle microscopy (BAM).
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and two barriers that move symmetrically from both ends toward the trough center. A subphase of 150 mM NaCl and 1 mM EDTA adjusted to pH 5.5 is used to float monolayers. Typically, ∼60−80 μL of lipid solution was spread at the air/water interface, and 15 min was allowed for solvent evaporation. Monolayers were then compressed with a constant barrier speed of ∼4 Å2 molecule−1 min−1 until the monolayers collapsed. Each compression took 10−15 min to finish. Comparable isotherms were obtained for pure OA monolayers either under air or argon purge. Subsequently, all of the experiments were conducted under air, and no sign of OA oxidation was observed. All isotherms used for miscibility calculations were acquired in duplicate, and differences in mean molecular area between runs are smaller than 2 Å/molecule. Averaged isotherms are presented. A high-resolution KSV NIMA BAM microscope (Biolin Scientific, Västra Fröluna, Sweden) was aligned to the trough center to view images of monolayers at 20 frames/s with a field of view of 720 μm (W) × 400 μm (H) and 2 μm resolution. The angle of incidence was set at 53.2°, and the polarizer and analyzer were both set to p polarization at 0° for best image contrast. BAM videos were recorded during continuous film compression simultaneously with π−A isotherm collection. Image frames at selected surface pressures were captured from the videos with Accurion Image (Accurion GmbH, Goettingen, Germany) and presented with brightness adjusted upward to the same level for all of the images with Adobe Photoshop for best visualization. IRRAS Measurements. Infrared (IR) spectra were acquired with a Bruker Instruments, Equinox 55 IR spectrometer, equipped with an external variable angle reflectance accessory, the XA51. The IRRAS accessory is coupled to a custom-designed Langmuir trough with a maximum surface area of 98 cm2 constructed by Nima Technology (currently, KSV NIMA). The IR beam was directed through a wire grid polarizer mounted in the optical path to the air/water interface, with the design allowing for an adjustable angle of incidence. The reflected light was collected and directed toward a HgCdTe detector. The entire accessory setup is enclosed and purged with nitrogen to maintain a stable low relative humidity to minimize water vapor interference with the IR spectrum. The trough can be shuttled to different positions relative to XA51 for interferogram collection from film-covered areas (sample with intensity R) and uncovered areas (background with intensity R0). Frequent updates of the background interferograms help to compensate for the residual water vapor. IRRAS spectra are reported as −log(R/R0) versus wavenumbers. Lipid monolayers were prepared in the same manner as for BAM measurements and compressed with one barrier moving from the end of the trough toward the surface pressure sensor at the other end at a speed of 2 cm2/min. The barrier was stopped at surface pressures of interest, and IRRAS spectra were collected during intermittent compression, while π−A isotherms were recorded. Following 5 min
EXPERIMENTAL SECTION
Materials and Sample Preparation. Bovine brain ceramide (type III), cholesterol, PA, PA-d31 (98 atom % d), OA, and OA-d34 (98 atom % d) were purchased from Sigma-Aldrich (St. Louis, MO) and used without additional purification. The ceramide used in this study belongs to the NS ceramide family with non-hydroxy fatty acid chains composed predominantly of stearic acid (C18:0) and nervonic acid (C24:1) attached to a sphingosine headgroup through an amide bond. Sodium chloride, ethylenediaminetetraacetic acid (EDTA), and chloroform were purchased from Fisher Scientific (Hampton, NH). All lipids were dissolved separately in chloroform at ∼1.00 mg/mL; individual solutions were combined volumetrically to make the mixed solutions desired. Mixtures containing OA are referenced by their molar percentages of OA. Solutions were kept in a −20 °C freezer, and all isotherms were collected within 3 weeks of sample preparation to minimize the effect of solvent evaporation and lipid oxidation. Monolayer and BAM Measurements. A KSV NIMA Langmuir trough (KN1006, Biolin Scientific, Västra Fröluna, Sweden) was used to acquire π−A isotherms. It has a maximum trough area of 840 cm2 4858
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of monolayer relaxation at each desired surface pressure, spectra were collected using s-polarized light at a 50° angle of incidence. For each spectrum, a total of 1024 scans were acquired at ∼8 cm−1 resolution in 2 blocks of 512 scans each, co-added, apodized with a Blackman− Harris 3-term function, and fast Fourier-transformed with one level of zero-filling to produce spectral data encoded at ∼4 cm−1 intervals. All experiments were duplicated, and the average results are reported here. IRRAS data were analyzed with Grams/32 software (Galactic Industries Corp., Salem, NH). Peak positions of methylene stretching modes were determined with a center-of-gravity algorithm written by Moffatt and provided by the National Research Council of Canada.
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RESULTS AND DISCUSSION π−A Isotherms and Monolayer Compressibility. Equimolar mixtures of ceramide/cholesterol/PA (referred to as Cer/Chol/PA or SC lipids) were used in this study to mimic the SC intercellular lipids. Figure 1A displays π−A isotherms for the SC lipids along with those for OA and their mixtures. Isotherms of SC lipids lifted off at ∼35.5 Å2/molecule and collapsed at ∼31.5 Å2/molecule and ∼50 mN/m, following a sharp surface pressure increase. These values are in good agreement with the literature29 and indicate that SC lipids form rigid and condensed monolayers at the air/water interface. In comparison, the OA monolayers were much more expanded. OA isotherms lifted off at ∼45 Å2/molecule and collapsed at ∼23 Å2/molecule with a pressure value of ∼37 mN/m. With a cis double bond in the middle of its acyl chain, OA cannot pack as tightly as saturated lipids and exists in a liquid-expanded phase at the air/water interface. A comparison of the series of π−A isotherms (Figure 1A) is made to study the miscibility and interaction between OA and SC lipids. The absolute values of isotherm slopes and monolayer collapse pressures decreased monotonically with increasing amounts of OA in the mixtures. The isotherm lift-off areas were also affected by monolayer composition. In comparison to monolayers of SC lipids, the liftoff areas first decreased with increasing OA percentages between 0 and 30% and then increased when OA compositions exceeded 40%; however, these values were always smaller than that of a pure OA monolayer. Overall, the addition of increasing amounts of OA caused SC lipid monolayers to shift from more condensed films to liquid-expanded films. The monolayer compressibility modulus (Cs−1) is a measure of the film stiffness/elasticity and can be calculated from π−A isotherms by eq 130 Cs−1 = {−1/A × (dA /dπ )T }−1
Figure 2. MMA for Cer/Chol/PA and OA mixtures at surface pressures of 1 mN/m (black circle), 5 mN/m (red circle), 10 mN/m (green circle), 20 mN/m (magenta circle), and 30 mN/m (blue circle). The solid lines with symbols depict the measured MMA from the isotherms, and the dashed lines are the calculated ideal mixing curve.
of the eight different SC lipid/OA mixtures as a function of the OA molar fraction at five surface pressures and provides a measure of the miscibility between SC lipids and OA. The symbols connected with solid lines are the experimental MMAs from the averaged isotherms shown in Figure 1A. The dashed lines depict the calculated MMAs according to the additivity rule, as shown in eq 2 A12 = X1A1 + X 2A 2 (2) where A1 and A2 represent the MMAs for the pure monolayers of component 1 (SC lipids) and component 2 (OA), respectively, under the same surface pressures, while X1 and X2 are the mole fractions of each component in the mixture. When the two components are ideally mixed or completely phase-separated, the MMA of their mixtures follow the additivity rule and lie on the dashed line. Most lipid mixtures studied in the literature show non-ideal behavior and deviate from the ideal mixing curve. Attractive or repulsive interactions in binary mixtures can be evaluated by means of negative or positive deviation from the additivity rule. In the current study, the SC lipids contain more than three components given that a naturally derived ceramide is used. We treat the equimolar mixture of SC lipids as a single component to evaluate collective interactions with OA. The interactions between OA and each SC lipid component and the effects of OA on interactions among SC lipid components cannot be delineated from the thermodynamic analysis and are addressed through IRRAS measurements in a later section. The SC lipid/OA mixtures were not ideally mixed at all compositions studied. Their MMAs were smaller than those calculated from eq 2 at each surface pressure, which indicates stronger attractive or weaker repulsive interactions between SC lipids and OA compared to the SC lipid/SC lipid and OA/OA interactions in their respective “single-component” monolayers. Because equimolar Cer/Chol/PA comprises the model SC lipids used in the mixture, OA becomes the most abundant single component at a molar percentage above 25%. When OA dominates the composition of the mixture, an inflection point in the MMA curve was observed at 40% OA. This may be the result of changes in the interactions between OA and SC lipids
(1)
where A is the mean molecular area (MMA) and π is the corresponding surface pressure under constant temperature, T. A lower compressibility modulus indicates a monolayer with lower interfacial stiffness or higher elasticity. Figure 1B plots the compressibility modulus as a function of the surface pressure for each monolayer shown in Figure 1A. SC lipids displayed a compressibility modulus up to ∼700 mN/m, while the compressibility modulus of OA was below 100 mN/m. The addition of 20% OA to SC lipid monolayers was sufficient to lower the monolayer compressibility modulus to half of its original value, indicating the significant fluidizing effects of OA on SC lipid monolayers. The compressibility modulus was further decreased with a higher OA percentage. Films containing 60 and 80% OA displayed compressibility moduli similar to that for the pure OA film. Miscibility of SC Lipids and OA. The interaction between SC lipids and OA was further evaluated with MMA plots and excess Gibbs energy of mixing analysis. Figure 2 plots the MMA 4859
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the density and acyl chain order of the lipids increases and the monolayer is visualized. In the first row of Figure 4, BAM images for SC lipids at surface pressures of 1, 5, 10, and 30 mN/m are shown. At molecular areas greater than the π−A isotherm lift-off area, monolayers of SC lipids appeared as a network structure (image not shown), consistent with the earlier report by Flach et al.29 At surface pressures of ∼1 mN/m, the entire field of view was covered with a condensed film of SC lipids, as shown in panel A1 of Figure 4. A few small defects in the monolayer appeared in black, as marked by the arrows, showing the exposed air/ water interface. The high resolution of the BAM, which was previously unavailable experimentally, enabled visualization of morphological details of the aqueous SC lipid monolayer films. To our knowledge, we are the first to report on these morphological aspects of SC lipid monolayer films. SC lipids do not form a uniform film at the air/water interface (panels A1−A4 of Figure 4). Domains with irregular branch-like structures in bright gray (domain A) are observed to be dispersed in a darker continuous domain (domain B). At higher surface pressures, the area of domain B contracted, while the area of domain A was observed to increase (as shown in panels A1−A4 of Figure 4). At surface pressures higher than ∼45 mN/m, the monolayer eventually collapsed into threedimensional (3D) structures, which were observed as very bright spots or stripes under the BAM microscope (images not shown). Because of the lack of chemical identification in BAM images, the composition of domains A and B cannot be ascertained directly. An assignment is suggested below. In earlier studies, domain separation between ceramides and free fatty acids was observed with 2H NMR and IR measurements in SC lipid dispersions.12,37,38 As discussed later in the IRRAS section (Figure 6B), ceramides and fatty acids in the SC lipid monolayers studied herein were not completely miscible within single domains either. The acyl chain order of PA is higher than that of ceramide, as indicated by methylene stretching frequencies acquired from IRRAS measurements, suggesting that the two components mostly exist in separate domains. Because domain brightness observed by BAM is affected by lipid density and packing, it seems reasonable to assume that the more densely packed and more ordered PA is enriched in the bright domain A and the relatively less ordered ceramide is enriched in the more compressible domain B. Panels H1−H4 of Figure 4 depict BAM images obtained from OA monolayers. Because of the liquid-expanded film formed by OA, no contrast was observed during monolayer compression until the monolayer collapsed into 3D aggregates (images not shown), which were visualized as very bright spots. Adding 10−30% OA to SC lipids did not dramatically modify monolayer morphology but decreased the coverage of domain A and slightly increased the uniformity of its dispersion in domain B (panels B−D of Figure 4). With 20 and 30% OA in the mixture, an additional, very dark domain (domain C) under the BAM was observed at low surface pressures (panels C1 and D1−D3 of Figure 4). Domain C was nearly black but could be differentiated from the exposed air/water interface observed at the beginning of monolayer compression. Domain C soon disappeared with compression at 5 and 20 mN/m, respectively, for mixtures containing 20 and 30% OA. The percentage of surface coverage and sustainable surface pressure of domain C increased with the OA concentration (up to 30%), and thus, it is possibly an OA-enriched domain.
as the OA concentration increases and the dominant species in the film switches from the ordered and rigid SC lipids to the more fluid OA. The surface excess Gibbs energy of mixing, ΔGex, offers a more quantitative evaluation of monolayer miscibility. It is calculated from eq 3 ΔGex = N
∫0
π
[A12 − (X1A1 + X 2A 2 )]dπ
(3)
where N is Avogadro’s constant. ΔGex represents the energy contribution from mixing of components in the monolayer. As noted above, A12 equals X1A1 + X2A2 when the film is either ideally mixed or completely phase-separated, and thus, ΔGex is zero under these conditions. Mixtures with more negative ΔGex are more thermodynamically stable. ΔGex values for the SC lipid/OA mixtures as a function of the OA molar fraction at several surface pressures are shown in Figure 3. ΔGex was
Figure 3. Excess Gibbs energy of mixing for Cer/Chol/PA and OA mixtures at surface pressures of 1 mN/m (black circle), 5 mN/m (red circle), 10 mN/m (green circle), 20 mN/m (magenta circle), and 30 mN/m (blue circle).
negative for mixtures at all of the OA levels investigated, and they were in the same range as those obtained for OA/ dipalmitoyl phosphatidylcholine (DPPC) and OA/cholesterol binary mixtures.31,32 The results indicate that the mixing of SC lipids and OA contributed to monolayer stability. More negative ΔGex values were observed at higher surface pressures, consistent with stronger hydrophobic interactions when molecules occupy smaller areas at the interface. Two minimum values were observed for monolayers containing 30 and 60% OA. According to standard thermodynamics, phase separation is expected in the region between these two minima,33,34 which is confirmed by the BAM images in the next section. BAM. When the bare air/water interface is illuminated with p-polarized light at the Brewster angle, no reflection occurs and the water surface appears black. When the refractive index is modified by the presence of a lipid monolayer, contrast arises. Different levels of brightness can also be observed within films from regions that vary in density or thickness, resultant from molecular interactions and/or acyl chain orientation. Monolayers and domain structure can thus be visualized through BAM.35,36 At the beginning of film compression, at large molecular areas, lipids are in a gaseous phase and the interface with low lipid density appears black. At smaller moleculer areas, 4860
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Figure 4. BAM images for Cer/Chol/PA, OA, and their mixtures at different surface pressures. The OA molar percentage and surface pressures are noted on the images. The actual size of each image is 720 μm (W) × 400 μm (H).
were well-separated and had either a circular or snowflake-like fractal shape. This shape change for domain A might be a result of an interaction between OA and PA in domain A, occurring at an OA content above 30%. Even though the difference in
At 40% OA (panels E1−E4 of Figure 4), the monolayer mixture still displayed phase separation into the bright domain A and darker domain B, and the density of the bright domains increased with surface pressure; however, the bright domains 4861
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lower coverages of domain A were observed for monolayers containing 40 and 60% OA. The dependence of domain A coverage upon the OA level is similar to the surface pressure− MMA curves in Figure 1A. Such similarity suggests a correlation between the macroscopic surface pressure measurement and the microscopic monolayer morphology observed in BAM images. The origin of such a correlation and identification of potential causal factors is not clear and requires further study. On the basis of the BAM images and IRRAS results (see the following section) for SC lipids, we suggest that SC lipids phase separate into PA- and ceramide-enriched domains (phases A and B, respectively). When OA was added to the mixture, it first incorporated into the ceramide-enriched domains, increasing the fluidity of this domain. When the OA concentration increased to 40% or above, OA started to disrupt the PA-enriched, more ordered domains. Once the concentration of OA reached 80%, SC lipids were completely dissolved in OA and no phase separation was observed with the BAM. IRRAS. IR spectroscopy has been widely used to study lipid acyl chain conformational and packing order in lipid vesicles, lamella, and monolayers.40−42 The methylene stretching modes, νCH2 between 2800 and 3000 cm−1, are used to monitor lipid acyl chain order. A lower stretching frequency corresponds to more ordered acyl chains. Cholesterol provides only a minor contribution to the methylene stretching band intensity compared to ceramide because of its structure.29 Acyl chain perdeuterated palmitic acid (PA-d31) was used in the IRRAS study. The use of the deuterium isotope shifts the methylene stretching bands of PA to the 2050−2250 cm−1 region, and thus, the acyl chain order of ceramide and PA can be monitored separately from the same monolayer through νCH2 and νCD2, respectively. The νCH2 and νCD2 regions of IRRAS spectra from Cer/ Chol/PA-d31 monolayers are shown in Figure 6A as a function of the increasing surface pressure from top to bottom. The methylene asymmetric stretching (νasymCH2) frequencies arising from the ceramide component were measured using a center-of-gravity routine and appear as the black symbols in Figure 6B. The νasymCH2 frequencies from both ceramide and PA in a Cer/Chol/PA monolayer are also shown for comparison as gray symbols. The νasymCH2 frequencies for both mixtures decreased with an increasing surface pressure, especially for surface pressure values less than 10 mN/m. It is also obvious that the νasymCH2 frequencies for ceramide in Cer/ Chol/PA-d31 were about 1.2 cm−1 higher than those for both ceramide and PA in Cer/Chol/PA monolayers, indicating that PA is more ordered than ceramide in the SC lipid monolayers. It is likely that ceramide and PA do not predominantly exist in the same phase because they would tend to have comparable conformational order if they were miscible in the same domain. Deuterated OA (OA-d34) was added to a Cer/Chol/PA-d31 monolayer at 30% to evaluate its effect on ceramide acyl chain order. The νasymCH2 frequencies for ceramide at different surface pressures with or without 30% OA-d34 are shown in Figure 7A. OA-d34 increased the νasymCH2 frequency for ceramide over the full surface pressure range with increases up to 1.7 cm−1 at low surface pressure. OA-d34 clearly disordered the ceramide acyl chains. Similarly, 30% protonated OA was added to Cer/Chol/PA-d31 to study the interaction between OA and PA. The νasymCD2 frequencies of PA-d31 in Cer/Chol/ PA-d31 monolayers with and without OA are shown in Figure 7B. It is noted that the νCD2 spectra from PA-d31 had a lower
brightness between the circular- and fractal-shaped domains is not significant enough to discriminate differences in composition, the appearance of the fractal domain clearly depends upon the OA level increase from 30 to 40%. The appearance of this additional fractal domain at 40% OA is within the range of the local maximum observed in the ΔGex−X(OA) curve (Figure 3). Standard thermodynamics points to the existence of phase separation in regions between two minima,33,34 consistent with the observation of the new fractal domain. The fractal domain was not present for mixtures containing 60% OA. We suggest that the fractal domains either contain a higher OA content than the circular domains or that OA is enriched at the fractal domain interface with the darker surrounding phases. An initial network-like domain structure (panel F1 of Figure 4) was observed at 1 mN/m but disappeared upon compression to ∼5 mN/m (panel F2 of Figure 4). Only bright circular domains appeared at 10 mN/m (panel F3 of Figure 4), and their size and density increased with further compression (panel F4 of Figure 4). Monolayers containing 80% OA also displayed a network structure at 1 mN/m (panel G1 of Figure 4) and showed a few circular domains at 5 mN/m (panel G2 of Figure 4). No contrast was observed at higher surface pressures, and black surfaces were observed as shown in panels G3 and G4 of Figure 4, similar to the OA monolayer (panel H of Figure 4). Surface coverage of domain A is plotted as a function of MMA in Figure 5 for monolayers containing 0−60% OA.
Figure 5. Surface coverage for domain A in monolayers for SC lipids (black line) and its mixtures with OA mole percent at 10% (gray line), 20% (cyan line), 30% (magenta line), 40% (blue line), and 60% (purple line).
Percent surface coverage of domain A was calculated by first determining a gray level threshold for each image stack and then calculating the ratio of pixels with gray levels higher than the threshold to total number of pixels in the image. The threshold was determined for each image stack by selecting an image at 80% of the maximum surface pressure and applying a histogram partitioning method, which minimizes within-class variance.39 A running average of the percent surface coverage of domain A over 30 neighboring frames was calculated, and the mean values from two separate experiments are plotted as a function of MMA in Figure 5. The percent surface coverage of domain A increased with compression for all of the monolayers. However, the slope of the percent surface coverage increase was dependent upon the amount of OA in the film, and significantly 4862
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Figure 6. (A) IRRAS spectra for Cer/Chol/PA-d31 at increasing surface pressures over a range of 2−40 mN/m (from top to bottom). (B) Frequencies for the νasymCH2 mode from ceramide in the Cer/Chol/PA-d31 monolayer (black circle) and ceramide and PA in the Cer/Chol/PA monolayer (gray circle).
Figure 7. (A) νasymCH2 frequencies from ceramide in Cer/Chol/PA-d31 (black circle) and Cer/Chol/PA-d31 + 30% OA-d34 (red square), along with those from ceramide and OA in Cer/Chol/PA-d31 + 30% OA (blue square). (B) νasymCD2 frequencies from PA-d31 in Cer/Chol/PA-d31 (black circle) and Cer/Chol/PA-d31 + 30% OA (red square), along with those from PA-d31 and OA-d34 in Cer/Chol/PA-d31 + 30% OA-d34 (blue square).
signal-to-noise ratio than the νCH2 from ceramide mainly because of fewer methylene groups in PA compared to ceramide; hence, there was more scatter in the data. The νasymCD2 frequencies were also higher in the presence of 30% OA at low surface pressures but decreased to around the same value as those of Cer/Chol/PA-d31 at 35−40 mN/m for the monolayer with protonated OA. Overall, PA was disordered by OA mostly at low surface pressure values. The interactions between OA and ceramide or PA were further studied by comparing their acyl chain stretching frequencies. The νasymCH2 from both ceramide and OA are plotted in Figure 7A, and the surface pressure dependence of these frequencies are shown to essentially overlap those arising only from the ceramide component in Cer/Chol/PA-d31 + 30% OA-d34 monolayers. Thus, the acyl chain order of ceramide and OA in the mixture was quite similar, indicating that they likely exist in the same domains. The frequencies for νasymCD2 from PA-d31 in Cer/Chol/PA-d31 + 30% OA and those from PA-d31 and OA-d34 from Cer/Chol/PA-d31 + 30% OA-d34 are compared in Figure 7B. The νCD2 frequencies for PA-d31 and OA-d31 were ∼0.5 wavenumber higher than those for PAd31 at most of the surface pressures measured, indicating that OA is more disordered than PA in the mixture. Thus, these two
components are not completely miscible in the monolayer and may be enriched in different domains. Overall, the IRRAS results show that OA disorders both ceramide and PA, albeit to a lesser extent for the latter. At 30% concentration in the mixture, OA was observed to be completely miscible with ceramide and partially phaseseparated from PA. This is consistent with the previous discussion of the BAM results that indicated preferential (at
