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Abstract: Fatty acids are recognised as lipophilic chemical penetration enhancers (CPEs) which might cause the fluidization and perturbation of stratum corneum (SC) lipid matrix. The prerequisite for fatty acid enhancing effect on drug permeation is its penetration into skin and following disruption of skin lipids arrangement. The aim of this study was to demonstrate the penetration of oleic, linoleic, lauric and capric acids into human skin layers by time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging after 12 hours of in vitro experiment and relate these data to enhancing effect of fatty acids on penetration of lipophilic model drug tolnaftate into human epidermis and dermis ex vivo. Visualization as well as spatial localization of fatty acids penetrating into human skin layers were performed using ToF-SIMS, operated with primary Bi3+ cluster ions. Statistical analysis revealed that only oleic acid significantly (P < 0.05) enhanced tolnaftate penetration into epidermis comparing to the control solution and the enhancing ratio was equal to 1.867. Treatment of human skin in vitro for 12 h with donor solutions resulted in significant penetration of oleic and lauric acid into human skin as compared to the control and was confirmed by ion images. Linoleic and capric acids were not significantly penetrating into the skin nor significantly enhancing tolnaftate penetration. In conclusion, ToF-SIMS imaging of oleic and lauric acids in the skin confirmed their penetration and possible interaction with lipid molecules in SC, but only oleic acid had significant enhancing effect on model drug penetration. Fatty acids vary in their physicochemical properties and thus in mechanisms of interaction with components of extracellular matrix of SC. Significant penetration of lauric acid into skin did not result in enhancement of lipophilic model drug penetration in vitro. Vice versa, negligible penetration of oleic acid causes significant enhancing effect on tolnaftate penetration into skin.
Keywords : Stratum corneum, fatty acids, penetration ehancers, ToF-SIMS imaging.
Stratum corneum (SC), composed of corneocytes and extracellular lipid matrix, is recognized as the main barrier layer for passive diffusion of drug molecules into and through the skin. The composition of extracellular lipids is already known, but SC lipid structural organization, which is mainly responsible for the barrier properties and which is described by several theoretical models, is still being under thorough elucidation. Knowledge about SC lipid organization allow for better understanding and interpretation of low permeability of drugs through SC and the modes of temporal and reversible action of chemical penetration enhancers (CPE). Lipophilic CPEs are supposed to alter structural organization of lipids in extracellular matrix and in this way increase the permeation of active drugs through the skin.
SC multilamellar lipid matrix is mainly composed of neutral lipids: ceramides (CER), cholesterol (CHOL) and long-chain saturated free fatty acids (FFA) (1-5) in an approximate molar ratio of 3:2:1 (6). Freeze-fracture (7-8) and ruthenium tetroxide post-fixation (9) electron microscopy studies revealed that lipids are arranged into bilayers (10-12). CER are formed of long-chain (mainly C20-C36) fatty acid (non-hydroxylated, Î±-hyroxylated or linoleic acid linked Ï‰-hydroxylated) bound to the amino group of hydrophilic sphingoid base (sphingosine, phytosphingosine, and 6-hydroxy-sphingosine) (1-2, 13-15). Amide-linked fatty acid chains in CER are nonbranched and saturated. FFAs predominantly have saturated and straight chains of 22 (docosanoic acid), 24 (lignocerin acid) and 26 (hexacosanoic acid) carbon atoms (2-3, 5, 16). Oleic and linoleic acids are the only unsaturated fatty acids detected in SC (17). Lipid chains tend to pack in tight lateral highly ordered packing (according to packing density: liquid < hexagonal (gel) < orthorhombic (crystalline) phases), which influences the barrier properties of SC and which has been studied using atomic force microscopy (18), Fourier transformed infrared spectroscopy (19), wide-angle X-ray diffraction (20-21) and electron diffraction (10). All three phases coexist, but it is believed that conformationally ordered orthorhombic packing of lipids is mainly responsible for the resistance to transdermal delivery of molecules (22).
Small-angle X-ray diffraction (23-25) as well as electron microscopy (26-27) studies demonstrated that two lamellar structures, namely long and short periodicity phases (LPP and SPP, respectively), are characteristic to lamellar ordering of lipid bilayers (10, 16, 28). The lipid lamella is oriented in parallel to corneocyte surface and its LPP has a repeat distance of 13 nm and SPP - of 6 nm (16, 29). LPP is organized in trilamellar repeat units of broad-narrow-broad electron lucent bands (6, 30) and is considered to highly impact the barrier properties of SC. Several theoretical SC lipid model systems were proposed in order to describe the ordering of lipids in lamella. These models, such as the stacked monolayer model (proposed by Swartzendruber et al. (27)), the domain mosaic model (by Forslind (31)), the sandwich model (by Bouwstra et al. (25)) and single gel phase model (by Norlen (32)) comprise the architecture of lipid molecules arrangement and the phase behaviour of lipid matrix (1, 4).
Lamellar but not lateral lipid organization is dependent on pH (13), thus pH of human SC is also maintaining skin barrier capacity and is in the range of 4.0-5.5 (3, 5).
Well-defined SC lipid composition, organization and phase behaviour of extracellular matrix allow for better interpretation of CPE interactions with lipid molecules. In order to relate the penetration of oleic, linoleic, lauric and capric fatty acids and their enhancing effect on lipophilic model drug penetration into human skin layers ex vivo, two techniques were applied: in vitro skin penetration studies and mass spectrometry imaging (MSI). In vitro skin penetration studies were carried out using Bronaugh-type flow-through diffusion cells mounted with full-thickness human skin. Donor solutions of polyethylene glycol 400 (PEG 400) having a hydrophobic model drug and CPE dissolved were applied on the skin surface. The amount of model drug penetrating into 1 cm2 of epidermis and dermis was quantified using a validated HPLC-UV method and then the enhancing ratio (ER) of fatty acid on model drug's penetration was calculated. In order to demonstrate the penetration of CPEs into human skin layers, MSI was applied after in vitro skin penetration studies. MSI is becoming a more and more widely used method for chemical mapping of organic and inorganic compounds from various surfaces, especially tissue sections. Among the various techniques aiming to map the surface of the sample, MSI is the only analytical method capable of providing in a single run the spatial distribution of a wide range of molecules over the surface of a biological sample (33). Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a technique of choice for MSI. This technology consists of the bombardment of the sample by a beam of mono- or polyatomic ions, which induces desorption/ionization of secondary ions from the sample surface (34-38). It also offers the possibility to localize various molecules, mainly lipids and metabolites, with a mass-to-charge ratio up to m/z 1000-1500 and a lateral resolution from 400 nm to 1-2 Âµm, which makes the technology particularly efficient for the analysis of tissue sections. The field of research of ToF-SIMS imaging is then rapidly expanding and more widely used in many applications, mainly in biological sciences and medicine (39-42). In the present work, we have used ToF-SIMS imaging in order to visualize and evaluate the penetration and location of externally applied fatty acids into human skin layers.
A thiocarbamate antifungal drug tolnaftate was chosen as a model compound for in vitro skin penetration experiments. High hydrophobicity (XLogP = 5.5), low molecular weight (307.4 Da), weak basic properties and melting point of 109-112Â°C (Eur. Pharm. 6.0; 01/2008:1158) are physicochemical properties which ensure tolnaftate's capability of passive diffusion through SC via lipoidal intercellular route and accumulation in superficial layers of skin. Hydrophilic skin layers (viable epidermis and dermis) form a barrier for tolnaftate deeper penetration.
MATERIALS AND METHODS
Chemicals and reagents
Tolnaftate (O-naphthalen-2-yl methyl(3-methylphenyl)thiocarbamate; Eur. Pharm. 6.0; purity of 99.7%) was obtained from pharmaceutical company Sanitas AB (Kaunas, Lithuania) as a gift. Polyethylene glycol 400 (PEG 400) was purchased from Carl Roth GmbH (Karlsruhe, Germany). Capric acid (decanoic acid, C10:0) was obtained from Merck Schuchardt OHG (Hohenbrunn, Germany). Oleic acid (cis-9-octadecenoic acid, C18:1) and methanol (ChromasolvÂ®) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Linoleic acid (cis, cis-9,12-octadecadienoic acid, C18:2) and lauric acid (dodecanoic acid, C12:0) were purchased from Alfa Aesar GmbH (Karlsruhe, Germany). Sodium azide (NaN3) was obtained from POCh (Gliwice, Poland). Ethanol (96.3%) was obtained from Stumbras AB (Kaunas, Lithuania). All other reagents were of analytical grade.
Human skin preparation
The studies with human skin were approved by Kaunas Region Bioethical Committee. Caucasian women (of age 25-40) abdominal skin was obtained after excision in the Department of Plastic and Reconstructive Surgery, Hospital of Lithuanian University of Health Sciences, appropriately treated, wrapped in aluminum foil and stored at -20 °C for not longer than 6 months before use.
In vitro skin penetration experiments were carried out using teflon-made Bronaugh-type flow-through diffusion cells deposited on the thermostated block. Acceptor medium was circulated by peristaltic pump (MasterflexÂ® L/SÂ®, Cole-Parmer Instrument Co., Illinois, USA). Extraction of human skin layers was performed using ultrasonication in the Bandelin Sonorex Digitec Ultrasonic Bath (DT 156, Bandelin electronic GmbH & Co. KG, Berlin, Germany).
High performance liquid chromatography (HPLC) analysis of skin layers extracts was carried out using Shimadzu Liquid Chromatograph (Shimadzu Corporation, Kyoto, Japan) coupled with UV-Vis detector.
Preparation of the donor phase
The donor solutions for in vitro skin penetration experiments were prepared by dissolving tolnaftate (1%, w/w) in PEG 400 and following addition of oleic, linoleic, lauric or capric acid to comprise 10% (w/w) of total amount. If necessary, slight heating (up to 50 °C) was employed. Tolnaftate 1% (w/w) solution in PEG 400 was used as a control for in vitro studies.
In vitro skin penetration experiments
Full-thickness human skin was mounted into Bronaugh-type flow-through diffusion cells having the effective diffusional area of 0.64 cm2 and the receptor volume of 0.13 mL. Circulating water from water bath maintained 37Â±1°C in the thermostated block, holding the cells. 12 hours equilibration period was followed circulating 0.9% NaCl+0.005% NaN3 underneath the skin. After the equilibration period the infinite dose (approximately 200 mg) of 1% (w/w) tolnaftate solution in PEG 400 with or without CPE was applied on the SC side of skin surface for 12 hours. The acceptor fluid (4 mL of 0.9% NaCl+0.005% NaN3) was pumped at a rate of 0.6 mL/min by the peristaltic pump and it was entirely replaced after 4, 8 and 12 hours. After 12 hours the donor phase was carefully removed and the skin surface was rinsed 2 times with 0.5 mL of 96.3% ethanol and then 2 times with 1 ml of 0.9% NaCl. The outer residuals of skin samples were trimmed off, leaving the central circles of 0.64 cm2 area.
After in vitro skin penetration experiment skin specimens were analyzed for tolnaftate content in epidermis and dermis separately, using a validated HPLC method (43), or were frozen and subjected for ToF-SIMS analysis.
HPLC analysis of human skin layers for model drug content
After in vitro skin penetration experiments epidermis was separated from the rest of the skin (dermis with residues of subcutis) using dry heat separation method (44). Heat separated layers were extracted with 1 ml of methanol, following bath sonication for 30 min. The supernatant was filtered through nylon membrane filter (0.45 Âµm, Carl Roth GmbH, Karlsruhe, Germany) and injected into HPLC.
Separation of tolnaftate from endogenous compounds, deriving from skin matrix, was accomplished on a LiChrospherÂ®100 RP-18 Endcapped column, 125 x 4 mm, i.d., packed with 5 Âµm size particles (Merck KGaA, Darmstadt, Germany). A LiChrospher 100 RP-18e (5 Âµm) (LiChroCART 4-4) was used as a guard column. Isocratic elution, using 70% methanol and 30% bi-distilled water as the mobile phase, resulted in tolnaftate retention time of 13.2 min. The flow rate of the mobile phase was 0.8 ml/min and the injection volume was 10 Âµl. The column was thermostated at 40 °C and tolnaftate detection was set at Î» = 257 nm.
ToF-SIMS imaging of human skin sections for CPE penetration visualization and ion imaging
Circle skin specimens, obtained after in vitro skin penetration experiments, were cut into two parts perpendicular to SC surface and immediately frozen at -60°C. The samples were stored frozen until cryosectioning procedure: skin specimens were embedded in OCT medium and sections of 12 Âµm thick were cut at -20°C using a CM3050-S cryostat (Leica Microsystems SA, Nanterre, France), and immediately deposited on a silicon wafer (2-in.-diameter polished silicon wafers, ACM, Villiers-Saint-Frederic, France) for ToF-SIMS experiments. The samples were dried under vacuum, at a pressure of a few hectopascals for 15 min before analysis, without any further treatment. Optical images were recorded with an Olympus BX51 microscope (Rungis, France) equipped with lenses Ã-1.25 to Ã-50 and a Color View I camera, monitored by CellB software (Soft Imaging System, GmbH, Münster, Germany).
The experiments have been performed using a commercial ToF-SIMS IV time-of-flight mass spectrometer (Ion-Tof GmbH, Münster, Germany), located at the Institut de Chimie des Substances Naturelles (Gif-sur-Yvette, France). The spectrometer is equipped with a liquid metal ion gun (LMIG) filled with bismuth. Bi3+ cluster ions were selected for all experiments. Primary ions extracted from the source emitted with a 25 kV potential reach the sample surface with a kinetic energy of 25 keV and at angle of incidence of 45Â°. Secondary ions are accelerated to an energy of 2 keV, fly through a field free region, and are reflected with a single stage reflector (effective flight path ~ 2 m) before being post accelerated to 10 keV just before hitting the entrance surface of the hybrid detector, which is made of one single micro-channel plate, followed by a scintillator and a photomultiplier. A low-energy electron flood gun is activated between two primary ions pulses in order to neutralize the sample surface with the minimum damage (45).
Only one mode of operation of the primary ion column has been used during the experiments, which is called "high-current bunched mode" (46-47), thus providing both a beam focus of 2 Âµm and a pulse duration of a less 1 ns. Such experimental conditions enabled an excellent mass resolution, M/Î”M = 8 Ã- 103 (full width at half maximum, FWHM), at m/z 500. The Bi3+ primary ion current, measured at 10 kHz with a Faraday cup on the grounded sample holder, is ~0.65 pA in the high-current bunched mode. For images of human skin sections, a large-area analysis (1.5 mm x 0.5 mm) was performed using these same LMIG conditions, i.e. high-current bunched mode, and the so-called stage scan. In this case, the sample is moved step by step to record three successive patches of 0.5 mm x 0.5 mm each. The number of pixel was 750 Ã- 250, each pixel having a size 2 Ã- 2 ÂµmÂ². Under these conditions, the fluence (also called primary ion dose density) is maintained to 5.1011 ions/cmÂ², which is below the so-called static SIMS limit (48).
Because of the very low kinetic energy distribution of the secondary ions, the relationship between the time-of-flight and the square root of m/z is always linear over the whole mass range. The calibration was always internal, and signals used for initial calibration were those of H-, C-, CH-, CH2-, C2-, C3-, and C4H- for the negative ion mode.
The name of the compound or the m/z value of the peak centroid, the maximal number of counts in a pixel (mc), and the total number of counts (tc) are presented below each figure. The color of scale corresponds to the [0, mc] intervals. The data acquisition and processing software was SurfaceLab 6 (ION-TOF GmbH, Münster, Germany).
For the statistical analysis, one-way analysis of variance (ANOVA) together with Tukey's HSD test were applied using SPSS software. The level of significance was determined as P < 0.05.
Effect of fatty acids on tolnaftate penetration into epidermis
The amounts of tolnaftate (T) (Âµg/cm2, n=3) penetrating into epidermis from the control solution and from solutions having 10% (w/w) of different fatty acids after in vitro skin penetration experiments of 12 hours duration are presented in Figure 1. The enhancing effect of four different fatty acids on tolnaftate penetration into 1 cm2 of epidermis (E) was expressed as the enhancing ratio (ER). ER was calculated using the following formula:
The obtained ER values are presented in Table 1.
Statistical analysis revealed that tolnaftate amount penetrating into 1 cm2 of epidermis from the solution having 10% of oleic acid was significantly greater (P < 0.05) than from the control solution. Other fatty acids - linoleic, lauric and capric - did not significantly enhance tolnaftate penetration into epidermis comparing to the control.
Tolnaftate was not penetrating into hydrophilic dermis (only traces below limit of quantitation were found after 12 hours of experiment) and no drug was detected in the acceptor fluid.
The appropriately chosen experimental conditions for in vitro studies were confirmed by means of mass balance calculation after extraction of the donor phase, remaining on the skin surface. The found amounts of tolnaftate were compared to the nominal amount of tolnaftate added on each skin sample. The relative errors were calculated for each skin sample and were ranging from -6.29% to 3.22%.
ToF-SIMS imaging of fatty acids penetration into human skin
Human skin sections were analyzed by ToF-SIMS imaging to demonstrate the penetration of fatty acids into skin. The fatty acids are labelled (CX:Y), which specifies the number of carbon atoms (X) and double bonds (Y) in the molecule. Figure 2 shows a typical mass spectrum recorded in the negative ion mode. This spectrum is dominated by lipid ion peaks, mainly fatty acids. The most intense ion peaks together with their formula are listed in Table 2.
Together with optical images of the sections, Figures 3-6 show ion images corresponding to the spatial localization of [M-H]- carboxylate ions of capric acid C10:0 (m/z 171.10, Figure 3), lauric acid C12:0 (m/z 198.96, Figure 4), linoleic acid C18:2 (m/z 279.28, Figure 5), and oleic acid C18:1 (m/z 281.29, Figure 6), in human skin after application of PEG 400 solution having 10% fatty acid corresponding during 12 hours (B) and control (C) For all these figures, optical images (A) are those of human skin sections corresponding to the ion images (B).
ToF-SIMS imaging revealed that lauric (Figure 4) and oleic (Figure 6D) acids did significantly penetrate into the human skin comparing to the control (Figure 4C and Figure 6C respectively). Capric (Figure 3) and linoleic (Figure 5) acids did not significantly penetrate into the skin sections (Figure 3C and Figure 5C respectively). These variations of concentration as a function are not obviously observed directly from the images shown in Figures 3 to 6, particularly because the skin sections are not exempt from holes or lipid droplets. Intensity profiles as a function of depth, followed by linear fits, have been drawn from each of these images. The results are presented in Suplementary data 1. It is clear from this figure that capric, oleic and lauric acids did penetrate into skin when compared to the control, while linoleic did not.
Intensity profiles might indicate that possibly back-difussion of fatty acids from the superficial layers of skin into hydrophilic PEG 400 solution occurred during 12 hours of in vitro experiment. The back-diffusion of capric acid might be related to the most hydrophilic properties of this fatty acid among others tested. Thus its concentration (corresponding to average intensity counts) in superficial layers of skin decreased comparing to the control, but almost reached the same level in the deeper layers of skin. It is obvious, that lauric acid significantly penetrated into skin layers and this might be confirmed by the highest affinity of this fatty acid to skin lipids as discussed below. It seems that linoleic acid neither penetrated into skin from 10% PEG 400 solution nor back-diffused from skin to PEG 400 solution. Explication of oleic acid back-diffusion and nevertheless its significant enhancing effect on tolnaftate penetration seems confusing and might be only correlated to oleic acid mechanism of action in skin. As discussed below, oleic acid is monounsaturated and forming a 'kink' (52), which fluidizes the skin lipids and might be responsible for higher penetration capabilities of our model drug.
Another possible explanation of fatty acid intensity counts decrease comparing to the blank might be related to the penetration of PEG 400 into skin. If PEG 400 penetrates into skin, then the relative area of skin section expands and the counts of fatty acid in the same area of skin section decrease.
The present study was aimed to demonstrate the penetration of oleic, linoleic, lauric and capric acids into human skin by ToF-SIMS imaging and relate to the enhancing effect of these fatty acids on model drug penetration into skin layers. PEG 400 was chosen as a hydrophilic vehicle, in which tolnaftate and fatty acids tested were soluble.
The diffusion cell method is a reliable method for measuring drug transport into/across the skin. Human skin was obtained from the same donor, thus inter-individual variance was reduced as the coefficients of variation (CV) were lower than 16.6% (calculations made after determination of tolnaftate levels in skin layers by HPLC).
ToF-SIMS analysis of skin specimens after external application of fatty acids might be interfered by several factors. Normally, skin surface is covered by sebum, composed of squalene, wax esters and triglycerides (13). Lipases acting on the surface of SC hydrolyze the sebaceous lipids to FFAs of C16 and C18 with monounsaturation or branched chains (5). Lauric and sapienic (C16:1) acids deriving from triglycerides (TGC) and covering the surface of human skin, are associated with antimicrobial action (14). Thus FFAs naturally present on skin surface might affect the ToF-SIMS analysis of externally applied fatty acids. In this case SC surface wipe procedures before and after in vitro skin penetration experiments are important for validity of results. Cleaning of SC surface with 0.9% NaCl before in vitro experiments and careful removal of donor phase followed by rinsing with ethanol and then 0.9% NaCl after in vitro experiments, ensured elimination of sebum traces from skin surface and there was no distortion of ToF-SIMS results.
On the other hand, cryosectioning of skin specimens for ToF-SIMS analysis caused contamination of skin sections with TGC from subcutaneous fat (5), as full-thickness human skin was used for in vitro skin penetration experiments. The contamination with lipid droplets was visually observed under the microscope and ToF-SIMS analysis revealed, that in these lipid droplets oleic, linoleic, palmitic and palmitoleic acids were present. This undesirable contamination did not change the quality of ToF-SIMS results. Despite above mentioned factors, ToF-SIMS analysis allowed for ion imaging and evaluation of fatty acid penetration into human skin after in vitro skin penetration experiments and valuable and reliable results were obtained.
Fatty acids increase the penetration of active drugs into the skin through the non-polar route, which is very important for the penetration of hydrophobic drugs, such as tolnaftate. The mechanism of fatty acids action in the extracellular matrix of the skin is widely investigated. Lipophilic CPEs cause the fluidization and perturbation of SC lipid matrix. CPEs partition and insert the hydrophobic tails into highly ordered packing of lipid bilayers. Differences in CPE head group and CER or CHOL structure (49) cause disruption of crystalline lipid packing. Formation of microcavities in SC lipids and increase of free volume fraction cause the enhancement of drugs diffusion coefficient (50) and, according to Fick's first law of diffusion, promotion of the permeation of molecules through SC occurs (51).
The enhancing effect of aliphatic acids has a parabolic dependence on the chain length and the maximum effect is exerted by the fatty acids with chain lengths around C12 (52-53). Lauric acid has a high affinity to skin due to its optimal partition coefficient and solubility parameter (54) and it also might acquire a spatial form, which is conformationally similar to CHOL framework and which affects the packing of lipids (53). During 12 hours of in vitro skin penetration experiments, lauric acid penetrated into the human skin from PEG 400 solution, but its enhancing effect on tolnaftate penetration was not significant.
Kravchenko et al. (53) states, that acids with shorter chains of less than C11 are not capable to disturb the packing of lipids in SC, as short chain acids are insufficiently lipophilic (55). This statement is consistent with our findings that capric acid of C10 is not penetrating and is not enhancing significantly the penetration of tolnaftate into human skin after 12 hours of experiment ex vivo. On the other hand, Nair et al. (49) proposed, that C10-C12 chain length acids disrupt CER-CHOL or CHOL-CHOL interaction and in this way should increase the permeability of drugs.
Oleic and linoleic fatty acids are both unsaturated, having one and two double bonds, respectively. Central location of double bond condition the formation of a 'kink' (52), and this is attributed to the most potential CPE - oleic acid. Oleic acid is considered to create fluid-like phase within intercellular space (52, 56-59). In our studies, only oleic acid significantly enhanced tolnaftate penetration into epidermis after 12 hours (ER=1.867) and the penetration of this fatty acid into human skin was confirmed by ToF-SIMS analysis. Linoleic acid did not have a significant enhancing effect on tolnaftate penetration and the penetration of the fatty acid itself did not occur as imaged by ToF-SIMS.
In conclusion, only oleic acid significantly enhanced the penetration of hydrophobic model drug into human skin layers during 12 hours of ex vivo experiment. Treatment of human skin with PEG 400 solution containing 10% (w/w) of fatty acids for 12 hours resulted in penetration of oleic and lauric acids into the skin as revealed by ToF-SIMS imaging, but linoleic and capric acids had not penetrated into the skin. According to intensity profiles the amount of fatty acid penetrating into skin is not related to the enhancement of model drug penetration. Differences in physicochemical properties of fatty acids might determine their different affinity to skin lipids and mechanisms of action, thus their penetration capabilities and enhancing effect on lipophilic model drug penetration are different.