Effect of Chemical Permeation Enhancers on Stratum Corneum

Article pubs.acs.org/molecularpharmaceutics

Effect of Chemical Permeation Enhancers on Stratum Corneum Barrier Lipid Organizational Structure and Interferon Alpha Permeability Shadi H. Moghadam, Evi Saliaj, Shawn D. Wettig, Chilbert Dong, Marina V. Ivanova, J. Torin Huzil, and Marianna Foldvari* School of Pharmacy, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada, N2L 3G1 ABSTRACT: The outermost layer of the skin, known as the stratum corneum (SC), is composed of dead corneocytes embedded in an intercellular lipid matrix consisting of ceramides, free fatty acids, and cholesterol. The high level of organization within this matrix protects the body by limiting the permeation of most compounds through the skin. While essential for its protective functions, the SC poses a significant barrier for the delivery of topically applied pharmaceutical agents. Chemical permeation enhancers (CPEs) can increase delivery of small drug compounds into the skin by interacting with the intercellular lipids through physical processes including extraction, fluidization, increased disorder, and phase separation. However, it is not clear whether these same mechanisms are involved in delivery of biotherapeutic macromolecules, such as proteins. Here we describe the effect of three categories of CPEs {solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes [menthol, nerol, camphor, methyl salicylate], and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)]} on the lipid organizational structure of human SC as determined by X-ray scattering studies. Small- and wide-angle X-ray scattering studies were conducted to correlate the degree of structural changes and hydrocarbon chain packing in SC lipids caused by these various classes of CPEs to the extent of permeation of interferon alpha-2b (IFNα), a 19 kDa protein drug, into human skin. With the exception of solvents, propylene glycol and ethanol, all classes of CPEs caused increased disordering of lamellar and lateral packing of lipids. We observed that the highest degree of SC lipid disordering was caused by surfactants (especially SDS, DDAB, and DTAB) followed by terpenes, such as nerol. Interestingly, in vitro skin permeation studies indicated that, in most cases, absorption of IFNα was low and that an increase in SC lipid disorder does not correspond to an increase in IFNα absorption. KEYWORDS: stratum corneum structure, chemical permeation enhancers, small-angle X-ray scattering, IFNα absorption

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INTRODUCTION

enhancers and prodrug design), and combinations of these methods.6−12 Chemical permeation enhancers (CPEs) are pharmacologically inactive compounds that are able to partition into the SC, interacting with the lipids to facilitate drug diffusion.13 While the mechanisms associated with CPE function are not well understood, absorption enhancement through lipid channels is generally thought to depend on the ability of a CPE to integrate with the lipids, creating a perturbed microenvironment through which a drug can freely diffuse.13−16 Several mechanisms related to this process have been proposed,17−25 such as (1) alteration of SC lipid structure and its fluidity, (2) enhancement of solubility characteristics of the skin for the drug to be delivered (increase in the partition coefficient of the drug into the skin as well as drug diffusivity in the SC), (3) creation of a

The skin is the largest and most easily accessible organ of the body, making it a desirable site for both topical and systemic delivery of drugs. However, efficient dermal and transdermal delivery of large hydrophilic molecules remains challenging because the skin poses a formidable barrier to these macromolecules. The stratum corneum (SC), the uppermost layer of the skin and the primary barrier to percutaneous penetration, is composed of 10−15 layers of dead, flattened corneocytes embedded in a lipid-rich matrix.1,2 The major pathway for percutaneous penetration of small drug molecules across the SC is thought to be diffusion through the intercellular lipid lamellae.3−5 In order to enhance skin permeability, various approaches to reversibly remove or weaken this barrier have been investigated. Some of these approaches include physical disruption (e.g., thermal, magnetic, pressure, laser or mechanical modulation, hydration, iontophoresis, phonophoresis, microneedles, skin abrasion and puncture), chemical disruption (permeation or penetration © 2013 American Chemical Society

Received: Revised: Accepted: Published: 2248

August 9, 2012 February 11, 2013 April 15, 2013 April 15, 2013 dx.doi.org/10.1021/mp300441c | Mol. Pharmaceutics 2013, 10, 2248−2260

Molecular Pharmaceutics

Article

from Gattefossé Canada Inc. (Toronto, ON). The water used in all experiments was of Milli-Q grade. Source of Human Skin. Full thickness human breast skin was obtained with permission from female donors undergoing elective mammoplasty surgeries at the Royal University Hospital, University of Saskatchewan (Saskatoon, SK, Canada). Approval for this study was granted by the University of Saskatchewan Human Ethics Committee. The skin was collected within two hours following surgery, trimmed of subcutaneous fat, and stored at −20 °C until use. Stratum Corneum Sample Preparation. SC sheets were isolated from skin samples as described previously,48 with the following modifications; strips of full-thickness human breast skin were thawed, rinsed with distilled water, and dermatomed (Integra, Padgett, Model S Slimline Dermatome, Plainsboro, NJ) from the subcutaneous side to approximately 5 mm thickness. The skin was submerged SC face up, in 1% porcine pancreas trypsin (Sigma Aldrich) in phosphate buffered saline (PBS) with 0.02% sodium azide and incubated overnight at 4 °C. Following trypsin digestion, the skin was washed three times with water. The SC was then carefully removed from the remaining epidermis using forceps and stored in 0.02% sodium azide. Permeation Enhancers. Three categories of CPEs were studied. (1) Solvents: ethanol (95%), propylene glycol (PG) (100%), oleic acid (unsaturated fatty acid; a liquid) (10% w/w in PG), and transcutol (10% v/w in PG). (2) Terpenes: menthol (1% w/w in PG), nerol (10% w/w in PG), camphor (1% w/w in PG), and methyl salicylate (10% w/w in PG). (3) Surfactants: Tween 80 (1% w/v in water), SDS (1% w/v in water), benzalkonium chloride (0.05% v/v in water), Cremophor RH40 (5% w/w in water), DDAB (1% w/v in water), and DTAB (1% w/v in water). UPLC Analysis of IFNα Stability in PE mixtures. The overall stability of IFNα when introduced to each of the CPEs was analyzed using ultraperformance liquid chromatography (UPLC). Each of the above-mentioned CPE solutions was spiked with a total of 125 ng/μL of IFNα and analyzed for protein stability immediately after sample preparation (T0) and following a 24 h incubation (T24) at 32 °C, simulating the conditions of the diffusion study. Following incubation, samples were first diluted into four volumes of [0.1% w/v Tween 20, 0.746 mg/mL methionine, 0.0005% v/v trifluoroacetic acid (TFA) in water/acetonitrile 72/28] and then filtered through a 0.2 μm Acrodisc syringe filter with a GH Polypro membrane (Pall, Ville St. Laurent, QC, Canada). The Acquity H-class chromatographic system, consisting of a quaternary pump, autosampler (sample manager flow-through needle), variable wavelength fluorescence detector and bio-column manager, was controlled by the Empower 3 software (Waters, Milford, MA). Analyses were performed on a 1.7 μm BEH300 C4 50 mm × 2.1 mm i.d. column (Waters) heated to 30 °C with an injection volume of 5 μL. The mobile phase (solvent A, 0.1% v/v TFA in 15% acetonitrile/85% water; solvent B, 0.1% v/v TFA in acetonitrile) was pumped at 0.25 mL/min according to the following gradient: 0−1 min 70A/30B, after 4 min 55A/45B, after 2 min 35A/65B for 1 min, after 1 min 70A/30B for 1 min. Fluorescence detection was done at 295 nm excitation and 360 nm emission. The protein of interest eluted at 4.2 min, and the total peak area was calculated using the Empower software. For PE samples resulting in very low IFNα levels following the 24 h incubation period, an additional time course incubation was

disordering effect among the alkyl chains of SC lipids, and (4) localized separation of lipid domains to create hydrophilic pores and/or establish a drug reservoir in the SC itself. Since drug molecules are primarily transported through intercellular lamellar regions in the SC,26 understanding the role of CPE disruption of lipid lamellar structure has been the subject of many studies. Techniques such as differential scanning calorimetry,27,28 infrared spectroscopy,29−31 Raman spectroscopy,32−35 and electron paramagnetic resonance or electron spin resonance spectroscopy36 can provide information on bulk phases and lipid organization in membranes, while confocal 37,38 and electron microscopy 39,40 can provide information about lipid distribution and fine membrane structures. For investigating the dynamic structural properties of lipid bilayers, small- and wide-angle X-ray scattering (SAXS and WAXS) studies are often the most useful.41−44 In SAXS studies, the scattered intensity of skin lipids can be measured at a low angle relative to the X-ray beam, typically from 0 to 5°, providing information about larger structural units in the sample, for example, the short and long periodicity phase (SPP and LPP) of SC samples.45 WAXS, on the other hand, measures X-ray scattering at wider angles and provides information about the lateral arrangement of the lipids, such as liquid, hexagonal, and orthorhombic. 45,46 The most significant benefit offered by both SAXS and WAXS is the ability to distinguish two types of disorder in SC lipid regions following application of CPEs: (1) disorder of the alkyl chains inside a single lipid bilayer (short-range disorder) and (2) disorder in the lipid bilayer arrangement, including any undulations that occur at the interface of the bilayer itself.47 Factors that alter the molecular structure of the SC, for example, changing the usual orthorhombic arrangement of lipids to a hexagonal or liquid phase or disturbing the lamellar distance of SC, may result in higher permeability of small molecular weight drugs. To examine any correlation between lipid organization in the SC and the degree of permeation enhancement for a representative protein drug, interferon alpha-2b (IFNα, 19 kDa), we conducted in vitro dermal absorption experiments using human skin in Bronaugh flowthrough diffusion cells. We have obtained quantitative dermal delivery data in the presence of three major classes of CPEs, including solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes (menthol, nerol, camphor and methyl salicylate), and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)].

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MATERIALS AND METHODS Materials. IFNα was provided by Schering Plough, Ireland. Propylene glycol, oleic acid, Tween 80, methyl salicylate, sodium dodecyl sulfate (SDS), and menthol [all national formulary (NF) grade] were purchased from Spectrum Chemicals and Laboratory Products (New Brunswick, NJ). Polyoxyl 40 hydrogenated castor oil (Cremophor RH40) was purchased from BASF (Ludwigshafen, Germany). Sodium azide, didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB), ethanol, nerol, and benzalkonium chloride were purchased from Sigma Aldrich (Oakville, ON, Canada). Menthol was purchased from Fisher Scientific (Toronto, ON, Canada). Diethylene glycol monoethyl ether (Transcutol P; pharmaceutical grade) was a gift 2249

dx.doi.org/10.1021/mp300441c | Mol. Pharmaceutics 2013, 10, 2248−2260


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Table 1. Profiles of SAXS and WAXS Peak Changes and Examples of Membrane Disordera

a

SPP: short periodicity phase. LPP: long periodicity phase. Stratum corneum lipid organizational diagram and SC TEM image were adapted with permission from ref 45. Copyright 2006 Elsevier.

sample (X21 beamline). All spectra were processed to remove background contributions by subtracting the scattering profile obtained for a water-filled capillary. The data was converted to radial average intensities for each diffraction vector, and the repeat distance (d) was calculated according to the Bragg equation (eq 1):

performed, with samples analyzed at intervals of 0, 1, 2, 4, 8, 12, and 24 h. Preparation of X-ray Scattering Samples. Full sheets of SC were cut to a final dimension of 1 × 1 cm and individually stored in 24-well plates in 0.02% w/v sodium azide in water at 4 °C before use. The storage solution was decanted, and the SC samples were treated for 24 h at room temperature with either CPE or the CPE/IFNα mixtures. Treatments were prepared with the same composition as the in vitro absorption study. Treated SC was rinsed three times with water and blot-dried prior to X-ray scattering studies. Small- and Wide-Angle X-ray Scattering Analysis. SAXS and WAXS studies were conducted using beamlines X21 and X27C at the National Synchrotron Light Source, Brookhaven National Laboratory, NY. Measurements were performed with 12 keV X-rays, and data covered a q-range from 0.008 Å−1 to 1.8 Å−1. SC sheets were mounted in a specially designed aluminum multiple-sample holder, and samples were randomly oriented to the primary beam. The scattering pattern was recorded using both small-angle and wide-angle detectors, in such a way that diffraction measurements were collected simultaneously (X27C beamline). Alternatively, samples were loaded into 1.5 mm capillaries (Charles Supper Company Inc. #15-BG, Natick, MA) and the scattering pattern was recorded using a 13 cm Mar CCD detector at 1.26 m (calibrated with the scattering pattern of silver behenate) downstream from the

nλ = 2d sin θ

(1)

where λ is the wavelength of the X-ray beam, θ is the scattering angle, and n is the order of the peak. For quantification of peak properties, such as peak position, height, area, full width at halfmaximum (fwhm), and nonlinear peak, fitting was performed using Fityk software and the Gaussian function. In vitro IFNα Permeation. In-line Bronaugh flow-through diffusion cells with a 9 mm orifice diameter (0.63 cm2) were mounted on a water insulated cell warmer (PermeGear, Inc., Hellertown, PA) maintaining a constant temperature of 32 °C. Prepared full thickness human breast skin was placed in the diffusion cells with the SC side facing up. Perfusion buffer (100 mM phosphate buffer with 100 μg/mL bovine serum albumin, 5 mM L-methionine, and Roche Complete protease inhibitor tablets) was circulated through the lower half of the diffusion cells at a rate of 1 mL/h using a peristaltic pump (PermeGear). Following 20 min equilibration, the SC surface of the skin was then dosed with 400,000 IU/cm2 of IFNα in a final volume of 100 μL for each CPE. Following 24 h incubation, any 2250



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Figure 1. Effect of chemical permeation enhancers (CPEs) on stratum corneum lipid structural organization evaluated by small- and wide-angle Xray scattering (SAXS and WAXS). (A) solvent CPEs, (B) terpene CPEs, and (C) surfactant CPEs. SAXS profiles of SC treated with three different groups of CPEs (column 1, A−C) and CPE + IFNα mixtures (column 2, A−C). WAXS profiles of SC treated with three different groups of CPEs in the absence of IFNα (column 3, A−C). PG, propylene glycol; MeSA, methyl salicylate; SDS, sodium dodecyl sulfate; DTAB, didecyltrimethylammonium bromide; DDAB, didecyldimethylammonium bromide; CR RH40, Cremophor RH40; BAK, benzalkonium chloride.

Analysis of in Vitro IFNα Permeation. Skin samples were individually homogenized using a Spex 6870 freezer mill (SPEX SamplePrep, Metuchen, NJ). Powdered skin was reconstituted in extraction buffer (100 mM phosphate buffer with 0.02% w/v Tween 80) to a final concentration of 0.1 g/mL. The homogenate was then incubated at 4 °C for 1 h to ensure complete extraction of absorbed IFNα. Homogenates were

remaining PE formulation in the receptor chamber was aspirated, the skin was removed from the cells, and the SC surface was washed 3 times with 10 mL of water. Each skin sample was immediately blot dried and tape-stripped twice using D-Squame stripping disks (CuDerm, Dallas, TX) to remove excess CPE and ensure removal of surface-adsorbed IFNα. 2251



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Table 2. Membrane Models for SC Interactions with (A) Solvent CPEs and IFNα, (B) Terpene CPEs and IFNα, and (C) Surfactant CPEs and IFNα

with a pinhole set at 63 μm. Emission of Alexa Fluor 594 was collected through a band-pass filter of 585−734 nm.

then centrifuged at 16000g, and the supernatant fractions were collected. A standard curve was generated by spiking untreated skin homogenates with increasing known amounts of IFNα prior to centrifugation. Supernatants from both samples and standards were then analyzed using a Bio-Plex 200 system (BioRad Laboratories, Inc., Mississauga, ON, Canada) following the manufacturer’s recommendations for the IFNα cytokine assay kit. Absorption was expressed as amount of IFNα per cm2 skin based on the surface area of the skin treated in the 9 mm diameter diffusion cells. Confocal Microscopy. As described above (in vitro IFNα permeation), skin samples were treated with each CPE formulation containing IFNα labeled with Alexa Fluor 594 dye (590ex/617em nm), prepared according to the manufacturer’s recommendations with the removal of unbound dye by dialysis (Invitrogen, Toronto, ON, Canada). Following a 24 h incubation the skin samples were removed from the diffusion cells and divided; one-half was stripped twice via D-Squame disks, and the other half was left unstripped. Confocal microscopy images of SC were collected on a Zeiss LSM 710 confocal laser scanning microscope with a HeNe laser (543 nm, 1 mW at 5.0% max power output) (Carl Zeiss, Jena, Germany). A 63× Plan-Apochromomat oil immersion objective was used

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RESULTS AND DISCUSSION Previous work by Bouwstra et al.47 demonstrated that, at physiological temperature, SC lipids are organized in two characteristic lamellar structures, characterized by repeat distances at 6.4 and 13.0 nm called SPP and LPP, respectively. This lamellar organization, as determined by the lateral packing of lipid head groups, plays a crucial role in the barrier properties of the SC45 (Table 1). Results reported here confirm this characteristic pattern, as demonstrated by the presence of two main reflections (peaks) at q = 0.098 and q = 0.14 Å−1 on the SAXS curves for untreated SC membranes (Figure 1). When repeat distances were calculated from these values, we obtained values of 6.6 and 4.6 nm, corresponding to a 6.6 nm repeat for the short-phase periodicity and 11 nm for the long-phase periodicity. The WAXS curve for untreated SC also produced two characteristic chain packing reflections at q = 1.50 and q = 1.63 Å−1, which correspond to an orthorhombic lateral packing of approximately 0.41 and 0.37 nm for the acyl chains in the bilayer (Figure 1). These parameters were used as a baseline to quantify membrane disorder caused by CPE formulations when used to deliver IFNα. 2252



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PE Effect on SC Membrane Structure. SAXS profiles for each treated SC sample were obtained and graphed showing the scattering intensity as a function of the scattering vector at low scattering angles (0° to 5°) (Figure 1). The degree of changes in SAXS profiles, such as shift in peak position, reduction of peak intensity, and change in peak width, indicates molecular changes to the lamellar structure of the SC and could be assigned to five different categories with increasing degree of disordering effect (Table 1). Category A was assigned to CPEs that cause no change in SAXS pattern, although these still may enhance permeation by facilitating increased partitioning of the drug through increased solubility in the SC. Category B indicates CPEs that cause slight decrease in the peak intensity at q = 0.098 and q = 0.14 Å−1, which results in slight disordering of the SC lamellar structure. Category C suggests a significant decrease in peak intensity at q = 0.098 and q = 0.14 Å−1 with disappearance of the peak at q = 0.19 Å−1, corresponding to a discernible disordering of the lamellar structure. Category D is assigned when disappearance of peaks at q = 0.098 and q = 0.14 Å−1 is observed resulting in disruption of the lamellar structure. Finally, category E is indicative of the presence of new peaks which can be explained by the incorporation of CPEs into the SC structure and presumably the creation of a novel lamellar structure. Structural Effect of Solvents. Detailed analysis of SAXS results demonstrated that PG and ethanol did not change the short or long lamellar spacing of the SC membrane structure (Figure 1A, column 1). Interestingly, even with very little observed change in the SAXS patterns for PG and ethanol, both have previously demonstrated increased skin permeation of drug molecules.49 Considering the unchanged SAXS pattern, the permeation enhancing ability could be attributed to changing the solubility of drug molecules followed by ease of drug partitioning into SC. Takeuchi et al. reported extended studies of the influence of PG on the penetration of hydrophilic and hydrophobic drugs, and showed no lipid disordering, but they did observe changes in the conformation of α- to βkeratin.19 Conflicting reports are available regarding ethanol’s effect on SC structure; some authors report an increase in the order and stabilization of the gel phase of the lipid bilayer in the presence of 100% ethanol, resulting in increased rigidity of the lipid bilayer.50,51 Infrared (IR) spectroscopy analysis by Bommannan et al. suggested that ethanol did not disorder the SC lipid structure, but extracted an appreciable amount of lipid from the SC and consequently increased drug permeation into the skin.52,53 The lipid extraction and pore formation mechanism of ethanol have been supported by some other studies.54−56 Similar to the SAXS results obtained for ethanol, transcutol also caused a slight disordering effect within the SC membrane. Treatment of SC samples with transcutol decreased the intensity of SAXS peaks, suggesting an overall change in membrane disorder, as well as the WAXS pattern, suggesting an increase in membrane fluidity. The most significant SAXS and WAXS effect was observed for oleic acid. The elimination of both short and long lamellar spacing peaks suggests that oleic acid completely disrupted the SC lamellar structure (Table 2A). After treatment with oleic acid, a new peak at q = 0.14 Å−1 was observed in the WAXS pattern (Figure 1A, column 3), which clearly supports the hypothesis of SC fluidization. Among other solvents, PG and ethanol did not change the overall WAXS peak characteristics of SC samples.

Treatment of SC by different solvents decreased SPP and LPP slightly, which might not be significant. In the case of oleic acid, a commonly used permeation enhancer, SPP decrease was more noticeable. Different studies have proven its ability to form a separate liquid phase inside the SC structure, and the boundary between the fluid and solid domains exhibits high permeability for drug molecules.57−59 These fluid domains could be responsible for decreasing the SPP distances. In the SAXS pattern of SC treated with oleic acid, a dramatic decrease in peak intensity was observed, which is a sign of SC lipid fluidization and destruction of lipid organizational structure (Table 2A). Results obtained by Naik et al.60 using IR spectroscopy demonstrated that the in vivo permeation enhancement by oleic acid also results from lipid phase separation and to a lesser extent from SC lipid fluidization. Structural Effect of Terpenes. A detailed analysis of SAXS and WAXS profiles demonstrated that terpenes and terpenoids produced a more pronounced effect on the short lamellar spacing than those observed for solvent CPEs, decreasing the spacing from 6.4 nm to about 5.7 nm (Figure 1B, column 1). Interestingly, the presence of IFNα in the formulations did not change this effect (Table 2B and Figure 1B, column 2). Fourier transform IR (FT-IR) studies done by Jain et al.61 suggested that terpenes exert their permeation enhancement effect by competitive hydrogen bonding, i.e., breaking of hydrogen bonds between the headgroup of ceramides in SC lipids and creating new bonds with their available moieties. Among the terpenes, we observed that nerol created the highest level of disruption in SC lamellae where the SPP and LPP peaks almost completely disappeared. One explanation for this observation may be due to the presence of a hydroxyl group, coupled with the alkene in nerol that can strongly donate the hydrogen bond which leads to disruption of existing hydrogen bonding between the ceramide head groups in the SC bilayer. In contrast, the increased membrane order observed for menthol and camphor is most likely due to the lessened electronegativity of the hydroxyl group in menthol and absence of a hydrogen bond donor in camphor altogether. A quantitative structure− activity relationship (QSAR) study by Kang et al. compared the permeation enhancement effect of 49 terpenes and terpenoids and showed that hydrophobic terpenes and those which are in liquid form at room temperature (such as nerol) are better permeation enhancers for haloperidol.62 Furthermore, FTIR studies by Zhang et al. demonstrated that extraction of the SC lipids by terpenes, similar to extraction by solvent CPEs, may be another mechanism of penetration enhancing effect.63 Similar to the WAXS results obtained for oleic acid, nerol resulted in a reduction of the height ratio of the peak at d = 3.77 Å to the peak at d = 4.17 Å. This observation supports a mechanism wherein disruption of the ceramide headgroup arrangement in the SC changes the membrane arrangement from orthorhombic to a more hexagonal phase. Furthermore, the intensity of peaks at d = 4.6 Å (q = 1.36 Å−1) in all cases of terpene CPEs points toward a mechanism wherein fluidization of SC membranes results in changes to the SC lipid lateral packing (Table 2B). The addition of IFNα to all CPE formulations resulted in an observable stabilizing effect in all SAXS plots, with the exception of nerol (Figure 1B, column 2). This effect may be a result of the high hydrogen bonding characteristic properties of nerol.64 Structural Effect of Surfactants. Benzalkonium chloride, DTAB, and SDS decreased both short and long lamellar periodicity distances. Nonionic surfactants, Tween 80 and 2253



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Figure 2. In vitro assessment of the permeation enhancement of IFNα by three different groups of chemical permeation enhancers: solvents, terpenes, and surfactants. IFNα absorption, determined by Bio-Plex immunoassay, is expressed as ng of IFNα/cm2 of skin (tape stripped) corresponding to images in Figure 4, column 2. Significant difference from control and other treatments was found for Tween 80 and Cremophor RH40, p < 0.05 by ANOVA and Tukey multiple comparison post-test. PBS, phosphate buffered saline; PG, propylene glycol; EtOH, ethanol; MeSA, methyl salicylate; BAK, benzalkonium chloride; CR RH40, Cremophor RH40; DTAB, didecyltrimethylammonium bromide; DDAB, didecyldimethylammonium bromide; SDS, sodium dodecyl sulfate.

induced by SDS,66,67 but this study is the first to demonstrate the appearance of this new peak. This difference might be a result of SDS concentration or the duration of SC treatment with the SDS solution (24 h). Data obtained from X-ray diffraction, differential scanning calorimetry (DSC), and FTIR analysis by other investigators confirmed the incorporation of surfactant molecules such as SDS and hexadecyltrimethylammonium bromide into the lipid bilayers resulting in the movement of packing and fluidity of the lipids but without changing their lamellar periodicities.28,65 Unlike our observations for solvent and terpene CPEs, surfactants all showed an increase in lipid disorder upon inclusion of IFNα in the formulations (Figure 1C). In case of Cremophor RH40, the presence of IFNα caused an uncharacteristic increase in this disordering effect. Overall, based on the SAXS and WAXS profiles for CPE alone and CPE-IFNα treated SC samples, several interesting observations emerged. As a general trend, both the solvent and terpene CPE groups exhibited similar disordering effect of SC lipids (Figure 1). However, upon addition of IFNα into samples, there was a general stabilization within the alkyl region of the membranes, as indicated by increasing definition of peaks at q = 0.098 and q = 0.14 Å−1 in the SAXS profiles (Figure 1, columns 1 and 2). Of the solvent CPEs, oleic acid demonstrated the greatest amount of fluidity in the alkyl region for both IFNα treated and untreated SC. In general, membrane disorder within the alkyl region followed the trend PG < ethanol < transcutol < oleic acid, exhibiting a similar degree of disorder for both the IFNα treated and untreated samples. This same trend was also observed when the WAXS profile was examined, indicating that the overall disorder and

Cremophor RH40, slightly decreased the peak intensity at q∼0.1 Å−1, which indicated a slight disordering effect on SC lamellar structure. Generally, nonionic surfactants have low toxicity, however; based on literature, this group of surfactants also has only a minor enhancement effect in human skin compared to ionic types,25 which could be explained by the low degree of SC structure disordering induction compared to ionic surfactants. All three of the cationic surfactants we examined (DDAB, DTAB, and benzalkonium chloride) disordered SC lamellar structure to a greater extent compared to nonionic surfactants as illustrated in the SAXS plots, and also decreased the WAXS peak intensity, a sign of disordering lateral packing (Figure 1C). In case of DTAB and benzalkonium chloride, this lateral packing disruption was so intense that the orthorhombic peak at d = 3.7 Å disappeared completely. The height ratio of peak d = 0.378 decreased to d = 0.417 after treating SC with all surfactants, showing that this group destroys orthorhombic packing of SC lipids. Furthermore, Cremophor RH40 fluidized the SC lipid structure to the lowest extent (appearance of a weak peak at d = 4.7 Å). Generally, surfactants affected the short lamellar spacing more as compared to the LPP (Table 2C). Interestingly, the anionic surfactant SDS destroyed the short and long lamellar phase, and a new broad peak appeared at around q = 0.2 Å−1 (d = 3.5 nm). This new peak could be explained as the interaction of SDS molecules with SC lipids followed by incorporation of SDS into the SC structure, creating a new lamellar structure. Ribaud et al.65 had previously observed lipid disorganization after SC treatment with SDS using X-ray diffraction studies. Several other studies also have shown a lipid fluidization effect 2254



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Figure 3. Chemical stability of IFNα in the CPE mixtures after 24 h at 32 °C, under conditions used in the in vitro absorption studies. IFNα concentration was determined by UPLC analysis. (A) Total IFNα stability in CPEs at T0 and T24. (B) Total IFNα stability in oleic acid and nerol over 4 h. The yellow line in both graphs represents the starting concentration of IFNα (125 ng/μL) that is added to each CPE. PBS, phosphate buffered saline; PG, propylene glycol; MeSA, methyl salicylate; BAK, benzalkonium chloride; CR RH40, Cremophor RH40; DTAB, didecyltrimethylammonium bromide; DDAB, didecyldimethylammonium bromide; SDS, sodium dodecyl sulfate.

differentially distributed fluid phases in human SC, however, this is still the subject of discussion.68 In contrast to the apparent stabilization of the SC membranes upon inclusion of IFNα into CPE formulations observed for the solvent and terpene families, surfactants demonstrated a significant destabilization upon addition of IFNα, according to the SAXS results (Figure 1C). In general the SAXS and WAXS effect of surfactants on SC membrane disorder and fluidity was more pronounced than that observed for solvent or terpenes (Figures 1A and 1B). Treatment of SC samples with surfactants resulted in a significant decrease in the orthorhombic to hexagonal lateral packing ratio, suggesting higher SC permeability (Table 2C). Cutaneous Absorption. In vitro cutaneous absorption studies showed that the permeation of IFNα into human skin was generally low in all of the CPE groups (Figure 2). This observation was in spite of the greater disorder and fluidization effects we observed on the lipid organizational structure for oleic acid, transcutol, nerol, and most of the surfactant CPEs. However, following the trend established for membrane disorder (Figure 1), the highest permeation into the skin was observed with surfactants, followed by the terpene and solvent

fluidity of the SC membrane followed the same trend upon treatment with the solvents. Changes in the WAXS ratio between intensities of the peaks at d = 3.7 Å and d = 4.1 Å are considered as changes in the orthorhombic to hexagonal phase ratio in isolated SC membranes. A notable difference between the SAXS and WAXS profiles was a significant deviation from the trend observed in the WAXS profile for oleic acid-treated SC samples (Figure 1, column 3). Laterally, lipids found in the SC are predominantly arranged in a crystalline orthorhombic arrangement, resulting in a very densely packed gel phase with slightly looser hexagonal structures (Table 2).20,46,47 In the orthorhombic phase, the distance between acyl chains is 4.1 and 3.7 Å, resulting in low membrane permeability.45 The hexagonal phase has a characteristic distance of 4.1 Å between hydrocarbon chains, resulting in moderate permeability of the membrane. Changing the membrane packing structure to a fluid phase (high permeability) produces a characteristic peak at 4.66 Å. Changes in lateral packing of SC lipids from orthorhombic to hexagonal to liquid phase are indicative of an increase in membrane permeability.24,43,46 Some studies have suggested the possibility of coexistence of solid and 2255



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Figure 4. continued

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Figure 4. Confocal microscopic imaging of human skin after treatment with CPE + IFNα mixtures in the in vitro diffusion cell study demonstrating the distribution of Alexa 594 (red) labeled IFNα on the skin surface (unstrapped skin, column 1) and upper stratum corneum and viable epidermal layer (stripped skin, column 2). (A) solvent CPEs, (B) terpene CPEs, and (C) surfactant CPEs. The 3D images show whole thickness and tapestripped human skin scanned in about 30 μm depth (z-scale); scales of depth are indicated on the y-axis of the graphs; the thickness of the stratum corneum (10−20 μm) was confirmed from light microscopic images. PBS, phosphate buffered saline; PG, propylene glycol; MeSA, methyl salicylate; BAK, benzalkonium chloride; CR RH40, Cremophor RH40; DTAB, didecyltrimethylammonium bromide; DDAB, didecyldimethylammonium bromide; SDS, sodium dodecyl sulfate. *Three different areas of the skin surface after stripping (I, II, III). Panel III showed some residue, which is probably due to the tape stripping method and representative of the incomplete removal of strongly bound DDAB-IFNα formulation. Bar = 20 μm.

CPEs (Figure 2). The highest permeation enhancement was achieved with Tween 80 and Cremophor RH40 with 6- and 3.7-fold enhancement compared with the IFNα solution without any CPE, which corresponds to 0.82% and 0.52% of IFNα dose, respectively. Cationic surfactants did not appear to promote permeation of IFNα to a greater extent. We have also evaluated the effect of CPEs on the stability of IFNα by UPLC coupled with fluorescence detection, to exclude the possibility that underestimation of IFNα absorption is not due to protein degradation. Our results demonstrate that, over a 24 h period, IFNα remained stable in most of the CPE formulations (Figure 3A). Therefore the detected levels of IFNα in treated skin samples are an accurate estimation of

absorption levels (Figure 2). Two exceptions were samples prepared with oleic acid and nerol, which demonstrated an absence of protein signal following the 24 h incubation. When we examined these two CPEs at earlier time points in the incubation, they both demonstrated significant loss of protein signal following 1 h of incubation at 32 °C (Figure 3B). Total protein levels in these formulations were reduced to below 10% after 4 h of incubation. In these two cases the IFNα levels in the skin are lower than expected due to the lower applied dose. IFNα distribution on the skin surface and within human skin after treatment with the various CPEs was imaged by confocal microscopy using Alexa 594-labeled IFNα (Figure 4). Samples in the “unstripped” column represent skin from which residual 2257



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Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, under Contract No. DE-AC0298CH10886. The research in this paper was supported by operating and equipment grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, and the Ontario Research Fund.

formulation was removed by pipetting off the remaining CPE/ IFNα solution, washing with water, and blotting dry but without tape stripping. In the “stripped” column, skin samples, cleaned as described above, were also tape-stripped twice to remove at least two layers of SC and any adhering formulation. In the unstripped samples, red fluorescence indicated the presence of IFNα in the upper layers of the skin in all cases, except when PBS or ethanol was used as the CPE. Tape stripping removed most surface-bound IFNα, and IFNα in the deeper layers of the skin could only be detected in the surfactant group (Figure 4). This was especially evident when examining Tween 80, Cremophor RH40, and SDS. This observation is consistent with the quantitative absorption data from homogenized skin samples (Figure 2). One exception was found in the case of DDAB treatment, when fluorescence could also be observed in the stripped sample (Figure 4C). Closer examination of different areas of the skin surface after stripping revealed that all other areas of the skin surface had minimal fluorescence as expected after tape stripping except one where there appeared to be noticeable residue (Figure 4C, * note). This residue is probably due to the tape stripping method and representative of the incomplete removal of the strongly bound DDAB-IFNα formulation.

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CONCLUSIONS In general, surfactants caused a greater degree of lipid disordering effect in SC and promoted higher levels of cutaneous absorption than did terpenes and solvents. Treatment of SC with various CPEs resulted in increased levels of disruption of either SPP or both SPP and LPP. For the solvent and terpene families of CPEs, the apparent degree of SC membrane disorder may be related to the overall size and degree of long chain alkyl functionality. Here, it seems that solvent and terpene CPEs having primarily ring structures, such as menthol and camphor, have less of an effect when compared to long chain alkyl containing compounds, such as nerol and oleic acid. This same trend also was observed when analyzing the long-range periodicity of the SC samples using WAXS. While this trend seems to hold for the solvent and terpene families, it does not apply as well for the surfactant family of CPEs. Among the solvents the order was transcutol < oleic acid (ethanol and PG had no effect), whereas among terpenes the order was menthol < camphor < nerol < methyl salicylate. Among surfactants permeation enhancement levels were benzalkonium chloride < SDS ≤ DDAB = DTAB < Cremophor RH40 < Tween 80. These studies indicated that overall the absorption of IFNα was relatively low and increased lipid disorder in the SC does not necessarily correspond to increased IFNα absorption.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (519) 888-4567, extension 21306. Fax: (519) 8837580. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Lin Yang for contribution to the SAXS experiments. Research involving SAXS was carried out at the National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY, which is supported by the U.S. 2258



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