Skin Research and Technology 2016; 22: 196–202 Printed in Singapore All rights reserved doi: 10.1111/srt.12250
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Skin Research and Technology
Effect of commercial cleansers on skin barrier permeability S. Hornby1, R. Walters1, N. Tierney1, Y. Appa1, G. Dorfman2 and Y. Kamath3 1
2
JOHNSON & JOHNSON Consumer Companies, Inc., Skillman, NJ, USA, Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA and 3Kamath Consulting Inc., Monmouth, NJ, USA
Background: Addition of hydrophobically modified polymers (HMPs) to cleansers can reduce the negative impact of surfactant-based cleansers. In this study, the effects of a cleanser containing HMPs, a gentle lotion cleanser (GLC), water, and 1% sodium lauryl sulfate (SLS) on barrier permeability, were evaluated in vitro in pig skin and in vivo in humans. Methods: Skin stratum corneum (SC) barrier function was quantitated by imaging fluorescence intensity of the sulforhodamine B (SRB) in a pig skin model system using 2-photon and conventional fluorescence confocal microscopy. Solutions containing SRB were applied to pig skin in Franz diffusion cells over a period of 2 h. Penetration of SRB into the skin was monitored from 2 lm to 38 lm. In vivo surfactant/cleanser penetration in human skin was determined using tape stripping.
contain at least one surfactant, or surface-acting-agent, that allows oils, dirt, sebum, and other unwanted substances to be washed from the skin more easily than with water alone (1, 2). However, surfactants can diffuse into the stratum corneum (SC) layer of the skin and disturb ordered lipid and cellular structures, leading to swelling (3). These effects lead to faster drying and cracking of the skin as a result of contractive stresses set up in the SC (4). The addition of hydrophobically modified polymers (HMPs) – large water soluble polymers that contain hydrophobic regions – to surfactant-based cleansers results in polymersurfactant complexes that are potentially less irritating to the SC (5–9). HMPs readily selfassemble with surfactants because of the attraction of the hydrophobic surfactant tail groups with the hydrophobic regions of the HMP; the hydrophobe on the surfactant is expelled by the water and onto the hydrophobe of the HMP. Addition of HMP to an aqueous solution of the surfactant sodium
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Results: After 2 h, water, 1% SLS, and GLC, significantly increased SRB intensity at all depths measured. SRB intensity was reduced in the HMP-cleanser group compared with other groups at each depth. In vivo, the presence of HMP reduced SLS penetration as measured by tape stripping. Conclusion: The cleanser containing HMP prevented changes in SC permeability and surfactant penetration, indicating a protective effect on skin barrier properties. Key words: hydrophobically modified polymers – surfactant – skin – cleanser – confocal microscopy – sulforhodamine B
Ó 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Accepted for publication 17 May 2015
dodecyl sulfate (SDS) reduces both SDS penetration into skin and SDS-induced swelling of the SC (8, 10). In this study, our primary objective was to visualize the effects on the SC barrier of a commercial mild cleanser formulation (CNF), an HMP-containing commercial formulation (NGC), a 1% solution of sodium lauryl sulfate (SLS), and water (no surfactant), using the transdermal diffusion of hydrophilic fluorescent dyes as a measure of SC barrier function. Dye diffusion into the SC was quantitated using two-photon fluorescence microscopy technique (11–14). In addition, the effect of HMP on SLS penetration into skin was quantitated using tape stripping.
Materials and Methods Skin Pig skin (Yucatan Miniature, white hairless, about 9 months old) was obtained from Sinclair Bio-Resources of Auxvasse, Missouri, cut into 2″ 9 2″ specimens, wrapped in aluminum foil,
Cleansers on skin barrier permeability
quick frozen in liquid nitrogen, and stored at 20°C.
Cleansers The tested cleansers used in this study were commercially available: Non-foaming lotion gentle cleanser (Cetaphilâ Gentle Skin Cleanser, Galderma Laboratories, L.P., Fort Worth, TX, USA; CNF); HMP-containing cleanser [(Neutrogena Ultra Gentle Daily Cleanser, Neutrogena Corp. Los Angeles CA (NGC)]. Solvents, phosphate buffered saline (PBS), anionic surfactant SLS, and the dye Sulforhodamine B (SRB) used in the permeation experiments were obtained from Sigma-Aldrich (St. Louis, MO, USA). Solutions of 1 wt% SLS and 1 wt% SLS with 1 wt% of the HMP potassium acrylates copolymer (Lubrizol) were created in deionized water.
Permeation experiments At the time of use, the skin sections were thawed overnight at ~5°C. The specimen surface was cleaned with PBS using a fine bristle sable brush, rinsed thoroughly with distilled water (DW), and mounted in PBS on a six cell array Franz diffusion cell apparatus (FDC, PermeGear Inc., Hellertown, PA, USA) dermis side down. SRB (0.02% final concentration) was added to DW, 1% SLS in water, and the test products CNF and NGC (diluted with DW to 80% of commercial concentration) and incubated on the SC side of the skin in the FDC for 2 h at 37°C. The solutions were then removed and the surface of the skin washed with DW several times until only a faint red color was visible in the washings. The round treated area of the skin sample was cut out with scissors and subjected to confocal fluorescence microscopy using an inverted confocal laser scanning microscope (Leica TCS SP2 with DMIRE2 base stand). Images were recorded at an excitation wavelength of 565 nm and emission wavelength of 586 nm at a magnification of 10X. Scans were performed at each depth 2, 8, 14, 20, 26, 32, and 38 lm to obtain an intensity vs. depth profile. Image analysis Four-to-six scans were made from different areas of each specimen and average intensities calculated using Macbiophotonics ImageJ soft-
ware (http://www.macbiophotonics.ca/downloads.htm). For each skin specimen, average intensities and standard deviations were calculated from replicate microscopic scans.
Time course of dye penetration For this experiment, the skin specimens were incubated in water without dye for 2 h in the FDC at 37°C followed by two washes with DW. Two drops of the DW, SLS (1%), CNF (80%), or NGC (80%) containing SRB (0.02%) were placed on a microscope slide. The SC side of the skin specimen was placed in contact with the test product and immediately mounted on the microscope and a suitable area was selected for imaging within 1 min after addition of the test products containing dye. Confocal scanning was performed every 2 min for 15 min. For each scan, 7 stacks of 21 images were obtained. Images at depths of 10 and 20 lm were taken at 1, 8, and 15 min after addition of the test product.
SLS penetration in vivo Tape stripping the SC is often used as a means to profile the depth of penetration of compounds into the SC (15, 16). For these studies, the volar forearms of two healthy adult female subjects were exposed to a solution of either SLS or SLS and HMP under a 25-millimeter diameter HillTopâ chamber occlusive patch (HillTop Research, St Petersburg, FL, USA) for 4 h. After removal of the occlusive patch, the test site was then rinsed with deionized water for 10 s. After 15 min, when the skin site was dry, 10 consecutive tape strips were taken from the test site using 22-millimeter diameter D-Squame Skin Sampling Discs (CuDerm Corporation, Dallas, TX, USA). Along with the two test sites exposed to a solution of SLS only, or SLS and HMP, 10 consecutive tape strips were taken of an adjacent unexposed, control site. The amount of SLS on each tape strip was quantified via colorimetric detection on each tape using a modified method based on previously published methodology (17). The total protein on each individual tape strip was also quantified as described previously (18). The SLS concentration on each tape strip (lg SLS/lg protein) was then calculated as amount of SLS/total protein for each tape
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strip. Each tape removes approximately 2 lm of the SC. The resulting depth profile is approximate because of the variation in the SC contour; thus each subsequent tape does not remove exclusively the next layer of cells of the SC (19).
Results Typical fluorescence photomicrographs of the hydrophilic dye SRB are shown below in Fig. 1 at penetration depths of 2, 10, 20, and 40 lm. From the images it is clear that the hydrophilic dye penetrates more easily and more extensively in specimens treated with DW, 1% SLS and CNF, a cleanser containing SLS in its formulation, than the HMP-containing NGC. The fluorescence intensity was highest in the intercellular spaces, areas of high SC lipid content, with the corneocytes exhibiting less fluorescence intensity. Cell faces appear red because the dye penetrates into the intercellular regions between the cells horizontally. In the case of NGC-treated specimens, both intercellular regions and cell faces exhibited lower fluorescence because the amount of hydrophilic dye penetrating into the specimen was apparently reduced.
Image analysis An image analysis approach was used to quantitate dye localization. For this purpose we conducted FDC experiments on five skin specimens for CNF and NGC and three each for DW and SLS. A typical area selected for image analysis in the photomicrograph is shown in Fig. 2. The image shows several corneocytes, often surrounded by bright lines. Because the bright lines are glyphs in the skin where the dye had accumulated, we selected corneocyte areas for analysis that excluded the bright red edge lines, as shown by the blue line. Averaged fluorescence intensity ( 95% confidence intervals) vs. depth from all experiments is shown in Table 1 and Fig. 3. The fluorescence intensity decreases with increasing depth into the skin (Fig. 3). DW, CNF, and SLS plots were almost coincident (not significantly different at the 95% confidence level), whereas the fluorescence intensity in the NGC group was much lower over all of the depths measured, clearly showing the preservation of barrier integrity.
Time course of dye penetration Images at depths of 10 and 20 lm were taken at 1, 8, and 15 min after addition of the test
Depth 2 µm
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Fig. 1. Fluorescence photomicrographs of SRB-penetrated SC are shown at penetration depths of 2, 10, 20, and 40 lm for the water, NGC, CNF, and SLS treated specimens.
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10 lm depth by 8 min. There were significant differences between CNF and water and SLS at all depths and at all times. However, the differences between water and SLS were minor. In contrast, dye penetration was slower after addition of NGC, with the 15-min time point at 10 lm depth appearing similar to the CNF 1min time point at 10 lm depth.
Fig. 2. Selected regions of corneocytes excluding the bright red edge lines were used for the calculations of averaged intensities. TABLE 1. Fluorescence intensity by depth from surface Fluorescence intensity Average 95% confidence interval Arbitrary units Depth from surface lm
Water
2 8 14 20 26 32 38
211 184 160 137 104 101 87
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SLS 1% 11 17 21 23 28 22 21
221 196 170 145 124 107 92
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Fig. 3. Average intensities (95% confidence interval) for water (control), NGC, CNF, and SLS treated skin specimens.
products are shown in Fig. 4. In all treatments, the dye filled the glyphs on the outside of the skin immediately and then penetrated much more slowly into the SC. In the case of CNF, the barrier penetration began within the first minute and was extensively penetrated at
Surfactant penetration Tape stripping was used to determine the depth of SLS penetration into SC of the human forearm in vivo. After 4 h of exposure (Fig. 5a), the presence of HMP resulted in a reduction in the SLS concentration found in the SC. As expected, the SLS concentration per tape strip decreased with increasing depth into the SC. By approximately the 7th tape (~14 lm), the SLS concentration found in the tape strip was not distinguishable from adjacent skin not exposed to SLS (unexposed control site). When re-plotted as the cumulative amount of SLS normalized to the protein removed on each tape (Fig. 5b), addition of HMP to the SLS solution resulted in a 47% reduction in the amount of SLS found in the SC.
Discussion The SC has often been referred to as a ‘brick and mortar’ structure (20). The disruption of the lipid bilayers and the loss of lipid molecules by surfactants leave regions through which permeants and water can diffuse more easily and to deeper levels in the SC and the viable epidermis (21). We surmise that increased permeability (barrier damage) in these experiments is caused by both water (22, 23) and surfactants (3) from the cleanser formulation. The lipid envelopes of adjacent corneocytes provide cellular adhesion by the formation of hydrophobic associations between lipids (24). The hydrophobicity of this lipid barrier provides resistance to water transport. As ionic surfactants diffuse into the SC, the surfactant can insert into the ordered SC lipids and thereby disrupt the lipid order and degrade the barrier (25). The ionic head group of the surfactant can attract water molecules into the structure to weaken it further by swelling. This leads to the formation of porous regions between cells, which can be occupied
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(c)
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Fig. 4. Representative fluorescence photomicrographs of skin specimens as a function of exposure time and tissue depth. (a) NGC, (b) CNF, (c) control, (d) SLS. (b)
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Fig. 5. Effect of HMP on SLS penetration in to human skin. After 4 h of exposure to SLS or SLS + HMP, the depth of SLS penetration was estimated by tape stripping. Consecutive strips were removed with the surface (tape strip #1) through the SC (tape strip #10). Absolute concentrations of SLS (a); Cumulative SLS concentration (b). Error bars show standard deviation. Control = SLS concentration in adjacent unexposed control test site.
by the fluorescent dye molecules. Closer to the surface we see bright fluorescence in the lamellae and a lighter intensity in the cell faces. Diffusion of the dye inside the corneocytes was significantly lower, especially at increased depths, as indicated by the darker cell faces.
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The better barrier protection efficacy of NGC is likely to be due to several components within the formulation, including glycerol and HMPs. Glycerol reduces the thermodynamic activity of water by forming hydrogen bonds with water molecules, rendering them less reactive toward
Cleansers on skin barrier permeability
barrier components, and has been demonstrated to inhibit SDS penetration of the skin barrier (14). By binding with surfactant, HMPs create a more stable environment for the surfactant, leading to slower surfactant dynamics, reducing transport of surfactant into the SC (7, 8) and reducing the ability of surfactants to disrupt the barrier. Thus, NGC acts as a barrier protector, limiting the barrier disruption by the surfactant and resulting in less SRB dye from penetrating the lamellae in the SC as compared with CNF. Interestingly, NGC appeared to attenuate the changes induced by water alone. Exposure of skin to water can result in changes to SC ultrastructure. Initial treatment of the skin samples in our study with DW for 2 h at 37°C may have initiated changes in the barrier similar to those observed by Warner and co-workers (26). After a 2-h incubation of porcine skin with DW at 46°C, they noted a disordered SC with greater spaces between cells than in skin not exposed to water. It may be that the initial exposure to water as part of the sample preparation may have potentiated and/or masked effects of the test cleansers. It might also be interpreted as a potential effect of repeated exposures. The transport of surfactants like SLS into the SC has both a kinetic and thermodynamic component. The SC is a heterogeneous material and the intercellular continuous lipids phase is different from the coenocytes and there is variability with depth. In a tape stripping study in rat skin, SLS was found to preferentially partition into the SC lipid phase after 6 h (27), and that the partitioning of SLS was maximal in the first five strips, similar to the results described herein. Furthermore, exposure of skin samples to SLS for 24 h did not result in an increase in the distribution of the surfactant to depths
References 1. Abbas S, Goldberg JW, Massaro M. Personal cleanser technology and clinical performance. Dermatol Ther 2004; 17: 35–42. 2. Ananthapadmanabhan KP, Moore DJ, Subramanyan K, Misra M, Meyer F. Cleansing without compromise: the impact of cleansers on the skin barrier and the technology
corresponding to tape strips 6–15 (27). These data are also consistent with studies imaging the penetration of SDS into porcine skin using confocal raman and infrared microspectroscopy (28). After a 3 h exposure, Mao et al. (28) demonstrated a notable distribution of SDS to at least 40 lm, with higher concentrations toward the surface of the SC. Increasing the time of exposure to 24 h or 40 h did not alter the relative distribution pattern. The results obtained in this work show conclusively that NGC protects the skin barrier over both short-term and long-term experiments. We surmise, indirectly, that the differences noted between cleansers could be due to the presence of SLS in CNF and its absence in NGC. Coincidence of the fluorescence intensity data for CNF and SLS lends support to this observation. NGC also contains HMPs, which interact rather strongly with surfactants, changing their micellar behavior, and associating with them to form larger micelles, thus preventing their entry into the lamellae between skin cells. Interestingly, NGC may protect the skin barrier from changes in barrier function induced by water alone.
Acknowledgement The authors thank Alex Loeb, PhD, CMPP of Evidence Scientific Solutions, Philadelphia, PA, for editorial and medical writing support, which was funded by JOHNSON & JOHNSON Consumer Companies, Inc. (Skillman, NJ, USA). These studies were fully funded by JOHNSON & JOHNSON Consumer Companies, Inc. (Skillman, NJ, USA). SH, RW, NT, and YA are, or were employees of JOHNSON & JOHNSON Consumer Companies, Inc. at the time this study was performed.
of mild cleansing. Dermatol Ther 2004; 17: 16–25. 3. Rhein LD, Robbins CR, Fernee K, Cantore R. Surfactant structure effects on swelling of isolated human stratum corneum. J Soc Cosmet Chem 1986; 37: 125–139. 4. Levi K, Weber RJ, Do JQ, Dauskardt RH. Drying stress and damage processes in human stratum corneum. Int J Cosmet Sci 2010; 32: 276–293.
5. LiBrizzi JJ, Walters RM, Fevola M, Tamareselvy K. Low-irritation compositions and methods of making the same. US Patent 7,803,403 September 28, 2010. 6. Draelos Z, Hornby S, Walters RM, Appa Y. Hydrophobically modified polymers can minimize skin irritation potential caused by surfactantbased cleansers. J Cosmet Dermatol 2013; 12: 314–321.
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Hornby et al. 7. Walters RM, Fevola MJ, Gandolfi L, LiBrizzi JJ, Tamareselvy K, Tierney N. Polymer-surfactant selfassembly for the design of mild skin cleansers. Polym Prepr 2011; 52: 697–698. 8. Fevola MJ, Walters RM, LiBrizzi JJ. A new approach to formulating mild cleansers: hydrophobically modified polymers for irritation mitigation. In: Morgan S, ed. Polymeric Delivery of Therapeutics. Washington, DC, USA: American Chemical Society; 2010: 221–242. 9. Walters RM, Mao G, Gunn ET, Hornby S. Cleansing formulations that respect skin barrier integrity. Dermatol Res Pract 2012; 2012: 495917. 10. Moore PN, Puvvada S, Blankschtein D. Challenging the surfactant monomer skin penetration model: penetration of sodium dodecyl sulfate micelles into the epidermis. J Cosmet Sci 2003; 54: 29–46. 11. Yu B, Kim KH, So PT, Blankschtein D, Langer R. Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy. J Invest Dermatol 2003; 120: 448–455. 12. Yu B, Dong CY, So PT, Blankschtein D, Langer R. In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy. J Invest Dermatol 2001; 117: 16–25. 13. Kushner JT, Kim D, So PT, Blankschtein D, Langer RS. Dual-channel two-photon microscopy study of transdermal transport in skin treated with low-frequency ultrasound and a chemical enhancer. J Invest Dermatol 2007; 127: 2832–2846. 14. Ghosh S, Kim D, So P, Blankschtein D. Visualization and quantification of skin barrier perturbation induced by surfactant-humectant systems using two-photon fluores-
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cence microscopy. J Cosmet Sci 2008; 59: 263–289. Mohammed D, Matts PJ, Hadgraft J, Lane ME. Depth profiling of stratum corneum biophysical and molecular properties. Br J Dermatol 2011; 164: 957–965. Boncheva M, Tay FH, Kazarian SG. Application of attenuated total reflection Fourier transform infrared imaging and tape stripping to investigate the three-dimensional distribution of exogenous chemicals and the molecular organization in Stratum corneum. J Biomed Opt 2008; 13: 064009. Rusconi F, Valton E, Nguyen R, Dufourc E. Quantification of sodium dodecyl sulfate in microliter-volume biochemical samples by visible light spectroscopy. Anal Biochem 2001; 295: 31–37. de Jongh CM, Jakasa I, Verberk MM, Kezic S. Variation in barrier impairment and inflammation of human skin as determined by sodium lauryl sulphate penetration rate. Br J Dermatol 2006; 154: 651– 657. Herkenne C, Alberti I, Naik A, Kalia YN, Mathy FX, Preat V, Guy RH. In vivo methods for the assessment of topical drug bioavailability. Pharm Res 2008; 25: 87–103. Petersen R. Ceramides-Key components for skin protection. Cosmetics & Toiletries 1992; 107: 45–49. Polat BE, Figueroa PL, Blankschtein D, Langer R. Transport pathways and enhancement mechanisms within localized and non-localized transport regions in skin treated with low-frequency sonophoresis and sodium lauryl sulfate. J Pharm Sci 2011; 100: 512–529. Bouwstra JA, de Graaff A, Gooris GS, Nijsse J, Wiechers JW, van Aelst AC. Water distribution and related morphology in human stratum corneum at different
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hydration levels. J Invest Dermatol 2003; 120: 750–758. Warner RR, Stone KJ, Boissy YL. Hydration disrupts human stratum corneum ultrastructure. J Invest Dermatol 2003; 120: 275–284. Wertz PW, Swartzendruber DC, Kitko DJ, Madison KC, Downing DT. The role of the corneocyte lipid envelopes in cohesion of the stratum corneum. J Invest Dermatol 1989; 93: 169–172. Saad P, Flach CR, Walters RM, Mendelsohn R. Infrared spectroscopic studies of sodium dodecyl sulphate permeation and interaction with stratum corneum lipids in skin. Int J Cosmet Sci 2012; 34: 36–43. Warner RR, Boissy YL, Lilly NA, Spears MJ, McKillop K, Marshall JL, Stone KJ. Water disrupts stratum corneum lipid lamellae: damage is similar to surfactants. J Invest Dermatol 1999; 113: 960– 966. Patil S, Singh P, Sarasour K, Maibach H. Quantification of sodium lauryl sulfate penetration into the skin and underlying tissue after topical application–pharmacological and toxicological implications. J Pharm Sci 1995; 84: 1240–1244. Mao G, Flach CR, Mendelsohn R, Walters RM. Imaging the distribution of sodium dodecyl sulfate in skin by confocal Raman and infrared microspectroscopy. Pharm Res 2012; 29: 2189–2201.
Address: S. Hornby JOHNSON & JOHNSON Consumer Companies, Inc. 199 Grandview Road Skillman NJ 08558 USA Tel: +908 874 2756 Fax: +908 874 1206 e-mail: [email protected]
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Skin Research and Technology
Effect of commercial cleansers on skin barrier permeability S. Hornby1, R. Walters1, N. Tierney1, Y. Appa1, G. Dorfman2 and Y. Kamath3 1
2
JOHNSON & JOHNSON Consumer Companies, Inc., Skillman, NJ, USA, Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA and 3Kamath Consulting Inc., Monmouth, NJ, USA
Background: Addition of hydrophobically modified polymers (HMPs) to cleansers can reduce the negative impact of surfactant-based cleansers. In this study, the effects of a cleanser containing HMPs, a gentle lotion cleanser (GLC), water, and 1% sodium lauryl sulfate (SLS) on barrier permeability, were evaluated in vitro in pig skin and in vivo in humans. Methods: Skin stratum corneum (SC) barrier function was quantitated by imaging fluorescence intensity of the sulforhodamine B (SRB) in a pig skin model system using 2-photon and conventional fluorescence confocal microscopy. Solutions containing SRB were applied to pig skin in Franz diffusion cells over a period of 2 h. Penetration of SRB into the skin was monitored from 2 lm to 38 lm. In vivo surfactant/cleanser penetration in human skin was determined using tape stripping.
contain at least one surfactant, or surface-acting-agent, that allows oils, dirt, sebum, and other unwanted substances to be washed from the skin more easily than with water alone (1, 2). However, surfactants can diffuse into the stratum corneum (SC) layer of the skin and disturb ordered lipid and cellular structures, leading to swelling (3). These effects lead to faster drying and cracking of the skin as a result of contractive stresses set up in the SC (4). The addition of hydrophobically modified polymers (HMPs) – large water soluble polymers that contain hydrophobic regions – to surfactant-based cleansers results in polymersurfactant complexes that are potentially less irritating to the SC (5–9). HMPs readily selfassemble with surfactants because of the attraction of the hydrophobic surfactant tail groups with the hydrophobic regions of the HMP; the hydrophobe on the surfactant is expelled by the water and onto the hydrophobe of the HMP. Addition of HMP to an aqueous solution of the surfactant sodium
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Results: After 2 h, water, 1% SLS, and GLC, significantly increased SRB intensity at all depths measured. SRB intensity was reduced in the HMP-cleanser group compared with other groups at each depth. In vivo, the presence of HMP reduced SLS penetration as measured by tape stripping. Conclusion: The cleanser containing HMP prevented changes in SC permeability and surfactant penetration, indicating a protective effect on skin barrier properties. Key words: hydrophobically modified polymers – surfactant – skin – cleanser – confocal microscopy – sulforhodamine B
Ó 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Accepted for publication 17 May 2015
dodecyl sulfate (SDS) reduces both SDS penetration into skin and SDS-induced swelling of the SC (8, 10). In this study, our primary objective was to visualize the effects on the SC barrier of a commercial mild cleanser formulation (CNF), an HMP-containing commercial formulation (NGC), a 1% solution of sodium lauryl sulfate (SLS), and water (no surfactant), using the transdermal diffusion of hydrophilic fluorescent dyes as a measure of SC barrier function. Dye diffusion into the SC was quantitated using two-photon fluorescence microscopy technique (11–14). In addition, the effect of HMP on SLS penetration into skin was quantitated using tape stripping.
Materials and Methods Skin Pig skin (Yucatan Miniature, white hairless, about 9 months old) was obtained from Sinclair Bio-Resources of Auxvasse, Missouri, cut into 2″ 9 2″ specimens, wrapped in aluminum foil,
Cleansers on skin barrier permeability
quick frozen in liquid nitrogen, and stored at 20°C.
Cleansers The tested cleansers used in this study were commercially available: Non-foaming lotion gentle cleanser (Cetaphilâ Gentle Skin Cleanser, Galderma Laboratories, L.P., Fort Worth, TX, USA; CNF); HMP-containing cleanser [(Neutrogena Ultra Gentle Daily Cleanser, Neutrogena Corp. Los Angeles CA (NGC)]. Solvents, phosphate buffered saline (PBS), anionic surfactant SLS, and the dye Sulforhodamine B (SRB) used in the permeation experiments were obtained from Sigma-Aldrich (St. Louis, MO, USA). Solutions of 1 wt% SLS and 1 wt% SLS with 1 wt% of the HMP potassium acrylates copolymer (Lubrizol) were created in deionized water.
Permeation experiments At the time of use, the skin sections were thawed overnight at ~5°C. The specimen surface was cleaned with PBS using a fine bristle sable brush, rinsed thoroughly with distilled water (DW), and mounted in PBS on a six cell array Franz diffusion cell apparatus (FDC, PermeGear Inc., Hellertown, PA, USA) dermis side down. SRB (0.02% final concentration) was added to DW, 1% SLS in water, and the test products CNF and NGC (diluted with DW to 80% of commercial concentration) and incubated on the SC side of the skin in the FDC for 2 h at 37°C. The solutions were then removed and the surface of the skin washed with DW several times until only a faint red color was visible in the washings. The round treated area of the skin sample was cut out with scissors and subjected to confocal fluorescence microscopy using an inverted confocal laser scanning microscope (Leica TCS SP2 with DMIRE2 base stand). Images were recorded at an excitation wavelength of 565 nm and emission wavelength of 586 nm at a magnification of 10X. Scans were performed at each depth 2, 8, 14, 20, 26, 32, and 38 lm to obtain an intensity vs. depth profile. Image analysis Four-to-six scans were made from different areas of each specimen and average intensities calculated using Macbiophotonics ImageJ soft-
ware (http://www.macbiophotonics.ca/downloads.htm). For each skin specimen, average intensities and standard deviations were calculated from replicate microscopic scans.
Time course of dye penetration For this experiment, the skin specimens were incubated in water without dye for 2 h in the FDC at 37°C followed by two washes with DW. Two drops of the DW, SLS (1%), CNF (80%), or NGC (80%) containing SRB (0.02%) were placed on a microscope slide. The SC side of the skin specimen was placed in contact with the test product and immediately mounted on the microscope and a suitable area was selected for imaging within 1 min after addition of the test products containing dye. Confocal scanning was performed every 2 min for 15 min. For each scan, 7 stacks of 21 images were obtained. Images at depths of 10 and 20 lm were taken at 1, 8, and 15 min after addition of the test product.
SLS penetration in vivo Tape stripping the SC is often used as a means to profile the depth of penetration of compounds into the SC (15, 16). For these studies, the volar forearms of two healthy adult female subjects were exposed to a solution of either SLS or SLS and HMP under a 25-millimeter diameter HillTopâ chamber occlusive patch (HillTop Research, St Petersburg, FL, USA) for 4 h. After removal of the occlusive patch, the test site was then rinsed with deionized water for 10 s. After 15 min, when the skin site was dry, 10 consecutive tape strips were taken from the test site using 22-millimeter diameter D-Squame Skin Sampling Discs (CuDerm Corporation, Dallas, TX, USA). Along with the two test sites exposed to a solution of SLS only, or SLS and HMP, 10 consecutive tape strips were taken of an adjacent unexposed, control site. The amount of SLS on each tape strip was quantified via colorimetric detection on each tape using a modified method based on previously published methodology (17). The total protein on each individual tape strip was also quantified as described previously (18). The SLS concentration on each tape strip (lg SLS/lg protein) was then calculated as amount of SLS/total protein for each tape
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strip. Each tape removes approximately 2 lm of the SC. The resulting depth profile is approximate because of the variation in the SC contour; thus each subsequent tape does not remove exclusively the next layer of cells of the SC (19).
Results Typical fluorescence photomicrographs of the hydrophilic dye SRB are shown below in Fig. 1 at penetration depths of 2, 10, 20, and 40 lm. From the images it is clear that the hydrophilic dye penetrates more easily and more extensively in specimens treated with DW, 1% SLS and CNF, a cleanser containing SLS in its formulation, than the HMP-containing NGC. The fluorescence intensity was highest in the intercellular spaces, areas of high SC lipid content, with the corneocytes exhibiting less fluorescence intensity. Cell faces appear red because the dye penetrates into the intercellular regions between the cells horizontally. In the case of NGC-treated specimens, both intercellular regions and cell faces exhibited lower fluorescence because the amount of hydrophilic dye penetrating into the specimen was apparently reduced.
Image analysis An image analysis approach was used to quantitate dye localization. For this purpose we conducted FDC experiments on five skin specimens for CNF and NGC and three each for DW and SLS. A typical area selected for image analysis in the photomicrograph is shown in Fig. 2. The image shows several corneocytes, often surrounded by bright lines. Because the bright lines are glyphs in the skin where the dye had accumulated, we selected corneocyte areas for analysis that excluded the bright red edge lines, as shown by the blue line. Averaged fluorescence intensity ( 95% confidence intervals) vs. depth from all experiments is shown in Table 1 and Fig. 3. The fluorescence intensity decreases with increasing depth into the skin (Fig. 3). DW, CNF, and SLS plots were almost coincident (not significantly different at the 95% confidence level), whereas the fluorescence intensity in the NGC group was much lower over all of the depths measured, clearly showing the preservation of barrier integrity.
Time course of dye penetration Images at depths of 10 and 20 lm were taken at 1, 8, and 15 min after addition of the test
Depth 2 µm
10 µm
20 µm
40 µm
Control
NGC
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Fig. 1. Fluorescence photomicrographs of SRB-penetrated SC are shown at penetration depths of 2, 10, 20, and 40 lm for the water, NGC, CNF, and SLS treated specimens.
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10 lm depth by 8 min. There were significant differences between CNF and water and SLS at all depths and at all times. However, the differences between water and SLS were minor. In contrast, dye penetration was slower after addition of NGC, with the 15-min time point at 10 lm depth appearing similar to the CNF 1min time point at 10 lm depth.
Fig. 2. Selected regions of corneocytes excluding the bright red edge lines were used for the calculations of averaged intensities. TABLE 1. Fluorescence intensity by depth from surface Fluorescence intensity Average 95% confidence interval Arbitrary units Depth from surface lm
Water
2 8 14 20 26 32 38
211 184 160 137 104 101 87
Controls
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SLS 1% 11 17 21 23 28 22 21
221 196 170 145 124 107 92
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NGC
8 9 10 11 12 13 13
114 86 64 49 37 28 23
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CNF 16 13 10 9 7 6 5
203 181 161 143 126 112 100
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Averaged intensity (a.u.)
200 180 160 140 120 100 80 60 40 20 0 2
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Penetration depth (µm)
Fig. 3. Average intensities (95% confidence interval) for water (control), NGC, CNF, and SLS treated skin specimens.
products are shown in Fig. 4. In all treatments, the dye filled the glyphs on the outside of the skin immediately and then penetrated much more slowly into the SC. In the case of CNF, the barrier penetration began within the first minute and was extensively penetrated at
Surfactant penetration Tape stripping was used to determine the depth of SLS penetration into SC of the human forearm in vivo. After 4 h of exposure (Fig. 5a), the presence of HMP resulted in a reduction in the SLS concentration found in the SC. As expected, the SLS concentration per tape strip decreased with increasing depth into the SC. By approximately the 7th tape (~14 lm), the SLS concentration found in the tape strip was not distinguishable from adjacent skin not exposed to SLS (unexposed control site). When re-plotted as the cumulative amount of SLS normalized to the protein removed on each tape (Fig. 5b), addition of HMP to the SLS solution resulted in a 47% reduction in the amount of SLS found in the SC.
Discussion The SC has often been referred to as a ‘brick and mortar’ structure (20). The disruption of the lipid bilayers and the loss of lipid molecules by surfactants leave regions through which permeants and water can diffuse more easily and to deeper levels in the SC and the viable epidermis (21). We surmise that increased permeability (barrier damage) in these experiments is caused by both water (22, 23) and surfactants (3) from the cleanser formulation. The lipid envelopes of adjacent corneocytes provide cellular adhesion by the formation of hydrophobic associations between lipids (24). The hydrophobicity of this lipid barrier provides resistance to water transport. As ionic surfactants diffuse into the SC, the surfactant can insert into the ordered SC lipids and thereby disrupt the lipid order and degrade the barrier (25). The ionic head group of the surfactant can attract water molecules into the structure to weaken it further by swelling. This leads to the formation of porous regions between cells, which can be occupied
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(c)
10 µm
1 min
(b)
8 min
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1 min
(d)
8 min
20 µm
15 min
10 µm
1 min
8 min
10 µm
20 µm
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1 min
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Fig. 4. Representative fluorescence photomicrographs of skin specimens as a function of exposure time and tissue depth. (a) NGC, (b) CNF, (c) control, (d) SLS. (b)
4.0
1% SLS
1.4
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Normalized SLS conc. (µg SLS/µg protein)
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~depth into skin, tape strip # (1 = surface, 2–10 = into skin)
Fig. 5. Effect of HMP on SLS penetration in to human skin. After 4 h of exposure to SLS or SLS + HMP, the depth of SLS penetration was estimated by tape stripping. Consecutive strips were removed with the surface (tape strip #1) through the SC (tape strip #10). Absolute concentrations of SLS (a); Cumulative SLS concentration (b). Error bars show standard deviation. Control = SLS concentration in adjacent unexposed control test site.
by the fluorescent dye molecules. Closer to the surface we see bright fluorescence in the lamellae and a lighter intensity in the cell faces. Diffusion of the dye inside the corneocytes was significantly lower, especially at increased depths, as indicated by the darker cell faces.
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The better barrier protection efficacy of NGC is likely to be due to several components within the formulation, including glycerol and HMPs. Glycerol reduces the thermodynamic activity of water by forming hydrogen bonds with water molecules, rendering them less reactive toward
Cleansers on skin barrier permeability
barrier components, and has been demonstrated to inhibit SDS penetration of the skin barrier (14). By binding with surfactant, HMPs create a more stable environment for the surfactant, leading to slower surfactant dynamics, reducing transport of surfactant into the SC (7, 8) and reducing the ability of surfactants to disrupt the barrier. Thus, NGC acts as a barrier protector, limiting the barrier disruption by the surfactant and resulting in less SRB dye from penetrating the lamellae in the SC as compared with CNF. Interestingly, NGC appeared to attenuate the changes induced by water alone. Exposure of skin to water can result in changes to SC ultrastructure. Initial treatment of the skin samples in our study with DW for 2 h at 37°C may have initiated changes in the barrier similar to those observed by Warner and co-workers (26). After a 2-h incubation of porcine skin with DW at 46°C, they noted a disordered SC with greater spaces between cells than in skin not exposed to water. It may be that the initial exposure to water as part of the sample preparation may have potentiated and/or masked effects of the test cleansers. It might also be interpreted as a potential effect of repeated exposures. The transport of surfactants like SLS into the SC has both a kinetic and thermodynamic component. The SC is a heterogeneous material and the intercellular continuous lipids phase is different from the coenocytes and there is variability with depth. In a tape stripping study in rat skin, SLS was found to preferentially partition into the SC lipid phase after 6 h (27), and that the partitioning of SLS was maximal in the first five strips, similar to the results described herein. Furthermore, exposure of skin samples to SLS for 24 h did not result in an increase in the distribution of the surfactant to depths
References 1. Abbas S, Goldberg JW, Massaro M. Personal cleanser technology and clinical performance. Dermatol Ther 2004; 17: 35–42. 2. Ananthapadmanabhan KP, Moore DJ, Subramanyan K, Misra M, Meyer F. Cleansing without compromise: the impact of cleansers on the skin barrier and the technology
corresponding to tape strips 6–15 (27). These data are also consistent with studies imaging the penetration of SDS into porcine skin using confocal raman and infrared microspectroscopy (28). After a 3 h exposure, Mao et al. (28) demonstrated a notable distribution of SDS to at least 40 lm, with higher concentrations toward the surface of the SC. Increasing the time of exposure to 24 h or 40 h did not alter the relative distribution pattern. The results obtained in this work show conclusively that NGC protects the skin barrier over both short-term and long-term experiments. We surmise, indirectly, that the differences noted between cleansers could be due to the presence of SLS in CNF and its absence in NGC. Coincidence of the fluorescence intensity data for CNF and SLS lends support to this observation. NGC also contains HMPs, which interact rather strongly with surfactants, changing their micellar behavior, and associating with them to form larger micelles, thus preventing their entry into the lamellae between skin cells. Interestingly, NGC may protect the skin barrier from changes in barrier function induced by water alone.
Acknowledgement The authors thank Alex Loeb, PhD, CMPP of Evidence Scientific Solutions, Philadelphia, PA, for editorial and medical writing support, which was funded by JOHNSON & JOHNSON Consumer Companies, Inc. (Skillman, NJ, USA). These studies were fully funded by JOHNSON & JOHNSON Consumer Companies, Inc. (Skillman, NJ, USA). SH, RW, NT, and YA are, or were employees of JOHNSON & JOHNSON Consumer Companies, Inc. at the time this study was performed.
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Address: S. Hornby JOHNSON & JOHNSON Consumer Companies, Inc. 199 Grandview Road Skillman NJ 08558 USA Tel: +908 874 2756 Fax: +908 874 1206 e-mail: [email protected]
