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Control of human skin wettability using the pH of anionic surfactant solution treatments
Accepted Manuscript Title: Control of human skin wettability using the pH of anionic surfactant solution treatments Authors: L. Bromberg, X. Liu, I. Wang, S. Smith, K. Schwicker, Z. Eller, G.K. German PII: DOI: Reference:
S0927-7765(17)30357-0 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.06.009 COLSUB 8619
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
29-11-2016 18-5-2017 8-6-2017
Please cite this article as: L.Bromberg, X.Liu, I.Wang, S.Smith, K.Schwicker, Z.Eller, G.K.German, Control of human skin wettability using the pH of anionic surfactant solution treatments, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.06.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Control of human skin wettability using the pH of anionic surfactant solution treatments. L. Bromberga, X. Liub, I. Wangb, S. Smithb, K. Schwickerb, Z. Ellerb, and G. K. Germanb* a b *Guy
Department of Chemistry, Binghamton University, Binghamton, New York, U.S.A.
Department of Biomedical Engineering, Binghamton University, Binghamton, New York, U.S.A. K. German, BI2609, Department of Biomedical Engineering, Binghamton University, 4400 Vestal Parkway East,
Binghamton, New York, 13902, Tel: +1 607-777-4270, Fax: +1 607-777-5780, Email: [email protected]
Graphical Abstract
Highlights
The wettability of human stratum corneum is examined. Changes in the pH of anionic surfactant treatments can alter skin wettability. Results suggest a reorientation of bound surfactant monomers. Lipids appear to reduce surfactant binding to the stratum corneum.
Abstract The outermost layer of human skin, or stratum corneum, acts as a protective barrier between underlying living tissue and the external environment. The wettability of this tissue layer can influence spreading of chemicals and the adhesion of pathogenic microorganisms. We show in this article that the wettability of isolated human stratum corneum can be controlled through treatment with solutions of the anionic surfactant, sodium lauryl sulfate, buffered to different pH values. Relative to control treatments with the buffer solution alone, surfactant solution treatments under acidic conditions cause delipidated stratum corneum to become more hydrophobic. In contrast, alkaline conditions cause the stratum corneum to become more hydrophilic; irrespective of lipid composition. This transition is consistent with a reorientation of bound surfactants at the tissue interface. Under acidic conditions, electrostatic binding of negatively charged surfactant head groups with positively charged keratin in the stratum corneum would increase tissue hydrophobicity due to the exposed hydrophobic tails. However, a hydrophobic based attraction of the apolar surfactant tails to the stratum corneum surface under alkaline conditions would leave the hydrophilic surfactant head groups exposed, causing increased tissue hydrophilicity. Changes in wettability with pH become diminished when lipids ordinarily found in stratum corneum are present, suggesting the lipids partially inhibit surfactant binding. Profilometry studies of the tissue topography highlight that surfactant induced changes in stratum corneum surface roughness cannot account for the observed changes in wettability.
Introduction Amphiphilic surfactants readily adsorb to liquid surfaces; whereupon they reduce the interfacial surface energy. This behavior makes them useful as foaming agents and emulsifiers. The ability of surfactants to emulsify lipids means that surfactants are widely used in cosmetic cleansing products[1]. Healthy skin conditions are regulated by the presence of lipids and hygroscopic natural moisturizing factors [2–4] that minimize water loss across the epidermis and keep the outermost layer, or stratum corneum (SC), hydrated. A depletion of SC lipids from cleansing [5,6] can cause barrier dysfunction that can lead to dry, xerotic skin that is more susceptible to cracking [7–10]. Surfactant monomers can also bind to keratin filaments in the SC; inducing irritation and erythema [6,11–13]. While both binding and lipid depletion from surfactant treatments have been examined extensively, few studies have examined surfactant induced changes in skin wettability [8,14,15]. Surface wettability is an important factor of skin protective function. Skin wettability can influence the contact inhibition of chemicals and the adhesion of pathogenic microorganisms [16,17]. Previous studies show that a depletion of SC lipids using solvents increases the hydrophobicity of the tissue interface. This wettability change is believed to be caused by the removal of the skin surface lipid film[18], exposing the underlying keratin [14]. Delipidation of SC results in reduced anionic surfactant binding for concentrations above 60 mM, however increased binding occurs below this concentration [19]. The pH at which SC is treated has also been shown to alter the potential for surfactants to bind to the SC [5,19,20]. While anionic surfactant monomers bind readily to SC in both acidic and alkaline environments, this binding is a minimum at pH 7-9 [19]. Previous studies have proposed that anionic surfactant binding to keratinous tissues such as wool, hair, and SC is governed by a combination of charge and hydrophobic binding [21–23]. In acidic environments, an electrostatic attraction between anionic surfactant monomer head groups and the positively charged keratin [16,24] substrate would cause binding that leaves the apolar tail groups exposed. In contrast, alkaline conditions would result in a hydrophobic based attraction of the surfactant tails to the SC surface; leaving the polar head groups exposed. This reorientation of bound surfactants suggests that a transition from a more hydrophobic to a more hydrophilic surface wettability is likely to occur as the pH of surfactant treatments is increased from acidic to alkaline. To date however, this has not been demonstrated. Wetting studies have only reported changes in the wetting time of surfactant treated wool fibers at pH 1 and 7 [23]. In this article, we take a first step towards understanding how the pH of anionic surfactant treatments can alter the wettability of human SC. We use contact angle goniometry to characterize changes in SC surface wettability after treatment with solutions of anionic surfactants in a pH buffer ranging between 5 and 9. By comparing the wettability of SC treated with an anionic surfactant solution with the wettability of SC treated only with a pH buffer, the direct effect of the surfactants is established. The potential for surfactant induced changes in SC surface roughness to cause changes in wettability is also examined.
Materials and Methods Preparation of stratum corneum samples. Full thickness human skin from elective surgery was received from Yale Pathology Tissue Services (New Haven, CT, U.S.A). An exempt approval (3002-13) was obtained to perform research using de-identified tissue samples; pursuant to the Department of Health and Human Services (DHHS) regulations, 45 CFR 46.101(b)(4). Female Caucasian thigh skin (44 year old) was used for surface wettability studies. Female Caucasian breast skin (40 year old) was used for profilometry studies. The choice of skin tissue was based on attainability from surgery. Isolation of SC was achieved using a standard water bath and trypsin solution based technique [8,9,25]. After separation, the isolated SC was rinsed twice for 10 minutes in
fresh deionized water (DIW, Milli-Q Integral 15, EMD Millipore, Billerica, MA, U.S.A), then allowed to dry to ambient conditions (23 °C, 25 % relative humidity (R.H.)) on plastic mesh for 24 hr. Individual circular SC samples with radii, 𝑅 = 5 mm, were cut from the isolated SC sheet with a hole punch (Harris Uni-core, Redding, CA, U.S.A). To highlight their topside, each SC sample was marked with a spiral using an indelible marker. No goniometry or profilometry studies were performed on, or in the vicinity of, the indelible marks. Lipid depleted SC samples (DLSC) were prepared by agitating SC samples in a chloroform and methanol solution (2:1 by volume) for 60 min, then DIW for 10 min. This chloroform/methanol treatment (CMT) fully depletes [26] intercellular and surface ceramides, cholesterol and free fatty acids ordinarily found in healthy human SC [27–30]. Control SC samples (REF SC) were prepared by agitating samples in DIW for 10 min. SC samples treated this way retain high levels of lipids [28]. Each SC sample was then allowed to dry in ambient conditions for 24 hr.
Sample treatment. Buffers with a pH of 5, 7, and 9 were prepared using standard protocols [31] and measured with a pH meter (sympHony B10p, VWR, Radnor, PA, U.S.A). Sodium lauryl sulfate (SLS, Stepanol WA-100 NF/USP, Stepan Company, Northfield, IL, U.S.A) was added to the buffers to create 1 % (by mass) mixtures, then agitated until the surfactant dissolved completely. These solutions were then measured with a pH meter. Table 1 displays the pH of each buffer and surfactant solution. The concentration of each surfactant solution was calculated to be 34.7 mM; significantly greater than the critical micelle concentration for SLS of 8.2 mM [32]. As such, treatment with the surfactant solutions were expected to emulsify some of the SC lipids [6]. After drying, both DLSC and REF SC samples were agitated either in a buffer solution or the surfactant solution for 60 min, then agitated in fresh DIW twice for 5 min to neutralize pH conditions and remove a majority of the emulsified surface and unbound surfactant monomers. Each SC sample was then laminated to a glass coverslip with their outermost face exposed. Drying the SC samples to ambient conditions for 24 hr strongly adhered samples to the glass substrate.
Contact Angle and Surface Tension Measurements Lateral profiles of small DIW drops deposited onto DLSC and REF SC surfaces were imaged 2, 30, 60 and 90 sec after initial drop contact with the SC using a contact angle goniometer. Drops were deposited using a screw driven syringe (Hamilton Company, Reno, NV, U.S.A) and 0.5 mm outer diameter (needle gauge 25) flat ended needle. The goniometer was comprised of a horizontally aligned CCD camera (PixeLINK Megapixel PL-A662, Gloucester, Canada) with zoom lens (Navitar Zoom 6000, Rochester, NY, U.S.A) facing a horizontal aluminium substrate upon which the glass substrates were placed. A humidity chamber containing saturated air covered the SC sample and drop to minimize evaporative effects over the deposition period. Drop contact angles, 𝜃𝑑 , were extracted from recorded imaged using ImageJ with the Bigdrop drop shape analysis plug-in [33]. Images (800 x 600 pixels) were recorded at a minimum resolution of 5.61 μm/pixel. Due to their size, each SC sample facilitated contact angle measurements of 2-3 drops; performed sequentially. The surface tension, 𝛾𝐿𝑉 , of water drops removed from the SC surface after a 90 sec deposition period was measured using a pendant drop method [34]. Here, the subscript LV denotes the liquid–vapor interface. Drops were removed from the SC surface using a clean screw syringe. Pendant drops were formed at the end of a 0.5 mm outer diameter flat ended needle and imaged using the CCD camera. Surface tension measurements are detailed in Table 2.
Drop mass The average mass of DIW drops formed by the screw driven syringe and 0.5 mm needle were established using an analytical balance (97035-620, Mettler-Toledo, Columbus, OH, U.S.A). In order to minimize evaporative effects from influencing the results, the average mass was established from ten measurements of 𝑛 = 4 drops.
Contact profilometry Prior to testing, each SC sample was treated, laminated to a glass coverslip and allowed to dry in laboratory conditions for 24 hr. Height profiles, 𝑧(𝑥), were recorded every ∆𝑥 = 200 nm along the diameter of the circular SC samples using a contact profilometer (Dektak 8, Veeco Instruments, Plainview, NY, U.S.A) with an 800 nm radius stylus.
Statistical analysis Statistical analyses were performed to test for significant differences in both surface roughness and wettability measurements. For each pH condition, GraphPad Prism 7.00 was used to perform an unpaired t-test between results from SC samples treated with the surfactant solution and those treated with the buffer alone. A statistically significant difference was recognized when 𝑝 ≤ 0.05. In the figures, * denotes 𝑝 ≤ 0.05, ** denotes 𝑝 ≤ 0.01 and *** denotes 𝑝 ≤ 0.001.
Results and Discussion We first verify the effects of lipid depletion on changes to the wettability of stratum corneum (SC). Changes in the average contact angle of deionized water (DIW) drops deposited on the outermost face of SC samples adhered to a glass cover slip are recorded over a 90 sec period. Adhered SC samples are equilibrated to laboratory conditions (25 % R.H., 23 °C) for 24 hr prior to testing. Fig. S1 (a) in the supplemental material shows a representative DIW drop contact angle, 𝜃𝑑 , on the surface of a control (REF SC) SC sample after a 60 sec deposition time. Fig. S1 (b) shows the equivalent contact angle on a lipid depleted (DLSC) SC sample after the same deposition period. A pendant drop technique [34] is then used to quantify the surface tension, 𝛾𝐿𝑉 , of the drops after they are removed from the SC. Surface tension measurements are made to account for the migration of residual surface lipids [35] and surfactants [36] to the liquid-vapor interface of the drop during deposition [37]. A typical image used to measure surface tension is shown in Fig. S1 (c). From the contact angle and surface tension, we determine the change in surface energy of the skin tissue upon wetting, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 . This energy difference provides a direct indicator of surface wettability and can be determined simply from Young's law, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 = 𝛾𝐿𝑉 cos(𝜃𝑑 ), where 𝛾𝑆𝑉 and 𝛾𝑆𝐿 are the solid–vapor and solid–liquid surface energies respectively. Fig. S1 (d) shows the change in average contact angle of DIW drops on control and lipid depleted SC over a 90 sec period. Drops exhibit larger contact angles on lipid depleted SC samples relative to the control samples. To ensure that hydrostatic pressure effects do not affect the contact angle of the drops, we quantify their equivalent radii and compare this with the capillary length. An average drop mass of 𝑚 = 9.68 ± 0.18 mg is recorded (𝑛 = 40 drops). This mass is used to establish the average equivalent drop 3 radius, 𝑅𝐸 = √3𝑚⁄4𝜋𝜌 = 1.323 ± 0.008mm, where 𝜌 = 998 kg/m3 is the density of DIW at 23 °C. This radius is significantly smaller than the capillary length, 𝑎 = √𝛾𝐿𝑉 ⁄𝜌𝑔 = 2.71 mm, signifying that the drops will retain a spherical cap shape upon deposition. Here, 𝑔, is the gravitational acceleration and the liquid-vapor surface energy of DIW is 𝛾𝐿𝑉 = 0.072 N/m. The corresponding surface wettability of the SC, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 , after a 90 sec deposition period is shown in Fig. S1 (e). Control SC samples show a large difference in surface energy between dry and wet states, indicating their moderate hydrophilicity. In contrast, lipid depleted samples show negligible changes in surface energy, indicating that lipid removal significantly decreases the wettability of the tissue. This result agrees with previous in-vivo and ex-vivo studies that observe similar reductions in wettability with surface and intercellular lipid depletion [8,14,15]. This is currently thought to be caused by increased exposure of underlying keratin fibers [15]. We next quantify changes in the surface wettability of SC retaining lipids (REF SC) after 60 min treatments with 1% solutions of sodium lauryl sulfate (SLS) in buffers varying in pH between 5 and 9. After treatment, SC samples are rinsed for 10 min in DIW, then laminated to a glass coverslip and allowed to dry for 24 hr to laboratory conditions. The contact angles of DIW drops on the treated SC are measured over a 90 sec period, removed from the SC surface and then used to quantify the drop surface
tension. Figs. 1 (a) and (b) respectively show the contact angles of DIW drops on SC samples treated with 1% SLS buffer solutions and samples treated with the buffer alone over a 90 sec period. Contact angles on buffer treated SC remain similar for all pH values, while a decrease in contact angle with increasing pH is observed for drops on the surfactant treated SC samples. Fig. 1 (c) shows that the wettability of the surfactant treated SC increases with pH while SC treated with only the buffer solutions show comparable magnitudes. Fig. 1 (d) shows the change in surface wettability, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), between the buffer and surfactant treated samples at each pH condition. This parameter provides a direct measure of the influence of SLS on the change in wettability of the SC surface. The small negative value observed at acidic conditions that would indicate an increase in surface hydrophobicity relative to the buffer treatment is not statistically significant. However, a statistically significant positive value at alkaline conditions denotes an increase in hydrophilicity. The impact of SC lipids on the ability of anionic surfactant treatments to alter the wettability of SC is further examined. The same experimental protocol used to obtain results in Fig. 1 is employed, however delipidated SC (DLSC) samples are now used. Delipidation of the SC removes a majority of surface and intercellular lipids [26], leaving only the covalently bound corneocyte lipid envelope [38]. Figs. 2 (a) and (b) respectively show the contact angles of DIW drops over a 90 sec period deposited on DLSC samples treated with surfactant solutions and the buffer alone. The contact angles of drops on the surfactant treated DLSC in Fig. 2 (a) are notably smaller than the corresponding contact angles of drops on the REF SC retaining lipids for pH 7 and 9 conditions, shown in Fig. 1 (a). Fig. 2 (c) displays the wettability of the delipidated SC for the three pH conditions after treatment with the surfactant solutions and the pH buffer alone. Comparisons of SC treated only with the buffer solutions reveal that relative to SC retaining lipids in Fig. 1 (c), delipidation causes an expected decrease in the wettability of the SC at pH 7. Little change however occurs at pH 5 and 9. While the wettability of buffer treated DLSC remains similar for all pH conditions, the wettability of surfactant treated DLSC increases in hydrophilicity with increasing pH; similar to those observed for SC retaining lipids in Fig. 1 (c). Moreover, differences in wettability between surfactant and buffer treated DLSC samples become statistically significant for all pH conditions. Fig. 2 (d) shows the change in wettability between the SLS and buffer treatments, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), for each pH condition. The results of wettability changes from Fig. 1 (d) have also been included in this figure for comparison. Surfactant treatments of DLSC are more pronounced than for SC retaining lipids. The transition from a more hydrophobic surface to a more hydrophilic surface occurs near neutral pH values. We note that this pH coincides with the mean iso-electric points of type I (4.9 ≤ 𝑝𝐼 ≤ 5.4 ) and type II (6.5 ≤ 𝑝𝐼 ≤ 8.5) keratin found in SC [39] (average 5.7 ≤ 𝑝𝐼 ≤ 7). The more pronounced change in wettability of DLSC with pH also suggests that an increased number of surfactant monomers bind to the tissue interface relative to SC tissue retaining lipids. Results from Figs. 1 and 2 indicate that anionic surfactant treatments can notably alter the wettability of human SC, with their pH influencing the resultant change. While this could be attributable to changes in surface chemistry emerging from interactions between surfactants and the SC, a surfactant induced change in SC roughness is also a potential candidate for altered wettability. It is widely recognized that surfactant treatments can induce corneocyte desquamation [27,40] and irreversible swelling of the SC [19]. Both of these factors could contribute to changes in SC surface roughness. A typical example of a topographical profile recorded with a contact profilometer is shown in Figs. 3 (a) and (b). The profile highlights the heterogeneous topography of the SC surface; consistent with the findings of previous studies [41,42]. Figs. 3 (c) and (d) respectively show the average relative diameter [43], 𝐷 ′ ⁄𝐷∗, of control REF SC and lipid depleted DLSC samples treated both with the surfactant solutions and the buffer alone. The apparent diameter,𝐷 ∗, is simply the sum of all longitudinal displacements ∆𝑥 along the diameter of
the sample. The true diameter, 𝐷 ′ = ∑𝑛𝑖=1 √(𝑧𝑖+1 − 𝑧𝑖 )2 + ∆𝑥 2 , is the sum of the absolute displacements between successive points on the profile, where 𝑧𝑖 denotes the vertical position of the 𝑖 th point. The only statistically significant difference in relative diameter between surfactant and buffer solution treatments occurs at pH 5 for the control REF SC. Here, the surfactant treatment induces a decrease in the relative diameter, consistent with a decrease in roughness. We employ a simple model to quantify how this surfactant induced change in surface roughness impacts the wettability of SC. The Wenzel model [44]; expressed by, 𝑐𝑜𝑠(𝜃𝑑 ) = 𝜑𝑐𝑜𝑠(𝜃′), relates the apparent contact angle, 𝜃𝑑 , on a rough surface to the ideal contact angle, 𝜃′, of a perfectly smooth surface with an identical surface energy. The roughness ratio, 𝜑 = 𝐴′ ⁄𝐴∗, defines the ratio of the true area of a substrate, 𝐴′ , to the projected area, 𝐴∗ . For two substrates with an identical surface energy but varying roughness, the ideal contact angle will remain constant. Therefore, 𝑐𝑜𝑠(𝜃𝑑𝑖 )⁄𝜑𝑖 = 𝑐𝑜𝑠(𝜃𝑑𝑗 )⁄𝜑𝑗 , where 𝑖 and 𝑗 denote substrate indices. The relationship between the contact angles of drops on the two substrates can therefore be approximated using the expression, 𝑐𝑜𝑠(𝜃𝑑𝑖 ) 𝑐𝑜𝑠(𝜃𝑑𝑗 )
=
𝜑𝑖 𝜑𝑗
=
∗
𝐴′𝑖 𝐴𝑗 𝐴∗𝑖 𝐴′𝑗
𝐷∗
2
𝐷′ 𝐷𝑖
2
≈ ( 𝑗′ ) ( 𝑖∗ ) . 𝐷𝑗
(1)
Using measurements of the average drop contact angles on buffer treated SC samples, 𝜃𝑑𝑖 , (Fig. 1 (b)) and the statistically significant difference in relative diameters between surfactant and buffer treated REF SC samples at pH 5 (Fig. 3(c)), we use Eq. (1) to solve for the change in the contact angle, ∆𝜃 = 𝜃𝑑𝑗 − 𝜃𝑑𝑖 caused by the variation in roughness. Fig. S2 plots ∆𝜃 against drop contact angle. The shaded region under the curve corresponds to the range of average contact angles, 𝜃𝑑𝑖 , recorded over the 90 sec drop deposition on the pH 5 buffer treated SC samples. We establish that the largest achievable change in contact angle due to this roughness variation is 0.035 degrees. This corresponds to a maximum absolute change in wettability of, |∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 )| = 0.038 mN/m. This difference is significantly smaller than any statistically significant change in wettability between corresponding surfactant and buffer treated SC samples. This indicates that while surfactants can marginally alter the surface roughness of SC samples, it is not the cause of the observed changes in wettability.
Summary and Conclusion Surfactants used in soaps and cleansers are effective at cleaning contaminants from the skin [5], but they can also remove natural skin lipids [5,6,27] and adsorb to the outermost stratum corneum layer [11,13,20,45,46]. These processes are relevant to skin wettability. Both changes in lipid/sebum levels and surfactant cleansing have previously been shown to impact the wettability of human stratum corneum [8,14,15]. To date however, it remains unclear whether surfactant binding or lipid emulsification is the primary cause of changes in skin wettability. The mechanisms by which surfactants bind to stratum corneum also currently remain unclear. By measuring the contact angle and surface tension of deionized water drops deposited onto the surface of isolated samples of human stratum corneum, we investigate how the pH of anionic surfactant treatments alter their wettability. Over the range 5 ≤ 𝑝𝐻 ≤ 9, differences between the wettability of stratum corneum samples treated with a 1% solution of sodium lauryl sulfate in a pH buffer, and samples treated with the pH buffer alone are established. For stratum corneum samples retaining skin lipids, surfactant treatments induce a more hydrophilic skin surface under alkaline conditions. With removal of skin lipids, surfactant treatments induce a hydrophobic surface under acidic conditions and a hydrophilic surface under alkaline conditions. Profilometry subsequently reveals that these changes in wettability are not caused by surfactant induced changes in stratum corneum roughness.
To our knowledge, the ability to control the wettability of human skin by altering the pH of an anionic surfactant solution treatment has not previously been reported. Moreover, the observed changes in human stratum corneum wettability we report in this article are consistent with a reorientation of bound surfactant monomers as the pH is varied [21]. Electrostatic binding of polar surfactant monomer head groups to positively charged keratin under acidic conditions [16] would leave the hydrophobic tails exposed. In contrast, a hydrophobic attraction of the surfactant tails to the tissue surface under alkaline conditions would leave the hydrophilic heads exposed. This mechanism has previously been employed to explain changes in wettability timescales for keratinous wool fibers [23]. Here we show this mechanism is also plausible for human skin. The more pronounced change in wettability that occurs with delipidated stratum corneum across the measured pH range further suggests that lipids present at the skin surface reduce interactions between the surfactant monomers and keratin in the tissue. This presumably occurs through direct obstruction of binding sites, or a preference for surfactants to adsorb to, then emulsify the lipids; with subsequent rinsing removing both lipids and surfactants from the tissue surface. A wide range of surfactants exist with numerous head group charges [22]. Future work should explore how changes in head group charge influence the wettability of stratum corneum after surfactant treatment at different pH conditions. This insight would help to further scrutinize the validity of a pH induced reorientation of bound surfactant monomers.
Acknowledgments We acknowledge funding from Schick. We also would like to thank Prof. David Cole-Hamilton for helpful discussions.
Figure captions Figure 1. Changes in wettability from SLS treatments of SC retaining lipids (REF SC). The average contact angle of deionized water drops over a 90 sec period after deposition on control REF SC samples treated for 60 min with (a) buffer solutions containing 1 % SLS and (b) buffer solutions alone. Square, triangle, and diamond symbols respectively denote buffer pH conditions of 5, 7, and 9. (c) The average change in surface energy of the REF SC samples upon wetting, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 , for buffer solution treatments (dark solid grey) and buffer solutions containing 1 % SLS (light solid grey) across the range 5 ≤ 𝑝𝐻 ≤ 9. Error bars denote standard deviations. (d) The average change in surface wettability, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), between SC samples treated with buffer solutions containing 1 % SLS and the buffer solutions alone. Error bars denote propagated standard deviations. Figure 2. Changes in wettability from SLS treatments of delipidated SC (DLSC). The average contact angle of deionized water drops over a 90 sec period after deposition on delipidated DLSC samples treated for 60 min with (a) buffer solutions containing 1 % SLS and (b) buffer solutions alone. Square, triangle, and diamond symbols respectively denote buffer pH conditions of 5, 7, and 9. (c) The average change in surface energy of the DLSC samples upon wetting, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 , for buffer solution treatments (dark solid grey) and buffer solutions containing 1 % SLS (light solid grey) across the range 5 ≤ 𝑝𝐻 ≤ 9. Error bars denote standard deviations. (d) The average change in surface wettability, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), between DLSC samples treated with buffer solutions containing 1 % SLS and the buffer solutions alone (dark solid grey). Changes in wettability of control REF SC samples from Fig. 1 (d) are plotted alongside for reference (light solid grey). Error bars denote propagated standard deviations. Figure 3. Changes in surface topography of SC with surfactant treatments. (a) Representative surface profile along the diameter of a circular REF SC sample after treatment with pH 5 buffer. A magnified view of the region outlined by the dashed box is shown in (b). (c) The average dimensionless relative diameter, 𝐷 ′ /𝐷 ∗ of control REF SC samples treated with buffer solutions containing 1 % SLS (blue solid bars, 𝑛 = 3 individual samples for each pH) plotted alongside equivalent measurements of samples treated with the buffer alone (open bars, 𝑛 = 3 individual samples for each pH). (d) The average relative diameter, 𝐷′ /𝐷 ∗ of lipid depleted DLSC samples treated
with buffer solutions containing 1 % SLS (blue solid bars, 𝑛 = 3 individual samples for each pH) plotted alongside equivalent measurement of samples treated with the buffer alone (open bars, 𝑛 = 3 individual samples for each pH). Error bars denote standard deviations.
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Figure Caption
Figr-1
Figr-2
Figr-3
Tables and Table Captions Table 1. Measured pH values of the buffer solutions alone and the buffer solutions containing 1% SLS. Buffer/Solution pH 5 buffer 1% SLS + pH 5 buffer pH 7 buffer 1% SLS + pH 7 buffer pH 9 buffer 1% SLS + pH 9 buffer
Measured pH 5.02 4.94 7.13 7.07 9.06 8.72
Table 2. The average surface tension of deionized water drops after a 90 sec deposition on SC samples treated with the different buffer and surfactant solutions.
Abbreviation
SC preparation
SC treatment and pH
REF SC REF SC REF SC REF SC DLSC DLSC DLSC DLSC REF SC REF SC REF SC DLSC DLSC DLSC
Control Control Control Control Delipidated Delipidated Delipidated Delipidated Control Control Control Delipidated Delipidated Delipidated
None pH 5 Buffer alone pH 7 Buffer alone pH 9 Buffer alone None pH 5 Buffer alone pH 7 Buffer alone pH 9 Buffer alone pH 5 Buffer + 1% SLS pH 7 Buffer + 1% SLS pH 9 Buffer + 1% SLS pH 5 Buffer + 1% SLS pH 7 Buffer + 1% SLS pH 9 Buffer + 1% SLS
Droplet 𝜸𝑳𝑽 after 90 sec deposition (mN/m) 70.7 ± 1.0 (n=3) 64.8 ± 0.5 (n=2) 68.9 ± 3.7 (n=4) 57.0 ± 3.7 (n=2) 70.4 ± 0.6 (n=3) 61.5 ± 3.1 (n=2) 62.6 ± 2.3 (n=2) 68.5 ± 3.5 (n=2) 44.4 ± 5.6 (n=3) 56.0 ± 5.6 (n=4) 49.2 ± 2.9 (n=3) 41.9 ± 2.2 (n=2) 53.8 ± 4.0 (n=2) 51.5 ± 12.4 (n=3)
S0927-7765(17)30357-0 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.06.009 COLSUB 8619
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
29-11-2016 18-5-2017 8-6-2017
Please cite this article as: L.Bromberg, X.Liu, I.Wang, S.Smith, K.Schwicker, Z.Eller, G.K.German, Control of human skin wettability using the pH of anionic surfactant solution treatments, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.06.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Control of human skin wettability using the pH of anionic surfactant solution treatments. L. Bromberga, X. Liub, I. Wangb, S. Smithb, K. Schwickerb, Z. Ellerb, and G. K. Germanb* a b *Guy
Department of Chemistry, Binghamton University, Binghamton, New York, U.S.A.
Department of Biomedical Engineering, Binghamton University, Binghamton, New York, U.S.A. K. German, BI2609, Department of Biomedical Engineering, Binghamton University, 4400 Vestal Parkway East,
Binghamton, New York, 13902, Tel: +1 607-777-4270, Fax: +1 607-777-5780, Email: [email protected]
Graphical Abstract
Highlights
The wettability of human stratum corneum is examined. Changes in the pH of anionic surfactant treatments can alter skin wettability. Results suggest a reorientation of bound surfactant monomers. Lipids appear to reduce surfactant binding to the stratum corneum.
Abstract The outermost layer of human skin, or stratum corneum, acts as a protective barrier between underlying living tissue and the external environment. The wettability of this tissue layer can influence spreading of chemicals and the adhesion of pathogenic microorganisms. We show in this article that the wettability of isolated human stratum corneum can be controlled through treatment with solutions of the anionic surfactant, sodium lauryl sulfate, buffered to different pH values. Relative to control treatments with the buffer solution alone, surfactant solution treatments under acidic conditions cause delipidated stratum corneum to become more hydrophobic. In contrast, alkaline conditions cause the stratum corneum to become more hydrophilic; irrespective of lipid composition. This transition is consistent with a reorientation of bound surfactants at the tissue interface. Under acidic conditions, electrostatic binding of negatively charged surfactant head groups with positively charged keratin in the stratum corneum would increase tissue hydrophobicity due to the exposed hydrophobic tails. However, a hydrophobic based attraction of the apolar surfactant tails to the stratum corneum surface under alkaline conditions would leave the hydrophilic surfactant head groups exposed, causing increased tissue hydrophilicity. Changes in wettability with pH become diminished when lipids ordinarily found in stratum corneum are present, suggesting the lipids partially inhibit surfactant binding. Profilometry studies of the tissue topography highlight that surfactant induced changes in stratum corneum surface roughness cannot account for the observed changes in wettability.
Introduction Amphiphilic surfactants readily adsorb to liquid surfaces; whereupon they reduce the interfacial surface energy. This behavior makes them useful as foaming agents and emulsifiers. The ability of surfactants to emulsify lipids means that surfactants are widely used in cosmetic cleansing products[1]. Healthy skin conditions are regulated by the presence of lipids and hygroscopic natural moisturizing factors [2–4] that minimize water loss across the epidermis and keep the outermost layer, or stratum corneum (SC), hydrated. A depletion of SC lipids from cleansing [5,6] can cause barrier dysfunction that can lead to dry, xerotic skin that is more susceptible to cracking [7–10]. Surfactant monomers can also bind to keratin filaments in the SC; inducing irritation and erythema [6,11–13]. While both binding and lipid depletion from surfactant treatments have been examined extensively, few studies have examined surfactant induced changes in skin wettability [8,14,15]. Surface wettability is an important factor of skin protective function. Skin wettability can influence the contact inhibition of chemicals and the adhesion of pathogenic microorganisms [16,17]. Previous studies show that a depletion of SC lipids using solvents increases the hydrophobicity of the tissue interface. This wettability change is believed to be caused by the removal of the skin surface lipid film[18], exposing the underlying keratin [14]. Delipidation of SC results in reduced anionic surfactant binding for concentrations above 60 mM, however increased binding occurs below this concentration [19]. The pH at which SC is treated has also been shown to alter the potential for surfactants to bind to the SC [5,19,20]. While anionic surfactant monomers bind readily to SC in both acidic and alkaline environments, this binding is a minimum at pH 7-9 [19]. Previous studies have proposed that anionic surfactant binding to keratinous tissues such as wool, hair, and SC is governed by a combination of charge and hydrophobic binding [21–23]. In acidic environments, an electrostatic attraction between anionic surfactant monomer head groups and the positively charged keratin [16,24] substrate would cause binding that leaves the apolar tail groups exposed. In contrast, alkaline conditions would result in a hydrophobic based attraction of the surfactant tails to the SC surface; leaving the polar head groups exposed. This reorientation of bound surfactants suggests that a transition from a more hydrophobic to a more hydrophilic surface wettability is likely to occur as the pH of surfactant treatments is increased from acidic to alkaline. To date however, this has not been demonstrated. Wetting studies have only reported changes in the wetting time of surfactant treated wool fibers at pH 1 and 7 [23]. In this article, we take a first step towards understanding how the pH of anionic surfactant treatments can alter the wettability of human SC. We use contact angle goniometry to characterize changes in SC surface wettability after treatment with solutions of anionic surfactants in a pH buffer ranging between 5 and 9. By comparing the wettability of SC treated with an anionic surfactant solution with the wettability of SC treated only with a pH buffer, the direct effect of the surfactants is established. The potential for surfactant induced changes in SC surface roughness to cause changes in wettability is also examined.
Materials and Methods Preparation of stratum corneum samples. Full thickness human skin from elective surgery was received from Yale Pathology Tissue Services (New Haven, CT, U.S.A). An exempt approval (3002-13) was obtained to perform research using de-identified tissue samples; pursuant to the Department of Health and Human Services (DHHS) regulations, 45 CFR 46.101(b)(4). Female Caucasian thigh skin (44 year old) was used for surface wettability studies. Female Caucasian breast skin (40 year old) was used for profilometry studies. The choice of skin tissue was based on attainability from surgery. Isolation of SC was achieved using a standard water bath and trypsin solution based technique [8,9,25]. After separation, the isolated SC was rinsed twice for 10 minutes in
fresh deionized water (DIW, Milli-Q Integral 15, EMD Millipore, Billerica, MA, U.S.A), then allowed to dry to ambient conditions (23 °C, 25 % relative humidity (R.H.)) on plastic mesh for 24 hr. Individual circular SC samples with radii, 𝑅 = 5 mm, were cut from the isolated SC sheet with a hole punch (Harris Uni-core, Redding, CA, U.S.A). To highlight their topside, each SC sample was marked with a spiral using an indelible marker. No goniometry or profilometry studies were performed on, or in the vicinity of, the indelible marks. Lipid depleted SC samples (DLSC) were prepared by agitating SC samples in a chloroform and methanol solution (2:1 by volume) for 60 min, then DIW for 10 min. This chloroform/methanol treatment (CMT) fully depletes [26] intercellular and surface ceramides, cholesterol and free fatty acids ordinarily found in healthy human SC [27–30]. Control SC samples (REF SC) were prepared by agitating samples in DIW for 10 min. SC samples treated this way retain high levels of lipids [28]. Each SC sample was then allowed to dry in ambient conditions for 24 hr.
Sample treatment. Buffers with a pH of 5, 7, and 9 were prepared using standard protocols [31] and measured with a pH meter (sympHony B10p, VWR, Radnor, PA, U.S.A). Sodium lauryl sulfate (SLS, Stepanol WA-100 NF/USP, Stepan Company, Northfield, IL, U.S.A) was added to the buffers to create 1 % (by mass) mixtures, then agitated until the surfactant dissolved completely. These solutions were then measured with a pH meter. Table 1 displays the pH of each buffer and surfactant solution. The concentration of each surfactant solution was calculated to be 34.7 mM; significantly greater than the critical micelle concentration for SLS of 8.2 mM [32]. As such, treatment with the surfactant solutions were expected to emulsify some of the SC lipids [6]. After drying, both DLSC and REF SC samples were agitated either in a buffer solution or the surfactant solution for 60 min, then agitated in fresh DIW twice for 5 min to neutralize pH conditions and remove a majority of the emulsified surface and unbound surfactant monomers. Each SC sample was then laminated to a glass coverslip with their outermost face exposed. Drying the SC samples to ambient conditions for 24 hr strongly adhered samples to the glass substrate.
Contact Angle and Surface Tension Measurements Lateral profiles of small DIW drops deposited onto DLSC and REF SC surfaces were imaged 2, 30, 60 and 90 sec after initial drop contact with the SC using a contact angle goniometer. Drops were deposited using a screw driven syringe (Hamilton Company, Reno, NV, U.S.A) and 0.5 mm outer diameter (needle gauge 25) flat ended needle. The goniometer was comprised of a horizontally aligned CCD camera (PixeLINK Megapixel PL-A662, Gloucester, Canada) with zoom lens (Navitar Zoom 6000, Rochester, NY, U.S.A) facing a horizontal aluminium substrate upon which the glass substrates were placed. A humidity chamber containing saturated air covered the SC sample and drop to minimize evaporative effects over the deposition period. Drop contact angles, 𝜃𝑑 , were extracted from recorded imaged using ImageJ with the Bigdrop drop shape analysis plug-in [33]. Images (800 x 600 pixels) were recorded at a minimum resolution of 5.61 μm/pixel. Due to their size, each SC sample facilitated contact angle measurements of 2-3 drops; performed sequentially. The surface tension, 𝛾𝐿𝑉 , of water drops removed from the SC surface after a 90 sec deposition period was measured using a pendant drop method [34]. Here, the subscript LV denotes the liquid–vapor interface. Drops were removed from the SC surface using a clean screw syringe. Pendant drops were formed at the end of a 0.5 mm outer diameter flat ended needle and imaged using the CCD camera. Surface tension measurements are detailed in Table 2.
Drop mass The average mass of DIW drops formed by the screw driven syringe and 0.5 mm needle were established using an analytical balance (97035-620, Mettler-Toledo, Columbus, OH, U.S.A). In order to minimize evaporative effects from influencing the results, the average mass was established from ten measurements of 𝑛 = 4 drops.
Contact profilometry Prior to testing, each SC sample was treated, laminated to a glass coverslip and allowed to dry in laboratory conditions for 24 hr. Height profiles, 𝑧(𝑥), were recorded every ∆𝑥 = 200 nm along the diameter of the circular SC samples using a contact profilometer (Dektak 8, Veeco Instruments, Plainview, NY, U.S.A) with an 800 nm radius stylus.
Statistical analysis Statistical analyses were performed to test for significant differences in both surface roughness and wettability measurements. For each pH condition, GraphPad Prism 7.00 was used to perform an unpaired t-test between results from SC samples treated with the surfactant solution and those treated with the buffer alone. A statistically significant difference was recognized when 𝑝 ≤ 0.05. In the figures, * denotes 𝑝 ≤ 0.05, ** denotes 𝑝 ≤ 0.01 and *** denotes 𝑝 ≤ 0.001.
Results and Discussion We first verify the effects of lipid depletion on changes to the wettability of stratum corneum (SC). Changes in the average contact angle of deionized water (DIW) drops deposited on the outermost face of SC samples adhered to a glass cover slip are recorded over a 90 sec period. Adhered SC samples are equilibrated to laboratory conditions (25 % R.H., 23 °C) for 24 hr prior to testing. Fig. S1 (a) in the supplemental material shows a representative DIW drop contact angle, 𝜃𝑑 , on the surface of a control (REF SC) SC sample after a 60 sec deposition time. Fig. S1 (b) shows the equivalent contact angle on a lipid depleted (DLSC) SC sample after the same deposition period. A pendant drop technique [34] is then used to quantify the surface tension, 𝛾𝐿𝑉 , of the drops after they are removed from the SC. Surface tension measurements are made to account for the migration of residual surface lipids [35] and surfactants [36] to the liquid-vapor interface of the drop during deposition [37]. A typical image used to measure surface tension is shown in Fig. S1 (c). From the contact angle and surface tension, we determine the change in surface energy of the skin tissue upon wetting, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 . This energy difference provides a direct indicator of surface wettability and can be determined simply from Young's law, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 = 𝛾𝐿𝑉 cos(𝜃𝑑 ), where 𝛾𝑆𝑉 and 𝛾𝑆𝐿 are the solid–vapor and solid–liquid surface energies respectively. Fig. S1 (d) shows the change in average contact angle of DIW drops on control and lipid depleted SC over a 90 sec period. Drops exhibit larger contact angles on lipid depleted SC samples relative to the control samples. To ensure that hydrostatic pressure effects do not affect the contact angle of the drops, we quantify their equivalent radii and compare this with the capillary length. An average drop mass of 𝑚 = 9.68 ± 0.18 mg is recorded (𝑛 = 40 drops). This mass is used to establish the average equivalent drop 3 radius, 𝑅𝐸 = √3𝑚⁄4𝜋𝜌 = 1.323 ± 0.008mm, where 𝜌 = 998 kg/m3 is the density of DIW at 23 °C. This radius is significantly smaller than the capillary length, 𝑎 = √𝛾𝐿𝑉 ⁄𝜌𝑔 = 2.71 mm, signifying that the drops will retain a spherical cap shape upon deposition. Here, 𝑔, is the gravitational acceleration and the liquid-vapor surface energy of DIW is 𝛾𝐿𝑉 = 0.072 N/m. The corresponding surface wettability of the SC, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 , after a 90 sec deposition period is shown in Fig. S1 (e). Control SC samples show a large difference in surface energy between dry and wet states, indicating their moderate hydrophilicity. In contrast, lipid depleted samples show negligible changes in surface energy, indicating that lipid removal significantly decreases the wettability of the tissue. This result agrees with previous in-vivo and ex-vivo studies that observe similar reductions in wettability with surface and intercellular lipid depletion [8,14,15]. This is currently thought to be caused by increased exposure of underlying keratin fibers [15]. We next quantify changes in the surface wettability of SC retaining lipids (REF SC) after 60 min treatments with 1% solutions of sodium lauryl sulfate (SLS) in buffers varying in pH between 5 and 9. After treatment, SC samples are rinsed for 10 min in DIW, then laminated to a glass coverslip and allowed to dry for 24 hr to laboratory conditions. The contact angles of DIW drops on the treated SC are measured over a 90 sec period, removed from the SC surface and then used to quantify the drop surface
tension. Figs. 1 (a) and (b) respectively show the contact angles of DIW drops on SC samples treated with 1% SLS buffer solutions and samples treated with the buffer alone over a 90 sec period. Contact angles on buffer treated SC remain similar for all pH values, while a decrease in contact angle with increasing pH is observed for drops on the surfactant treated SC samples. Fig. 1 (c) shows that the wettability of the surfactant treated SC increases with pH while SC treated with only the buffer solutions show comparable magnitudes. Fig. 1 (d) shows the change in surface wettability, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), between the buffer and surfactant treated samples at each pH condition. This parameter provides a direct measure of the influence of SLS on the change in wettability of the SC surface. The small negative value observed at acidic conditions that would indicate an increase in surface hydrophobicity relative to the buffer treatment is not statistically significant. However, a statistically significant positive value at alkaline conditions denotes an increase in hydrophilicity. The impact of SC lipids on the ability of anionic surfactant treatments to alter the wettability of SC is further examined. The same experimental protocol used to obtain results in Fig. 1 is employed, however delipidated SC (DLSC) samples are now used. Delipidation of the SC removes a majority of surface and intercellular lipids [26], leaving only the covalently bound corneocyte lipid envelope [38]. Figs. 2 (a) and (b) respectively show the contact angles of DIW drops over a 90 sec period deposited on DLSC samples treated with surfactant solutions and the buffer alone. The contact angles of drops on the surfactant treated DLSC in Fig. 2 (a) are notably smaller than the corresponding contact angles of drops on the REF SC retaining lipids for pH 7 and 9 conditions, shown in Fig. 1 (a). Fig. 2 (c) displays the wettability of the delipidated SC for the three pH conditions after treatment with the surfactant solutions and the pH buffer alone. Comparisons of SC treated only with the buffer solutions reveal that relative to SC retaining lipids in Fig. 1 (c), delipidation causes an expected decrease in the wettability of the SC at pH 7. Little change however occurs at pH 5 and 9. While the wettability of buffer treated DLSC remains similar for all pH conditions, the wettability of surfactant treated DLSC increases in hydrophilicity with increasing pH; similar to those observed for SC retaining lipids in Fig. 1 (c). Moreover, differences in wettability between surfactant and buffer treated DLSC samples become statistically significant for all pH conditions. Fig. 2 (d) shows the change in wettability between the SLS and buffer treatments, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), for each pH condition. The results of wettability changes from Fig. 1 (d) have also been included in this figure for comparison. Surfactant treatments of DLSC are more pronounced than for SC retaining lipids. The transition from a more hydrophobic surface to a more hydrophilic surface occurs near neutral pH values. We note that this pH coincides with the mean iso-electric points of type I (4.9 ≤ 𝑝𝐼 ≤ 5.4 ) and type II (6.5 ≤ 𝑝𝐼 ≤ 8.5) keratin found in SC [39] (average 5.7 ≤ 𝑝𝐼 ≤ 7). The more pronounced change in wettability of DLSC with pH also suggests that an increased number of surfactant monomers bind to the tissue interface relative to SC tissue retaining lipids. Results from Figs. 1 and 2 indicate that anionic surfactant treatments can notably alter the wettability of human SC, with their pH influencing the resultant change. While this could be attributable to changes in surface chemistry emerging from interactions between surfactants and the SC, a surfactant induced change in SC roughness is also a potential candidate for altered wettability. It is widely recognized that surfactant treatments can induce corneocyte desquamation [27,40] and irreversible swelling of the SC [19]. Both of these factors could contribute to changes in SC surface roughness. A typical example of a topographical profile recorded with a contact profilometer is shown in Figs. 3 (a) and (b). The profile highlights the heterogeneous topography of the SC surface; consistent with the findings of previous studies [41,42]. Figs. 3 (c) and (d) respectively show the average relative diameter [43], 𝐷 ′ ⁄𝐷∗, of control REF SC and lipid depleted DLSC samples treated both with the surfactant solutions and the buffer alone. The apparent diameter,𝐷 ∗, is simply the sum of all longitudinal displacements ∆𝑥 along the diameter of
the sample. The true diameter, 𝐷 ′ = ∑𝑛𝑖=1 √(𝑧𝑖+1 − 𝑧𝑖 )2 + ∆𝑥 2 , is the sum of the absolute displacements between successive points on the profile, where 𝑧𝑖 denotes the vertical position of the 𝑖 th point. The only statistically significant difference in relative diameter between surfactant and buffer solution treatments occurs at pH 5 for the control REF SC. Here, the surfactant treatment induces a decrease in the relative diameter, consistent with a decrease in roughness. We employ a simple model to quantify how this surfactant induced change in surface roughness impacts the wettability of SC. The Wenzel model [44]; expressed by, 𝑐𝑜𝑠(𝜃𝑑 ) = 𝜑𝑐𝑜𝑠(𝜃′), relates the apparent contact angle, 𝜃𝑑 , on a rough surface to the ideal contact angle, 𝜃′, of a perfectly smooth surface with an identical surface energy. The roughness ratio, 𝜑 = 𝐴′ ⁄𝐴∗, defines the ratio of the true area of a substrate, 𝐴′ , to the projected area, 𝐴∗ . For two substrates with an identical surface energy but varying roughness, the ideal contact angle will remain constant. Therefore, 𝑐𝑜𝑠(𝜃𝑑𝑖 )⁄𝜑𝑖 = 𝑐𝑜𝑠(𝜃𝑑𝑗 )⁄𝜑𝑗 , where 𝑖 and 𝑗 denote substrate indices. The relationship between the contact angles of drops on the two substrates can therefore be approximated using the expression, 𝑐𝑜𝑠(𝜃𝑑𝑖 ) 𝑐𝑜𝑠(𝜃𝑑𝑗 )
=
𝜑𝑖 𝜑𝑗
=
∗
𝐴′𝑖 𝐴𝑗 𝐴∗𝑖 𝐴′𝑗
𝐷∗
2
𝐷′ 𝐷𝑖
2
≈ ( 𝑗′ ) ( 𝑖∗ ) . 𝐷𝑗
(1)
Using measurements of the average drop contact angles on buffer treated SC samples, 𝜃𝑑𝑖 , (Fig. 1 (b)) and the statistically significant difference in relative diameters between surfactant and buffer treated REF SC samples at pH 5 (Fig. 3(c)), we use Eq. (1) to solve for the change in the contact angle, ∆𝜃 = 𝜃𝑑𝑗 − 𝜃𝑑𝑖 caused by the variation in roughness. Fig. S2 plots ∆𝜃 against drop contact angle. The shaded region under the curve corresponds to the range of average contact angles, 𝜃𝑑𝑖 , recorded over the 90 sec drop deposition on the pH 5 buffer treated SC samples. We establish that the largest achievable change in contact angle due to this roughness variation is 0.035 degrees. This corresponds to a maximum absolute change in wettability of, |∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 )| = 0.038 mN/m. This difference is significantly smaller than any statistically significant change in wettability between corresponding surfactant and buffer treated SC samples. This indicates that while surfactants can marginally alter the surface roughness of SC samples, it is not the cause of the observed changes in wettability.
Summary and Conclusion Surfactants used in soaps and cleansers are effective at cleaning contaminants from the skin [5], but they can also remove natural skin lipids [5,6,27] and adsorb to the outermost stratum corneum layer [11,13,20,45,46]. These processes are relevant to skin wettability. Both changes in lipid/sebum levels and surfactant cleansing have previously been shown to impact the wettability of human stratum corneum [8,14,15]. To date however, it remains unclear whether surfactant binding or lipid emulsification is the primary cause of changes in skin wettability. The mechanisms by which surfactants bind to stratum corneum also currently remain unclear. By measuring the contact angle and surface tension of deionized water drops deposited onto the surface of isolated samples of human stratum corneum, we investigate how the pH of anionic surfactant treatments alter their wettability. Over the range 5 ≤ 𝑝𝐻 ≤ 9, differences between the wettability of stratum corneum samples treated with a 1% solution of sodium lauryl sulfate in a pH buffer, and samples treated with the pH buffer alone are established. For stratum corneum samples retaining skin lipids, surfactant treatments induce a more hydrophilic skin surface under alkaline conditions. With removal of skin lipids, surfactant treatments induce a hydrophobic surface under acidic conditions and a hydrophilic surface under alkaline conditions. Profilometry subsequently reveals that these changes in wettability are not caused by surfactant induced changes in stratum corneum roughness.
To our knowledge, the ability to control the wettability of human skin by altering the pH of an anionic surfactant solution treatment has not previously been reported. Moreover, the observed changes in human stratum corneum wettability we report in this article are consistent with a reorientation of bound surfactant monomers as the pH is varied [21]. Electrostatic binding of polar surfactant monomer head groups to positively charged keratin under acidic conditions [16] would leave the hydrophobic tails exposed. In contrast, a hydrophobic attraction of the surfactant tails to the tissue surface under alkaline conditions would leave the hydrophilic heads exposed. This mechanism has previously been employed to explain changes in wettability timescales for keratinous wool fibers [23]. Here we show this mechanism is also plausible for human skin. The more pronounced change in wettability that occurs with delipidated stratum corneum across the measured pH range further suggests that lipids present at the skin surface reduce interactions between the surfactant monomers and keratin in the tissue. This presumably occurs through direct obstruction of binding sites, or a preference for surfactants to adsorb to, then emulsify the lipids; with subsequent rinsing removing both lipids and surfactants from the tissue surface. A wide range of surfactants exist with numerous head group charges [22]. Future work should explore how changes in head group charge influence the wettability of stratum corneum after surfactant treatment at different pH conditions. This insight would help to further scrutinize the validity of a pH induced reorientation of bound surfactant monomers.
Acknowledgments We acknowledge funding from Schick. We also would like to thank Prof. David Cole-Hamilton for helpful discussions.
Figure captions Figure 1. Changes in wettability from SLS treatments of SC retaining lipids (REF SC). The average contact angle of deionized water drops over a 90 sec period after deposition on control REF SC samples treated for 60 min with (a) buffer solutions containing 1 % SLS and (b) buffer solutions alone. Square, triangle, and diamond symbols respectively denote buffer pH conditions of 5, 7, and 9. (c) The average change in surface energy of the REF SC samples upon wetting, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 , for buffer solution treatments (dark solid grey) and buffer solutions containing 1 % SLS (light solid grey) across the range 5 ≤ 𝑝𝐻 ≤ 9. Error bars denote standard deviations. (d) The average change in surface wettability, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), between SC samples treated with buffer solutions containing 1 % SLS and the buffer solutions alone. Error bars denote propagated standard deviations. Figure 2. Changes in wettability from SLS treatments of delipidated SC (DLSC). The average contact angle of deionized water drops over a 90 sec period after deposition on delipidated DLSC samples treated for 60 min with (a) buffer solutions containing 1 % SLS and (b) buffer solutions alone. Square, triangle, and diamond symbols respectively denote buffer pH conditions of 5, 7, and 9. (c) The average change in surface energy of the DLSC samples upon wetting, 𝛾𝑆𝑉 − 𝛾𝑆𝐿 , for buffer solution treatments (dark solid grey) and buffer solutions containing 1 % SLS (light solid grey) across the range 5 ≤ 𝑝𝐻 ≤ 9. Error bars denote standard deviations. (d) The average change in surface wettability, ∆(𝛾𝑆𝑉 − 𝛾𝑆𝐿 ), between DLSC samples treated with buffer solutions containing 1 % SLS and the buffer solutions alone (dark solid grey). Changes in wettability of control REF SC samples from Fig. 1 (d) are plotted alongside for reference (light solid grey). Error bars denote propagated standard deviations. Figure 3. Changes in surface topography of SC with surfactant treatments. (a) Representative surface profile along the diameter of a circular REF SC sample after treatment with pH 5 buffer. A magnified view of the region outlined by the dashed box is shown in (b). (c) The average dimensionless relative diameter, 𝐷 ′ /𝐷 ∗ of control REF SC samples treated with buffer solutions containing 1 % SLS (blue solid bars, 𝑛 = 3 individual samples for each pH) plotted alongside equivalent measurements of samples treated with the buffer alone (open bars, 𝑛 = 3 individual samples for each pH). (d) The average relative diameter, 𝐷′ /𝐷 ∗ of lipid depleted DLSC samples treated
with buffer solutions containing 1 % SLS (blue solid bars, 𝑛 = 3 individual samples for each pH) plotted alongside equivalent measurement of samples treated with the buffer alone (open bars, 𝑛 = 3 individual samples for each pH). Error bars denote standard deviations.
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Figure Caption
Figr-1
Figr-2
Figr-3
Tables and Table Captions Table 1. Measured pH values of the buffer solutions alone and the buffer solutions containing 1% SLS. Buffer/Solution pH 5 buffer 1% SLS + pH 5 buffer pH 7 buffer 1% SLS + pH 7 buffer pH 9 buffer 1% SLS + pH 9 buffer
Measured pH 5.02 4.94 7.13 7.07 9.06 8.72
Table 2. The average surface tension of deionized water drops after a 90 sec deposition on SC samples treated with the different buffer and surfactant solutions.
Abbreviation
SC preparation
SC treatment and pH
REF SC REF SC REF SC REF SC DLSC DLSC DLSC DLSC REF SC REF SC REF SC DLSC DLSC DLSC
Control Control Control Control Delipidated Delipidated Delipidated Delipidated Control Control Control Delipidated Delipidated Delipidated
None pH 5 Buffer alone pH 7 Buffer alone pH 9 Buffer alone None pH 5 Buffer alone pH 7 Buffer alone pH 9 Buffer alone pH 5 Buffer + 1% SLS pH 7 Buffer + 1% SLS pH 9 Buffer + 1% SLS pH 5 Buffer + 1% SLS pH 7 Buffer + 1% SLS pH 9 Buffer + 1% SLS
Droplet 𝜸𝑳𝑽 after 90 sec deposition (mN/m) 70.7 ± 1.0 (n=3) 64.8 ± 0.5 (n=2) 68.9 ± 3.7 (n=4) 57.0 ± 3.7 (n=2) 70.4 ± 0.6 (n=3) 61.5 ± 3.1 (n=2) 62.6 ± 2.3 (n=2) 68.5 ± 3.5 (n=2) 44.4 ± 5.6 (n=3) 56.0 ± 5.6 (n=4) 49.2 ± 2.9 (n=3) 41.9 ± 2.2 (n=2) 53.8 ± 4.0 (n=2) 51.5 ± 12.4 (n=3)