A sequential tape stripping approach for the assessment of the impact of personal cleansing products on the stratum corneum surface layers' acid mantle properties and antimicrobial defense

Stratum corneum (SC) plays a critical role in skin barrier function for protection and defense in nature. The acidic skin pH, which is also known as the acid mantle, is very important in fighting against outer environmental threats, especially, bacteria. Furthermore, recent research has shown that the transient bacteria could potentially penetrate into deeper layer of the SC down to a few micrometers while posing an additional threat to the deeper layers of the skin.


| INTRODUC TI ON
The stratum corneum (SC) is organized into a dual-compartment structure, consisting of a layer of strong, dead corneocytes embedded in a lipid-enriched matrix that helps to keep water from entering or leaving the body.This tight structure acts to protect the skin from harm via several barrier functions, including supporting cohesion (integrity), permeability limitations, antimicrobial defenses, chemical exclusion, and anti-oxidant resistance. 1,2e acidity of the skin plays a vital role in enabling the defensive functions of the SC, such as: (1) Beta-glucocerebrosidase (GLC) and sphingomyelinase, two key enzymes that generate ceramide, show optimum performance at acidic pH. 3,4The generated ceramide is the dominant lipid to enable the intact structure of the SC. 5 (2) Acidic pH is required for the formation of multi-lamellar structures assembled from ceramide, free fatty acids, and cholesterol which are critical for the skin to restrict water loss and protect against other harmful factors from the environment. 5(3) Skin pH is also critical in fighting bacteria; that is, the acidity of the skin inhibits the proliferation of transient bacteria and opportunists, such as Staphylococcus aureus. 6Additionally, the antimicrobial hostility of the free fatty acids is also regulated by pH.It is found that the minimum inhibitory concentration (MIC) values of acetic and propionic acids for resident bacteria are much higher than for transient bacteria at low pH. 7(4) The integrity or cohesion of the SC is also related to the acidic pH.Hachem et al. found that a change of the skin pH from acidic to neutral increases the activity of serine protease which leads to the degradation of corneodesmosomes. 3The consequences of this include damage to the skin permeability barrier, and similarly to the cohesion or integrity of the SC with resulting higher trans-epidermal water loss (TEWL) and the removal of more protein from same number of tape-strips. 3][10] Among cleansing products, washing with alkaline soap (pH >9) has been shown to render the harshest skin results, that is, pH value increase, lipid removal, and decrease in hydration. 9ch alkaline changes in skin pH have also been linked with bacteria-related skin issues; whereby several studies also demonstrated the benefit of using acidic cleansing products to inhibit bacterial growth and improve the related skin problems. 11,12Consequently, it is reasonable to believe that the daily use of cleansing products not only impact the skin surface, but also compel deeper changes in the SC as shown in the protein removal value from sequential tape stripping method. 8This leads to a further concern relative to the ability of bacteria to travel and infect more deeply within the SC layer, as suggested by recent investigations where the potential penetration of opportunistic/transient bacteria such as, Staphylococcus aureus was highlighted in three-dimensional human reconstructed skin models and human explant skin. 13,14Therefore, there is a need to investigate the impact of various cleansing products on the skin defense properties at the surface and more deeply within the SC.
Previously, a cup scrub-based residual efficacy testing (RET) clinical approach was established to evaluate the residual antimicrobial efficacy of personal cleansing products. 15This method has significantly simplified the evaluation process under a simulated product-use condition rather than a chronic washing procedure.This approach, however, still bears safety concerns, as a high level of germ inoculation on the forearm is needed.Recently, a tape stripping-based approach was developed where the germ was inoculated on the SC layers of the tape 16 with antimicrobial hostility being evaluated based on the delayed growth of bacteria using Soleris detection time (DT) as the endpoint.As such, the DT is inversely proportional to the number and/or metabolic health of the germs carried over on the tape strip; that is, a longer DT indicates a stronger level of antibacterial efficacy. 17,18This method alleviates the safety concerns for potential method-induced infection on the human body and further greatly improves testing efficiency because both inoculation and recovery of germs from human forearms constitute a tedious process that requires strict training of the operator.In this paper, this method has been further advanced to understand the skin acid mantle properties at deeper layers of the SC surface.
In order to probe the acid mantle properties of the deeper layers of the SC surface, a sequential tape-stripping approach was employed to obtain the skin samples from the same location.
0][21][22][23] Leveraging this approach, two personal cleansing products with different compositions and varied pH were evaluated through a clinical study.High pH product is a soap combo formula containing mainly soap-based surfactants.The low pH product is made of synthetic surfactants including sulfate and betaine.These two types of formulations are very representative of the current in-market low and high pH products and have been thoroughly investigated in the academic field. 9,10,15,24Skin pH and antibacterial defense properties were assessed at baseline before product treatment and 12 h post-washing under simulated use conditions.Importantly, these measurements were performed several layers (Layer 1 to Layer 3) into the SC surface.

| Test products and materials
The key compositions of the two-test liquid bodywash formulations are described below.The high pH (pH = 9.35) Product 1 comprised the following ingredients: sodium fatty acid soap, water, glycerin, fragrance, and minors.The Low pH (pH = 5.7) Product 2 bodywash contained water, sodium laureth sulphate sodium chloride, cocamidopropyl betaine, fragrance, pyrrolidone carboxylic acid (PCA) and minors.The sampling tape was purchased from D100-D-Squame Standard Sampling Discs (CuDerm Corp., USA).

| Application of test products
After the 7-day prewash phase, subjects arrived at the clinical site and acclimated for 30 min in a controlled environment (RH = 50 ± 10%, Temp = 21 ± 1°C).The two test products were assigned to subjects' left and right volar forearms, according to computer-generated randomization.Following the 7-day washout period, subjects performed a supervised washing procedure for three times on both forearms with the assigned liquid bodywash product, respectively.Starting from the right side, the subject's forearm was wetted under running water for 15 s, before 0.7 g of product was dispensed at the center of the forearm.This was followed by a 45-s spreading and rubbing time, using a back-and-forth motion.The forearm was then rinsed with running water for 15 s and patted dry.This procedure was repeated on the left forearm.Each of subject's forearms was washed three times in total.This is the same procedure used in clinical RET (ASTM E2752).

| Tape stripping
Once the target forearm site was ready for sampling, at baseline and after the third wash, the first strip of adhesive tape was adhered to the designated site on the forearm, avoiding folds.The skin site of interest was marked in advance for consistency.To keep uniform pressure and reach optimal adhesive bond, a roller was used to press the tape onto the skin surface (e.g., twice on each site) immediately after the tape application.The tapes were peeled off from the skin using tweezers.Next, the second strip of adhesive tape was adhered to the same location on the skin, pressed, and peeled off, like above.
Finally, another strip of adhesive tape was adhered to the same location and processed as was done twice before.

| Skin anti-bacteria defense property measurement
Eleven out of the 55 study subjects were selected for skin antibacterial defense property evaluation.The tapes from those selected subjects were placed on the surface of a TSA agar plate with the skin sample side up, at which time, 10 μL of the Staphylococcus aureus culture was inoculated onto the skin sample side of each tape strip and spread evenly over the tape surface with a sterile inoculation loop or pipette tip.The inoculum was allowed to visually dry on the surface of the tape strip (approximately ~3-5 min).
The inoculated tape strip residing on the TSA agar plate was then placed into a humidified incubator at 35°C and 60% ± 20% relative humidity for 4 h.Subsequently, each tape was aseptically transferred into one NF-TVC (non-fermenting total viable count) culture vial for continuous monitoring for 24 h to determine the DTs through Soleris (Neogen) optical measurements.The DT was determined as the time when color change of the indicator portion of the Soleris vial reached a certain magnitude, as determined by the Soleris software, which was inversely proportional to the number and/or metabolic health of the germs carried over on the tapes. 17,18

| Skin pH measurement
Skin pH measurements were performed for all 55 subjects, before any strip of adhesive tape was taken and after the third strip was taken.
All skin pH values were measured by a pH meter HI 99181 (Hanna Instruments, USA) with a flat glass electrode.The measurements were taken before forearm washing (baseline) and 12 h after the washing.

| Data analysis
The pH value is measured for each subject at each time point (base-

| Skin anti-bacteria defense property
The skin antibacterial property was explored on the SC layers stripped using D-squama tape for 11 out of the 55 subjects.Five to six logs of bacteria were inoculated onto the SC layers and the DT was measured.Compared with the well-established clinical RET method (ASTM 2752), this approach avoids direct germ contact with human arms, thereby reducing safety concerns and also significantly increasing the testing efficiency as the collection of the residual germs is not needed. 15Our method has a baseline measurement deemed necessary to assess the antibacterial hostility of the skin before use of any product treatment.

| Antibacterial efficacy of baseline skin
As shown in Figure 1, before any treatment, the DT was similar among three consecutive layers (around 3.5 h) with no statistically significant difference.This indicates that the baseline skin, after washout period, was under the resting stage with no meaningful difference in the antibacterial property for all the subjects and that the contribution of the skin innate antibacterial property was normalized.Consequently, these observations suggest that this normalized antibacterial property obtained using the sequential tape stripping approach may be contributing significantly to the reduction in noise that often occurs when measuring the skin innate antibacterial property between different subjects, thereby increasing the overall sensitivity of the method.

| Product treatment impact
After treatment, at the 12 h time point, the low pH Product 2 significantly increased the DTs to 7.22, 6.08, and 4.75 h from the first to the third skin layer, which indicated an improvement in skin antibacterial efficacy from top to deeper skin, as shown in Figure 1.For the high pH Product 1, however, the DTs are 3.84, 3.63, and 3.43 h at first, second and third skin layer, respectively, revealing no significant improvement in skin antibacterial efficacy.Delta DTs were calculated by subtracting the baseline value by the 12 h time point value per subject per site basis, as shown in Figure 2. Statistical analyses showed a significant difference at each layer between the two products (p < 0.01).In this investigation, a longer DT using the Soleris microbial quantification method was indicative of a slower bacterial growth curve and a better skin antibacterial defense property. 17,18As a result, it appears that the skin antibacterial defense property was stronger after using the low pH Product 2 treatment when compared to the forearms cleansed with the high pH Product 1.Additionally, the skin's defense power of the low pH cleansing product was also measurably stronger across at least three skin layers.This suggests that the low pH cleansing product provided deeper antibacterial support to the SC, which was also evident 12 h post treatment.as evidenced in Figure 1, thereby also revealing its longer-lasting skin defense benefit, as well.

| Skin pH
The skin pH value was first measured at baseline following a 7-day wash out period prior to product treatment.The skin pH value distribution was performed for the 55 subjects on both the first and third skin layer.According to the literature, 19,21 these three layers' sequential tape stripping would likely only remove one to a few μms of skin corneocytes from the SC.As shown in Figure 3A,B, the skin pH values are mainly distributed in the range of four to six with males having a slightly lower pH than females on both the first and third layer.As skin pH has been extensively studied and found to vary between genders, body sites, and age groups, 6,25,26 the skin pH value range in this study is consistent with much of the literature. 6me studies; however, have shown that women can have a lower pH than men, which seemingly contradicts our observations. 25,27is difference might be attributed to the study population in which the subjects were often Caucasian. 25,27Recently, a large-scale study of the Chinese population has shown that men have a lower skin pH than women on the forearm, which is consistent with our findings.Skin pH value changes from the top surface to the deeper layer in SC.The pH value decreased first and then increased both from in vitro and in vivo studies. 28,29As shown in Table 1, the pH value of the third layer is lower than the first layer on average value.
Statistical analysis, based on each site of each subject, showed a significant difference between the first and third layer with p < 0.01 and mean difference value of 0.18.This result is consistent with the literature 28,29 and likely indicates that the third layer, in our study, has approached the deeper skin SC layer.
The skin pH value was also measured at 12 h time point after three times repeated wash treatments.Table 2 showed that the skin pH value, at 12 h time point, after the high pH Product 1 treatment is significantly higher than low pH Product 2. This is also true for the third skin layer as indicated by Table 2.

| DISCUSS ION
The present clinical study investigated a sequential tape stripping approach for assessing the impact of personal cleansing products on the SC surface layers' acid mantle properties and antimicrobial defense.The antimicrobial defense property increased significantly at the 12 h timepoint comparing the treatment of the low pH Product 2 as compared to the high pH Product 1.It is interesting to note that the delta DT decreased from 3.69 h for the top skin surface to 1.27 h for the third layer showing a significant decay in defense power across the three skin layers.This may be attributed to skin pH difference at different layer of the SC.Another factor could be due to differences of deposition/penetration of specific formulation ingredients (e.g., surfactants and PCA etc.).Further studies are needed to understand the fundamental mechanisms.
Results from this work clearly demonstrated that the low pH Product 2 showed less disturbance to the skin pH than high pH Product 1.As shown in Table 2, the mean skin pH value after use of the low pH Product 2 treatment is significantly lower than the resulting skin pH after use of the high pH cleansing Product 1. Seemingly, this could be attributed to a compositional difference in formulations, the low pH Product 2 used in this study contains PCA, which is a skin innate material that has been shown to contribute to skin acidity. 1 To demonstrate that the PCA from the low pH Product 2 could be deposited after wash, a similar washing protocol was followed using a human cadaver skin ex vivo model (see supporting information), where five consecutive tape stripes were taken following product application and rinse.To distinguish the PCA applied to the skin from our product from the innate PCA in skin, the PCA added in the product was radio-labeled with carbon-14, which could be distinctively detected via liquid scintil- Another factor, for the lower skin pH from Product 2, could be attributed to its buffering capacity.In fact, there is published literature 1,3,30,31 that points to the biochemical investments of the epidermis in supporting the acidification of the skin, such as by: (1) the generation of urocanic acid and PCA through the filaggrin-histidine pathway in the cornified layer, (2) the conversion of phospholipids to free fatty acids during the cornification process, and (3) the sodium-proton exchanger (NHE1) that exports H + ions across cell membranes in the outer layers of the SC.Among these endogenous factors, free fatty acids have been considered as key components in regulating the acid buffer capability due to their location and also their pKa value. 3,32Free fatty acids typically have a pKa value of ~4.9, which is very close to the acidic skin pH.Thus, it is believed that free fatty acids behave as a strong buffer to help resist skin pH changes, when exposed to an acid or alkaline environment.The increase in pH of the skin that was observed after treatment, therefore, could be attributed to the washing away of those skin acidity-contributing components, compelling this effect to be more obvious for the top surface than in the deeper skin layer, as evident in Table 2.As the deeper surface skin layers are afforded protection by the tight structural configuration of the SC, it was not surprising that the low pH cleansing Product 2 significantly limited its disturbance to the skin pH, regardless of number of tape strips, with faster recovery after 12 h versus use of the high pH soap Product 1 during washing and rinsing.As shown in Figure S2, the dilution curves for these two cleansing Products highlight the changes in pH in response to increased product dilution.This is a mimic of the real rinsing process during washing.The high pH Product 1 showed much higher pH than Product 2-even at 50 times of the product dilution.Given the pKa of free fatty acids is around 4.9, 32,33 at this high pH, one could postulate that the fatty acids would quickly turn to salt and be washed away rapidly.Consequently, it is also just as likely that more skin pH buffer agents also would be washed away after using the high pH soap combination Product 1 when compared to the low pH cleansing Product 2. In this line of thinking, the higher skin pH value disturbance across surface to deeper layers, also may weaken or lower the skin's natural defense power.Specifically, the long-lasting, 12 h pH increase may be indicative of the damage to the skin barrier function that can occur from chronic usage of high pH soap-based cleansing products.This corroborates the published literature, where the increase and upper shift of skin pH profile across the SC has been linked with diseased skin. 31,34in hydration state is another factor that may impact skin natural defense property.Previous study reported that NMF level increased for the first few SC layers to reach the maximum and then decreased in the deeper SC region. 35Similar trend was also observed for PCA (a key component of NMF). 36It has been proposed that, at the upper SC layer, NMF is most responsible for water binding. 35As PCA plays an important role in maintaining acidic skin pH and the skin hydration, an increase of PCA level will result in a lower skin pH and higher water content.Interestingly, the defense power across the three skin layers was also significantly stronger following use of the low pH Product 2 versus the high pH Product 1.In addition, the deeper layer analysis (third layer tape strip) showed a slightly but statistically significant lower pH value than the top SC surface, which is in agreement with previous literature findings. 28,29This could be attributed to less removal of the skin's pH buffer agents, as revealed by the product dilution curve analyses versus pH.Net, these results suggest that the low pH cleansing Product 2 not only provided deeper antibacterial support to the SC, but as the skin pH was still lower 12 h post treatment, it also rendered a longer-lasting skin defense benefit.Data sharing is not applicable to this article as no new data were created or analyzed in this study.

K 2 . 1 |
E Y W O R D S personal cleansing, skin acid mantle, skin antimicrobial defense, tape stripping 2 | MATERIAL S AND ME THODS Clinical study design and execution 2.1.1 | Study population Fifty-five healthy Chinese males and females who met the inclusion/ exclusion criteria with ages from 20 to 60 years old were recruited.Informed consent was obtained from all subjects.Subjects were provided with a commercially available mild baby wash product (pH 5.21) and haircare products for daily application during a 7-day prewash phase prior to the baseline measurement.This study protocol complies with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review committee of Chinese Academy of Inspection and Quarantine (per the U.S. Code of Federal Regulations, Title 21, Part 56, and applicable ICH guidelines).
line and 12 h after washing) on the skin surface and the third layer after tape-stripping.Delta pH value was calculated by subtraction of the 12 h by baseline value.The DT was measured for 11 out of 55 subjects at each time point.Delta DT was calculated by subtraction of the baseline by 12 h.The delta DT value was calculated by the difference between the two testing legs.Statistical analyses were performed with JMP 14 pro software.Pair comparison method was used for testing the significance with α value (threshold) set as 0.05.All error bars are calculated based on standard error.

F I G U R E 1
Detection time results at baseline (left part) and 12 h time point (right part) across three skin SC layers with two cleansing products.Product 1 (red color) is with high pH and product 2 (blue color) is with low pH.
lation counting.Deposition amount was normalized to microgram (μg) of protein on each tape.As shown in Figure S1, PCA from the product was deposited across the five consecutive tape strips with the amount delivered between 0.15 and 0.25 ng per μg protein.DI water was used as a negative control and was also measured, with no radio labeled PCA detected, as expected.The measured deposition/penetration of PCA across the different skin layers of low pH Product 2 may contribute to the reduced skin pH versus the high pH Product 1.

F I G U R E 2
Delta detection time results across three skin SC layers with two cleansing products.Product 1 (red color) and product 2 (blue color) are a high pH and a low pH product, respectively.F I G U R E 3 Skin pH value distribution of the study population on (A) first and (B) third skin layer.
Leveraging a sequential tape-stripping approach, the deeper body skin SC acid mantle property was compared, following use of two different personal cleansing products-a high pH soap-based Product 1 and a low pH synthetic surfactant-based Product 2. It is shown that the skin's antibacterial defense power was increased significantly after the treatment with the low pH Product 2 at the 12 h time point versus treatment with the high pH Product 1.
This study protocol complies with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review committee of Chinese Academy of Inspection and Quarantine (per the U.S. Code of Federal Regulations, Title 21, Part 56 and applicable ICH guidelines).O RCI D Lesheng Zhang https://orcid.org/0000-0001-8813-3112TA B L E 1 pH value and statistical analysis of first and third layer of skin at baseline.

2
pH value and statistical analysis of first and third layer of skin after treatment at 12 h time point.