Development of an in vitro Functional Assay to Evaluate the Occlusive Properties of Moisturizers on Dry Skin

Maintaining proper skin hydration is essential for healthy, flawless-looking skin. Skin hydration is not only of cosmetic importance but also necessary for the skin to fulfill “la raison d'être” as described by Madison: acting as the body’s primary permeability barrier [1]. The outermost layer, the stratum corneum (SC), consists of a network of tightly packed corneocytes surrounded by intracellular lipids and hygroscopic materials that restrict the passage of water [2, 6]. The permeability of SC is significantly influenced by the lamellae lipid components and their structural organization [7, 9]. In normal skin, a relatively low level of water is lost to the environment, which is constantly replenished by the underlying viable tissue. This homeostatic level of moisture is vital to preserve the integrity and smooth appearance of the SC, as insufficient hydration can compromise its barrier function.

Xerosis, or dry skin, is a common problem that results from damage to the barrier and excessive water loss. The underlying causes of xerosis are diverse and complex and can result from several individual or environmental factors. Moisturizers play a central role in a basic skin-care regimen, and a myriad of products are available claiming to address dry skin. Active moisturizing ingredients may work by acting as occlusives, which coat the skin surface as a hydrophobic film, humectants, which increase SC hydration by attracting and binding to water, or emollients that can fill gaps between corneocytes and smooth the skin [10]. In addition, some products include actives that stimulate endogenous barrier repair. Moisturizers are formulated to fulfill one or several of these functions, but in general, they aim to prevent further evaporation (occlusives) or allow water to replenish the SC from within the deeper layers of the skin (humectants). This increase in water content can both improve skin appearance and provide sufficient hydration for the restoration of barrier function.

Despite the wide range of products and formulations available, effective moisturizers represent an area of high consumer need [11]. However, the lack of reliable measurements of barrier function using in vitro models poses a challenge for the preclinical development of new products. There is a paucity of clinical studies evaluating the efficacy of moisturizers and even fewer describing their development and optimization. Comparative assessment of trans-epidermal water loss (TEWL) is the most common method used to assess the effect of moisturizers on the skin, under the premise that a compromised barrier is more permeable to water. Several different devices exist to measure TEWL; these have found widespread clinical use because they are noninvasive. However, because these measurements rely on measuring vapor density, they are prone to interference from the environment and require very careful, controlled conditions [12, 13]. Moreover, these measurements may not be able to detect more minor changes in barrier integrity that produce clinically significant results [14]. Advanced spectral and imaging techniques have been recently introduced to measure skin hydration. While they can provide a vast amount of information regarding the structure and composition of the skin, the instrumentation is expensive, and complex data deconvolution and analysis techniques are required [13]. There is a need for simple, robust, and higher throughput methods that can be used to test the effect of moisturizers on skin barrier function. Thus, the purpose of the study was to establish a functional assay to measure occlusive properties of moisturizers in a 3D reconstructed human epidermis (RHE) model.

EZ-Link^TM NHS-LC-Biotin (N-hydroxysulfosuccinimide [NHS], cat#21335) was obtained from Thermo Fisher (Waltham, MA, USA), and 5 mg/mL solution (tracer solution) was prepared in PBS. Lucifer Yellow (LY) CH dilithium salt (cat#L0259) was purchased from Sigma (St. Louis, MO, USA) and reconstituted to 1mm in water. Dimethylpolysiloxane, hyaluronic acid (HA), poly-L-glutamic acid (PGA) were obtained from Sigma (cat#DMPS5C, cat#53163, and cat#P4886, respectively). HA and PGA were solubilized to 2% w/v and 4% w/v in water, respectively. Glycerol was purchased from VWR (cat#0854, Randor, PA, USA), and petroleum jelly (petrolatum, Vaseline) was sourced from Unilever (Englewood Cliffs, NJ, USA). Ultra Sheer Moisturizer (USM) and HA5 Rejuvenating Hydrator (HA5) that contain both HA and dimethylpolysiloxane were obtained from SkinMedica (Allergan Aesthetics, an AbbVie Company, Irvine, CA, USA). The ingredients for USM are as follows: water, cetyl ethylhexanoate, sorbitan stearate, methyl gluceth-20, polysorbate 60, dimethicone, tocopherol, tocopheryl acetate, tetrahexyldecyl ascorbate, panthenol, sodium hyaluronate, cetearyl alcohol, Ceteareth-20, aminomethyl propanol, carbomer, disodium EDTA, phenoxyethanol, ethylhexylglycerin, potassium sorbate. The ingredients for HA5 are as follows: water, dimethicone, HDI/trimethylol hexyllactone crosspolymer, glycerin, butylene glycol, polysilicone-11, Bis-PEG-8 dimethicone, sodium acrylate/sodium acryloyldimethyl taurate copolymer, sodium hyaluronate crosspolymer, hydrolyzed HA, sodium hyaluronate, Vitis vinifera (grape) flower cell extract, Vibrio alginolyticus ferment filtrate, Alteromonas ferment extract, Porphyridium cruentum extract, whey protein, plankton extract, trehalose, urea, serine, algin, caprylyl glycol, pullulan, disodium phosphate, potassium phosphate, pentylene glycol, polymethylsilsesquioxane, glyceryl polyacrylate, sodium citrate, sea water, sucrose palmitate, tocopheryl acetate, hydroxyacetophenone, polysorbate 60, propanediol, potassium sorbate, citric acid, isohexadecane, polysorbate 80, silica, decyl glucoside, tromethamine, ethylhexylglycerin, phenoxyethanol, disodium EDTA.

Epiderm™ (3D epidermis human skin model) and EpidermFT™ (3D full thickness human skin model) tissues were obtained and maintained in culture according to the manufacturer’s recommendation (MatTek Corp., Ashland, MA, USA). Briefly, after being equilibrated overnight, tissues were first treated topically with either water (control) or 2% sodium dodecyl sulfate (SDS) for 2 h in a 37°C/5% CO2 incubator. Next, 10 µL of water, single active ingredients, or commercial moisturizing products were applied to the surface for an additional 1 h incubation before being subjected to the tracer assay.

where X = height of tracer and Y = height of SG.

The relative distance per biological replicate was expressed as an average of 5 randomly chosen points per tissue.

Twenty microliters of an LY solution (1 mm) was placed on the top of EpidermFT™ tissues treated as described above. After incubation for 1 h, tissues were fixed with 10% neutral buffered formalin, embedded into paraffin blocks, sectioned (4 µm), and then mounted on a glass slide. Fluorescent and phase-contrast images were taken with a REVOLVE microscope equipped with ×10 objective (Echo, San Diego, CA, USA).

Water content in the skin was measured using the gen2-SCA performance confocal Raman system (RiverD International, Rotterdam, The Netherlands) [15, 16]. Raman spectra in the high wave number region (2,500–4,000 cm-1) were acquired with ×60 oil-immersion objective using an excitation wavelength of 671 nm and 2 s exposure time. Measurements were taken in increments of 2 µm from the skin’s surface down to a depth of 70 µm. The average of 4 readings was obtained from each sample, which was repeated across 3 different skin samples. Raman spectra were analyzed using the embedded software (SkinTools 3, RiverD).

Data analysis was conducted in R, and figures were produced using the package ggplot2 [17].

To quantify the occlusive ability of moisturizers, an “inside-out” tracer assay was adapted from the surface biotinylation method to indirectly measure the migration of water through 3D organotypic skin culture [18]. The tracer molecule (NHS-Biotin) is dissolved in PBS that is in contact with the basal keratinocyte layer, where it can then migrate upward due to the moisture evaporation from the top surface (Fig. 1a). Since the NHS-LC-biotin tracer is cell impermeable, it may also react with proteins containing N-terminal amino groups and exposed lysine residues during its migration through the paracellular track. They can be structural transmembrane proteins that constitute adherens junctions (E-cadherin), desmosomes (desmoglein), tight junctions (claudin), and extracellularly secreted proteins, such as perlecan. Although tight junctions halt the movement of NHS-Biotin at the SG, a shift in the water gradient will affect how fast the tracer moves paracellularly. To demonstrate the link between water loss and tracer migration, a forced evaporation apparatus was designed to blow compressed air at the rate of 4 L/min directly on the top center surface of RHE (Fig. 1a). Differences in permeability were calculated as the relative distance traveled by the tracer (Fig. 1b, bottom). Natural evaporation did not induce a noticeable tracer movement (Fig. 1b, c). In contrast, forced evaporation significantly induced tracer migration toward the SC-SG layer, indicating that topical water loss promoted tracer migration (Fig. 1b, c). Considering that the migration in dry skin would be higher, and topical occlusion would reduce migration, a 4 min experimental duration was chosen to evaluate dry skin barrier function and improvement (Fig. 1d).

To mimic dry skin, the skin barrier function was acutely compromised by applying a strong anionic detergent SDS to the SC at the air-liquid interface [19]. The SDS-treated tissue displayed an increased distance traveled by the tracer media compared to control-treated tissue (Fig. 2a, b). Thus, tissue barrier function can be comparatively measured by the tracer flux in this system. Independent experiments from the “outside-in” LY assay and confocal Raman microspectroscopy were performed to support the observation that SDS treatment disrupts SC barrier integrity (Fig. 2c, d) [19, 21]. With LY, a deeper and brighter fluorescent signal in SDS-treated tissue was observed compared to control, representing the compromised outer barrier (Fig. 2c). Confocal Raman microspectroscopy was then used to measure the water content in the SC and its below layers, as previously described in the literature [16, 22]. Both control- and SDS-treated skin were exposed to forced evaporation, without contact with PBS. Whereas normal skin maintained 70% of water content (I_water/I_protein) below ~30 µm skin depth, in SDS-treated skin it dropped to between 50 and 60% (Fig. 2d).

If the movement of the tracer is reflective of water loss, then the passage of the tracer should be influenced by the properties of topically applied material. To validate the assay, both a humectant (glycerol) and an occlusive (petrolatum) were tested on control- and SDS-treated skin. In normal skin, the petrolatum reduced the relative distance of tracer uptake, while that of glycerol was greater than the water-treated condition (Fig. 3a, b). As expected, SDS treatment induced higher tracer migration, whereas topical application of petrolatum reduced migration in both control- and SDS-treated tissue. In contrast, the humectant only provided marginal improvement to normal skin tissue barrier integrity (Fig. 3a, c).

To demonstrate the utility of the tracer assay, the occlusive capabilities of either single ingredients or comprehensive products were tested. An occlusive, dimethicone, as well as 2 humectants HA and PGA were tested on control- and SDS-treated skin as single ingredients (Fig. 4). For comparison, 2 commercial cosmetic moisturizers USM and HA5 that contain one or more of the above ingredients were also tested using this platform. As expected, dimethicone reduced the relative distance in both control- and SDS-treated skin. However, while both 2% HA and 4% PGA increased the tracer migration in control skin, PGA additionally decreased that in SDS-treated skin. Despite these differences, both USM and HA5 reduced tracer migration on control- and SDS-treated skin.

Having a robust in vitro platform can facilitate the optimization of moisturizer formulations. The approach described here represents a simple and accessible methodology to evaluate the physical effects of moisturizing products in an in vitro model of dry skin. SDS, a severe irritant previously established to interfere with the SC, was used to disrupt skin barrier integrity [19, 23], and the degree of damage was measured by the migration of an aqueous small molecule tracer. It was shown that the application of an occlusive moisturizing product was able to reduce the uptake of the tracer and thus water loss (Fig. 3, 4). Ideal moisturizers not only physically prevent the water loss from the top surface but also replenish water molecules from deep within the skin to maintain the physiological condition up to the SC. For this reason, most moisturizers contain a mixture of ingredients of two opposite properties – occlusives and humectants, but fine-tuning to maximize each property is a difficult task. While dimethicone hindered the tracer migration in both normal and dry skin models, the humectants HA and PGA increased tracer migration in the control skin condition (Fig. 4). It is reasonable to postulate that topical water evaporation drew the tracer solution by capillary action and water-holding humectant properties facilitate its continuity. However, it is not clear why in the SDS-treated model higher tracer uptake was not observed. One possible explanation is that the disruption of SC enlarged the paracellular space in SC and interfered with capillary action. The opposite effects from humectants and occlusives and their dosage level determine overall efficacy of the final formulation as shown in the tracer migration of USM and HA5, although other ingredients likely play a role. Individual ingredient assessment using this tracer assay could give insights on how to balance these two properties.

This system has several advantages over previously used methodologies to evaluate barrier integrity [24]. First, the advent of RHE removes the ethical constraints of in vivo experiments and the tedious recruitment of human volunteers. The test system can thus be customized to include other methods of barrier disruption, and the use of RHE from donors of different skin types and ethnicities can address some of the biological variables that would affect future clinical results [25]. Second, while the classic, gold standard measurements such as TEWL and skin capacitance have utility in the clinic because they are noninvasive, they are prone to interference and require careful standardization between conditions [12]. This system uses reliable cellular and molecular biology techniques and controlled laboratory conditions, removing many of the variables associated with experimental error. In this tracer assay, the imaging measurements and calculations are simple and straightforward. Therefore, many samples can be processed in parallel, making this approach highly amenable to structure-activity relationship studies and rapid screening of different formulations.

While this methodology was developed for cosmetic dry skin, it can be extended to other in vitro models of conditions that disrupt barrier function. In addition to what was shown here, the tracer assay can be combined with other endpoints for a more detailed analysis of changes to the skin. For example, biophysical, analytical, and atomic simulation techniques used to evaluate the structure, morphology, and molecular composition would complement the measurements of barrier integrity by the tracer assay described herein [26, 30]. Especially since SDS treatment was reported to disrupt long periodicity phase of lipid assembly significantly, rather than short periodicity phase, it would be interesting to demonstrate the functional roles of long periodicity phase and short periodicity phase in skin permeability [7, 8, 23]. If there are reagents to specifically alter the nanostructure of the lipid matrix in SC, it can be explored how the lipid matrix contributes to overall SC barrier function with the tracer assay [31]. Altogether, these complementary assays can be excellent tools to further optimize the properties of moisturizers for cosmetic dry skin and possibly other indications.

There are some limitations to the methodology as presented here, namely, due to fundamental differences between 3D in vitro models as compared to native skin. Cultured 3D skin may be more permeable compared to human epidermis due to differences in lipid composition and organization, but consequences of this may be mitigated because the results from this assay are comparative [32]. The possible differences can be addressed in the future by using additional models, such as ex vivo skin explants or cultured biopsies [33]. Importantly, proper management of dry skin often requires repeated, long-term application of a product. Some formulations can improve barrier function in the short term, but they may cause long-term damage when used repeatedly [34]. In this study, the effect of a single application was examined but could potentially be extended up to several days. Because the tracer assay cannot be employed in patients, it is currently unclear if the results from this assay would be predictive of efficacy in vivo. In the future, it will be important to relate the results from the barrier assay in vitro to those noninvasively obtained in complementary clinical studies.

In summary, a simple and practical method was developed to assess moisturizing ingredients and formulations in terms of occlusive effects in vitro. This approach may be a valuable tool for the preclinical development and optimization of new moisturizing formulations to treat cosmetic dry skin.

Writing assistance and editorial support were provided by Liza Selwan-Lewis, PhD, an employee of AbbVie, Inc.

Ethical approval was not required for this study type, and no human or animal subjects or materials were used.

Soonjin Hong, Prithwiraj Maitra, Audrey Nguyen, Kuniko Kadoya, and Rahul Mehta are employees of AbbVie, Inc., and may own stock in the company.

This research was supported by Allergan Aesthetics, an AbbVie company. Employees of AbbVie participated in the research, interpretation of data, review of the manuscript, and the decision to submit for publication.

Soonjin Hong, Prithwiraj Maitra, Kuniko Kadoya, and Rahul Mehta were responsible for the conception and design of the work. Soonjin Hong was responsible for the acquisition, analysis, and interpretation of the results. Audrey Nguyen was responsible for the histological sample preparation. All authors provided critical feedback for the manuscript as developed, reviewed and approved the manuscript, and have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.