Research Article

Skin Pharmacology
and Physiology Received: January 5, 2022
Skin Pharmacol Physiol 2023;36:140–148
Accepted: February 1, 2023
DOI: 10.1159/000529630 Published online: March 2, 2023

Development of an in vitro Functional

Assay to Evaluate the Occlusive
Properties of Moisturizers on Dry Skin
Soonjin Hong Prithwiraj Maitra Audrey Nguyen Kuniko Kadoya

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Rahul C. Mehta
Skincare RandD, Allergan Aesthetics, an AbbVie Co., Irvine, CA, USA

Keywords Introduction
Dry skin · Moisturizers · Three-dimensional skin model · In vitro
models · Bioassay 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
Abstract fulfill “la raison d’être” as described by Madison: acting as
Introduction: Dry skin is a hallmark of impaired skin the body’s primary permeability barrier [1]. The outer-
barrier function. Moisturizers are a mainstay of treat- most layer, the stratum corneum (SC), consists of a
ment to help the skin retain moisture, and there is a high network of tightly packed corneocytes surrounded by
consumer demand for effective products. However, the intracellular lipids and hygroscopic materials that restrict
development and optimization of new formulations are the passage of water [2–6]. The permeability of SC is
hampered due to lack of reliable efficacy measures using significantly influenced by the lamellae lipid components
in vitro models. Methods: In this study, a microscopy- and their structural organization [7–9]. In normal skin, a
based barrier functional assay was developed using an relatively low level of water is lost to the environment,
in vitro skin model of chemically induced barrier damage which is constantly replenished by the underlying viable
to evaluate the occlusive activity of moisturizers. tissue. This homeostatic level of moisture is vital to
Results: The assay was validated by demonstrating the preserve the integrity and smooth appearance of the SC,
different effects on barrier function between humectant as insufficient hydration can compromise its barrier
(glycerol) and occlusive (petrolatum). Significant function.
changes in barrier function were observed upon tissue Xerosis, or dry skin, is a common problem that results
disruption, which was ameliorated by commercial from damage to the barrier and excessive water loss. The
moisturizing products. Conclusion: This newly devel- underlying causes of xerosis are diverse and complex and
oped experimental method may be helpful to develop can result from several individual or environmental
new and improved occlusive moisturizers for the factors. Moisturizers play a central role in a basic skin-
treatment of dry skin conditions. care regimen, and a myriad of products are available
© 2023 S. Karger AG, Basel claiming to address dry skin. Active moisturizing

karger@karger.com © 2023 S. Karger AG, Basel Correspondence to:

www.karger.com/spp Rahul C. Mehta, Rahul.mehta @ abbvie.com
ingredients may work by acting as occlusives, which coat and 5 mg/mL solution (tracer solution) was prepared in PBS. Lucifer
the skin surface as a hydrophobic film, humectants, which Yellow (LY) CH dilithium salt (cat#L0259) was purchased from
Sigma (St. Louis, MO, USA) and reconstituted to 1mM in water.
increase SC hydration by attracting and binding to water, Dimethylpolysiloxane, hyaluronic acid (HA), poly-L-glutamic acid
or emollients that can fill gaps between corneocytes and (PGA) were obtained from Sigma (cat#DMPS5C, cat#53163, and
smooth the skin [10]. In addition, some products include cat#P4886, respectively). HA and PGA were solubilized to 2% w/v
actives that stimulate endogenous barrier repair. Mois- and 4% w/v in water, respectively. Glycerol was purchased from VWR
turizers are formulated to fulfill one or several of these (cat#0854, Randor, PA, USA), and petroleum jelly (petrolatum,
functions, but in general, they aim to prevent further Vaseline) was sourced from Unilever (Englewood Cliffs, NJ, USA).
Ultra Sheer Moisturizer (USM) and HA5 Rejuvenating Hydrator
evaporation (occlusives) or allow water to replenish the (HA5) that contain both HA and dimethylpolysiloxane were obtained
SC from within the deeper layers of the skin (humec- from SkinMedica (Allergan Aesthetics, an AbbVie Company, Irvine,
tants). This increase in water content can both improve CA, USA). The ingredients for USM are as follows: water, cetyl
skin appearance and provide sufficient hydration for the ethylhexanoate, sorbitan stearate, methyl gluceth-20, polysorbate 60,
restoration of barrier function. dimethicone, tocopherol, tocopheryl acetate, tetrahexyldecyl ascor-
bate, panthenol, sodium hyaluronate, cetearyl alcohol, Ceteareth-20,
Despite the wide range of products and formulations aminomethyl propanol, carbomer, disodium EDTA, phenoxyethanol,
available, effective moisturizers represent an area of high ethylhexylglycerin, potassium sorbate. The ingredients for HA5 are as
consumer need [11]. However, the lack of reliable mea-

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follows: water, dimethicone, HDI/trimethylol hexyllactone cross-
surements of barrier function using in vitro models poses a polymer, glycerin, butylene glycol, polysilicone-11, Bis-PEG-8
challenge for the preclinical development of new products. dimethicone, sodium acrylate/sodium acryloyldimethyl taurate co-
polymer, sodium hyaluronate crosspolymer, hydrolyzed HA, sodium
There is a paucity of clinical studies evaluating the efficacy of hyaluronate, Vitis vinifera (grape) flower cell extract, Vibrio algino-
moisturizers and even fewer describing their development lyticus ferment filtrate, Alteromonas ferment extract, Porphyridium
and optimization. Comparative assessment of trans- cruentum extract, whey protein, plankton extract, trehalose, urea,
epidermal water loss (TEWL) is the most common serine, algin, caprylyl glycol, pullulan, disodium phosphate, potassium
method used to assess the effect of moisturizers on the skin, phosphate, pentylene glycol, polymethylsilsesquioxane, glyceryl pol-
yacrylate, sodium citrate, sea water, sucrose palmitate, tocopheryl
under the premise that a compromised barrier is more acetate, hydroxyacetophenone, polysorbate 60, propanediol, potas-
permeable to water. Several different devices exist to sium sorbate, citric acid, isohexadecane, polysorbate 80, silica, decyl
measure TEWL; these have found widespread clinical use glucoside, tromethamine, ethylhexylglycerin, phenoxyethanol,
because they are noninvasive. However, because these disodium EDTA.
measurements rely on measuring vapor density, they are
prone to interference from the environment and require Normal and Dry-Skin Mimicking 3D Human Skin Model
very careful, controlled conditions [12, 13]. Moreover, these Epiderm™ (3D epidermis human skin model) and Epi-
measurements may not be able to detect more minor dermFT™ (3D full thickness human skin model) tissues were
changes in barrier integrity that produce clinically signifi- obtained and maintained in culture according to the manufac-
cant results [14]. Advanced spectral and imaging techniques turer’s recommendation (MatTek Corp., Ashland, MA, USA).
Briefly, after being equilibrated overnight, tissues were first treated
have been recently introduced to measure skin hydration. topically with either water (control) or 2% sodium dodecyl sulfate
While they can provide a vast amount of information re- (SDS) for 2 h in a 37°C/5% CO2 incubator. Next, 10 μL of water,
garding the structure and composition of the skin, the single active ingredients, or commercial moisturizing products
instrumentation is expensive, and complex data deconvo- were applied to the surface for an additional 1 h incubation before
lution and analysis techniques are required [13]. There is a being subjected to the tracer assay.
need for simple, robust, and higher throughput methods
that can be used to test the effect of moisturizers on skin Tracer Assay (Surface Biotinylation Assay)
barrier function. Thus, the purpose of the study was to After topical treatment, tissues were placed in the tracer
establish a functional assay to measure occlusive properties solution for 4 min at room temperature while blowing air (4 L/
min) on top of the tissue to induce fast water evaporation.
of moisturizers in a 3D reconstructed human epidermis
Tissues were then fixed with 10% neutral buffered formalin,
(RHE) model. embedded into paraffin blocks, and processed for immuno-
histochemistry. Tissues were sectioned (4 µm), mounted,
stained with streptavidin-HRP, and tracer migration was vi-
sualized with 3,3’-diaminobenzidine (cat#k3468, Agilent
Materials and Methods Technologies, Santa Clara, CA, USA). Images of whole tissue
sections were captured by the NanoZoomer digital slide scanner
Materials (model C9600-02, Hamamatsu Photonics, Hamamatsu, Japan).
EZ-LinkTM NHS-LC-Biotin (N-hydroxysulfosuccinimide [NHS], The uptake height of the 3,3’-diaminobenzidine signal and the
cat#21335) was obtained from Thermo Fisher (Waltham, MA, USA), distance from the bottom of the tissue to the stratum

In vitro Assay Evaluating Moisturizers on Skin Pharmacol Physiol 2023;36:140–148 141

Dry Skin DOI: 10.1159/000529630
granulosum (SG) were measured using NDP view2 software at the SG, a shift in the water gradient will affect how fast
(Hamamatsu) and expressed as the relative distance traveled the tracer moves paracellularly. To demonstrate the link
using the following equation:
between water loss and tracer migration, a forced
X evaporation apparatus was designed to blow compressed
Relative distance (%)  100
Y* air at the rate of 4 L/min directly on the top center surface
where X = height of tracer and Y = height of SG. of RHE (Fig. 1a). Differences in permeability were cal-
The relative distance per biological replicate was expressed as culated as the relative distance traveled by the tracer
an average of 5 randomly chosen points per tissue. (Fig. 1b, bottom). Natural evaporation did not induce a
noticeable tracer movement (Fig. 1b, c). In contrast,
LY Barrier Assay forced evaporation significantly induced tracer migration
Twenty microliters of an LY solution (1 mM) was placed on the
toward the SC-SG layer, indicating that topical water loss
top of EpidermFT™ tissues treated as described above. After
incubation for 1 h, tissues were fixed with 10% neutral buffered promoted tracer migration (Fig. 1b, c). Considering that
formalin, embedded into paraffin blocks, sectioned (4 µm), and the migration in dry skin would be higher, and topical
then mounted on a glass slide. Fluorescent and phase-contrast occlusion would reduce migration, a 4 min experimental
images were taken with a REVOLVE microscope equipped with duration was chosen to evaluate dry skin barrier function
×10 objective (Echo, San Diego, CA, USA). and improvement (Fig. 1d).

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Confocal Raman Microspectroscopy
Water content in the skin was measured using the gen2-SCA Measuring Tracer Flux in a 3D Human Skin Model
performance confocal Raman system (RiverD International, Can Show Compromised Skin Barrier Function
Rotterdam, The Netherlands) [15, 16]. Raman spectra in the high To mimic dry skin, the skin barrier function was
wave number region (2,500–4,000 cm-1) were acquired with acutely compromised by applying a strong anionic de-
×60 oil-immersion objective using an excitation wavelength of 671 tergent SDS to the SC at the air-liquid interface [19]. The
nm and 2 s exposure time. Measurements were taken in incre-
ments of 2 μm from the skin’s surface down to a depth of 70 μm. SDS-treated tissue displayed an increased distance trav-
The average of 4 readings was obtained from each sample, which eled by the tracer media compared to control-treated
was repeated across 3 different skin samples. Raman spectra were tissue (Fig. 2a, b). Thus, tissue barrier function can be
analyzed using the embedded software (SkinTools 3, RiverD). comparatively measured by the tracer flux in this system.
Independent experiments from the “outside-in” LY assay
Statistical Analysis
Data analysis was conducted in R, and figures were produced
and confocal Raman microspectroscopy were performed
using the package ggplot2 [17]. 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
Results tissue was observed compared to control, representing
the compromised outer barrier (Fig. 2c). Confocal Raman
Tracer Assay (Surface Biotinylation) Visualizes the microspectroscopy was then used to measure the water
Water Loss from the Top Layer of RHE content in the SC and its below layers, as previously
To quantify the occlusive ability of moisturizers, an described in the literature [16, 22]. Both control- and
“inside-out” tracer assay was adapted from the surface SDS-treated skin were exposed to forced evaporation,
biotinylation method to indirectly measure the migration without contact with PBS. Whereas normal skin main-
of water through 3D organotypic skin culture [18]. The tained 70% of water content (Iwater/Iprotein) below ~30 µm
tracer molecule (NHS-Biotin) is dissolved in PBS that is skin depth, in SDS-treated skin it dropped to between 50
in contact with the basal keratinocyte layer, where it can and 60% (Fig. 2d).
then migrate upward due to the moisture evaporation If the movement of the tracer is reflective of water loss,
from the top surface (Fig. 1a). Since the NHS-LC-biotin then the passage of the tracer should be influenced by the
tracer is cell impermeable, it may also react with proteins properties of topically applied material. To validate the
containing N-terminal amino groups and exposed lysine assay, both a humectant (glycerol) and an occlusive
residues during its migration through the paracellular (petrolatum) were tested on control- and SDS-treated
track. They can be structural transmembrane proteins skin. In normal skin, the petrolatum reduced the relative
that constitute adherens junctions (E-cadherin), des- distance of tracer uptake, while that of glycerol was
mosomes (desmoglein), tight junctions (claudin), and greater than the water-treated condition (Fig. 3a, b). As
extracellularly secreted proteins, such as perlecan. Al- expected, SDS treatment induced higher tracer migration,
though tight junctions halt the movement of NHS-Biotin whereas topical application of petrolatum reduced

142 Skin Pharmacol Physiol 2023;36:140–148 Hong/Maitra/Nguyen/Kadoya/Mehta

DOI: 10.1159/000529630
 a
b

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c d

Fig. 1. Surface biotinylation assay visualizes the water loss from top (X) was expressed as a relative distance traveled toward the SG (Y).
layer of skin, which represents skin barrier function. a A schematic This was quantified from 5 distinct points per tissue. c Quanti-
depicting the well containing the tissue, tracer, and air-liquid fication of the conditions in (b). d With the forced evaporation,
interface for topical administration (left, figure created on EpiDerm™ tissues were treated for different and the relative
Biorender.com). The forced evaporation system constructed for distances were calculated as described in (c). Significant differences
the EpiDerm™ tissue culture format (right). b Topical moisture were determined using Students’ T test; **p ≤ 0.01, ***p ≤ 0.001,
evaporation naturally (left) or induced by blowing air (4L/min) for ****p ≤ 0.0001, n.s. indicates not significant. Data are shown as
10 min of linker treatment, processed for immunohistochemistry mean ± standard deviation; N = 3 tissues per condition. DAB, 3,3’-
with streptavidin-HRP and DAB. The uptake height of the tracer diaminobenzidine.

migration in both control- and SDS-treated tissue. In tested on control- and SDS-treated skin as single in-
contrast, the humectant only provided marginal im- gredients (Fig. 4). For comparison, 2 commercial cos-
provement to normal skin tissue barrier integrity metic moisturizers USM and HA5 that contain one or
(Fig. 3a, c). more of the above ingredients were also tested using this
platform. As expected, dimethicone reduced the relative
Both Single Ingredients and Commercial Products distance in both control- and SDS-treated skin. However,
Modulate Skin Barrier Function while both 2% HA and 4% PGA increased the tracer
To demonstrate the utility of the tracer assay, the migration in control skin, PGA additionally decreased
occlusive capabilities of either single ingredients or that in SDS-treated skin. Despite these differences, both
comprehensive products were tested. An occlusive, USM and HA5 reduced tracer migration on control- and
dimethicone, as well as 2 humectants HA and PGA were SDS-treated skin.

In vitro Assay Evaluating Moisturizers on Skin Pharmacol Physiol 2023;36:140–148 143

Dry Skin DOI: 10.1159/000529630
 a

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c
d

Fig. 2. Measuring tracer flux in a 3D human skin model is indicative c Tissues were treated with either control (water) or SDS (2%), then
of compromised skin barrier function. a Control tissue (left) or LY to measure the skin barrier integrity. d Confocal Raman
tissue treated with SDS (2%) after 4 min of linker treatment, spectroscopic measurements were performed for water content in
processed for immunohistochemistry with streptavidin-HRP and EpiDerm™ tissues treated either with water or SDS (2%) under
DAB. b Quantification of the conditions in (a). Significant differ- forced evaporation. Water contents were depicted along the skin
ences were determined using Students’ T test; ****p ≤ 0.0001. Data depth (~70 µm). Mean value was represented by dotted line and
shown as mean ± standard deviation; n = 3 tissues per condition. standard error by shades. DAB, 3,3’-diaminobenzidine.

Discussion with the SC, was used to disrupt skin barrier integrity
[19, 23], and the degree of damage was measured by the
Having a robust in vitro platform can facilitate the migration of an aqueous small molecule tracer. It was
optimization of moisturizer formulations. The ap- shown that the application of an occlusive moisturizing
proach described here represents a simple and acces- product was able to reduce the uptake of the tracer and
sible methodology to evaluate the physical effects of thus water loss (Fig. 3, 4). Ideal moisturizers not only
moisturizing products in an in vitro model of dry skin. physically prevent the water loss from the top surface
SDS, a severe irritant previously established to interfere but also replenish water molecules from deep within the

144 Skin Pharmacol Physiol 2023;36:140–148 Hong/Maitra/Nguyen/Kadoya/Mehta

DOI: 10.1159/000529630
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a b

Fig. 3. Topical applications of humectants and occlusives not significant. c Relative distance of the conditions in (a),
modulate skin barrier function. a The tracer assay outlined in comparing tracer migration in control- and SDS-treated skin in
Fig. 2 was performed, using SDS treatment followed by the the presence of glycerol or petrolatum. Significant differences
additional topical application of water, glycerol (humectant), or were determined using Students’ T test; **p ≤ 0.01, ****p ≤
petrolatum (occlusive). b Relative distance of the conditions in 0.0001, n.s. indicates not significant. In figure b, c, the relative
(a), comparing the effects of glycerol or petrolatum, in either distance was calculated from 5 different points per tissue. For all
control- or SDS-treated skin. Significant differences were de- figures, data are shown as mean ± standard deviation. N = 3
termined using Students’ T test; ****p ≤ 0.0001, n.s. indicates tissues per condition.

skin to maintain the physiological condition up to the continuity. However, it is not clear why in the SDS-
SC. For this reason, most moisturizers contain a treated model higher tracer uptake was not observed.
mixture of ingredients of two opposite properties One possible explanation is that the disruption of SC
– occlusives and humectants, but fine-tuning to max- enlarged the paracellular space in SC and interfered
imize each property is a difficult task. While dime- with capillary action. The opposite effects from hu-
thicone hindered the tracer migration in both normal mectants and occlusives and their dosage level deter-
and dry skin models, the humectants HA and PGA mine overall efficacy of the final formulation as shown
increased tracer migration in the control skin condition in the tracer migration of USM and HA5, although
(Fig. 4). It is reasonable to postulate that topical water other ingredients likely play a role. Individual ingre-
evaporation drew the tracer solution by capillary action dient assessment using this tracer assay could give
and water-holding humectant properties facilitate its insights on how to balance these two properties.

In vitro Assay Evaluating Moisturizers on Skin Pharmacol Physiol 2023;36:140–148 145

Dry Skin DOI: 10.1159/000529630
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Fig. 4. Degree of skin barrier function varies upon topical applications of single ingredient and commercial
products. The tracer assay outlined in Fig. 2 was performed, using SDS (2%) treatment, followed by the additional
topical application of silicone, HA, polyglutamic acid, or commercial moisturizer/hydrator (USM or HA5). The
non-treated average levels of water and SDS-treated skin were noted by red and orange dotted lines. Data are
shown as mean ± standard deviation. N = 3 tissues per condition.

This system has several advantages over previously structure, morphology, and molecular composition
used methodologies to evaluate barrier integrity [24]. would complement the measurements of barrier integrity
First, the advent of RHE removes the ethical constraints by the tracer assay described herein [26–30]. Especially
of in vivo experiments and the tedious recruitment of since SDS treatment was reported to disrupt long peri-
human volunteers. The test system can thus be cus- odicity phase of lipid assembly significantly, rather than
tomized to include other methods of barrier disruption, short periodicity phase, it would be interesting to dem-
and the use of RHE from donors of different skin types onstrate the functional roles of long periodicity phase and
and ethnicities can address some of the biological vari- short periodicity phase in skin permeability [7, 8, 23]. If
ables that would affect future clinical results [25]. Second, there are reagents to specifically alter the nanostructure of
while the classic, gold standard measurements such as the lipid matrix in SC, it can be explored how the lipid
TEWL and skin capacitance have utility in the clinic matrix contributes to overall SC barrier function with the
because they are noninvasive, they are prone to inter- tracer assay [31]. Altogether, these complementary assays
ference and require careful standardization between can be excellent tools to further optimize the properties of
conditions [12]. This system uses reliable cellular and moisturizers for cosmetic dry skin and possibly other
molecular biology techniques and controlled laboratory indications.
conditions, removing many of the variables associated There are some limitations to the methodology as
with experimental error. In this tracer assay, the imaging presented here, namely, due to fundamental differences
measurements and calculations are simple and between 3D in vitro models as compared to native skin.
straightforward. Therefore, many samples can be pro- Cultured 3D skin may be more permeable compared to
cessed in parallel, making this approach highly amenable human epidermis due to differences in lipid composition
to structure-activity relationship studies and rapid and organization, but consequences of this may be
screening of different formulations. mitigated because the results from this assay are com-
While this methodology was developed for cosmetic parative [32]. The possible differences can be addressed in
dry skin, it can be extended to other in vitro models of the future by using additional models, such as ex vivo skin
conditions that disrupt barrier function. In addition to explants or cultured biopsies [33]. Importantly, proper
what was shown here, the tracer assay can be combined management of dry skin often requires repeated, long-
with other endpoints for a more detailed analysis of term application of a product. Some formulations can
changes to the skin. For example, biophysical, analytical, improve barrier function in the short term, but they may
and atomic simulation techniques used to evaluate the cause long-term damage when used repeatedly [34]. In

146 Skin Pharmacol Physiol 2023;36:140–148 Hong/Maitra/Nguyen/Kadoya/Mehta

DOI: 10.1159/000529630
this study, the effect of a single application was examined Conflict of Interest Statement
but could potentially be extended up to several days.
Because the tracer assay cannot be employed in patients, Soonjin Hong, Prithwiraj Maitra, Audrey Nguyen, Kuniko
Kadoya, and Rahul Mehta are employees of AbbVie, Inc., and may
it is currently unclear if the results from this assay would own stock in the company.
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 comple- Funding Sources
mentary clinical studies.
This research was supported by Allergan Aesthetics, an AbbVie
company. Employees of AbbVie participated in the research,
Conclusion interpretation of data, review of the manuscript, and the decision
to submit for publication.
In summary, a simple and practical method was de-
veloped to assess moisturizing ingredients and formu-
Author Contributions
lations in terms of occlusive effects in vitro. This
approach may be a valuable tool for the preclinical de-

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Soonjin Hong, Prithwiraj Maitra, Kuniko Kadoya, and Rahul
velopment and optimization of new moisturizing for- Mehta were responsible for the conception and design of the work.
mulations to treat cosmetic dry skin. Soonjin Hong was responsible for the acquisition, analysis, and in-
terpretation 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 man-
Acknowledgment uscript, and have agreed to be accountable for all aspects of the work
in ensuring that questions related to the accuracy or integrity of any
Writing assistance and editorial support were provided by Liza part of the work are appropriately investigated and resolved.
Selwan-Lewis, PhD, an employee of AbbVie, Inc.

Data Availability Statement

Statement of Ethics
All data generated or analyzed during this study are included in
Ethical approval was not required for this study type, and no this article. Further inquiries can be directed to the corresponding
human or animal subjects or materials were used. author.

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DOI: 10.1159/000529630