In Vitro and In Vivo Confocal Raman Study of Human Skin Hydration:
Assessment of a New Moisturizing Agent, pMPC
L. Chrit,1,2P. Bastien,1B. Biatry,1J.-T. Simonnet,1A. Potter,1A. M. Minondo,1F. Flament,1
R. Bazin,1G. D. Sockalingum,2F. Leroy,1M. Manfait,2C. Hadjur1
1L’Ore ´al Recherche, France
2Unite ´ Me ´DIAN, CNRS UMR 6142, UFR Pharmacie, IFR 53, Universite ´ de Reims Champagne-Ardenne,
51 rue Cognacq-Jay, 51096 Reims Cedex, France
Received 6 September 2006; revised 21 November 2006; accepted 21 November 2006
Published online 1 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20644
This article was originally published online as an accepted
version. You can request a copy of the preprint by emailing the
5–10% lipids (ceramides, cholesterol, and fatty acids), and
5–10% of other materials (amino acids, NMF ...).5
Skin plays a major part both in the aesthetics and in the
health fields. In this standpoint, the cutaneous hydration
he skin is a heterogeneous organ made of tissues that
differ in morphology and molecular composition.
The uppermost layer contains mainly the molecular
composition of Stratum corneum, that is, 75–80% fi-
brous protein (mostly in ?-keratin conformation),1–4
The hydration capacities of a biomimetic polymer, 2-
methacryloyloxethylphosphorylcholine polymer (pMPC),
alone and microencapsulated, in association with another
well known hydrating polymer, Hyaluronic acid, were
investigated in vitro on skin models and in vivo on
volunteers by using confocal Raman microspectroscopy.
The hydration impact and the relative water content in
the Stratum corneum were calculated from the Raman
spectra using the OH (water)/CH3(protein) ratio.
Moreover, the follow-up of the presence of pMPC through
the Stratum corneum was possible with confocal Raman
microspectroscopy, using a characteristic vibration of
pMPC, different from that of the encapsulating material.
From our in vitro measurements, the improved hydration
of the Stratum corneum was confirmed by the use of the
encapsulated form of pMPC, which was higher when
combined with Hyaluronic acid. On the basis of these in
vitro findings, we validated this trend in in vivo
measurements on 26 volunteers, and found a good
correlation with the in vitro results. Mechanical and
ultrastructural studies have been carried out to
demonstrate the positive effects of the pMPC on the
Stratum corneum function, namely the interaction with
lamellar lipids and the plasticizing effects, which are both
supposed to spell out the moisturizing effect. This study
demonstrates the efficiency of a original hydrating agent,
pMPC, entrapped with Hyaluronic acid in a new type of
microcapsules by the use of a novel tool developed for
both in vitro and in vivo approaches. This indicates
a new step to evaluate and improve new moisturizers
in response to the cosmetics or dermatologic
demands.#2006 Wiley Periodicals, Inc. Biopolymers
Keywords: skin hydration; in vivo confocal Raman
microprobe; in vitro confocal Raman; test evaluation; 2-
In Vitro and In Vivo Confocal Raman Study of Human Skin Hydration:
Assessment of a New Moisturizing Agent, pMPC
Correspondence to: C. Hadjur; e-mail: firstname.lastname@example.org
C2006 Wiley Periodicals, Inc.
Biopolymers Volume 85 / Number 4 359
holds a dominating place in dermatology and in cosmetic
applications. A skin hydration ‘‘disorder’’ will be for the for-
mer, a discomfort due to the ageing, resulting from climatic
effects or sun exposures, whereas for the latter the clinical
sign of an ill-defined pathogenic dysfunction, for instance
lack of barrier function. In fact, the most important aspect of
skin function is to maintain a good homeostasis of the skin.6
The concern of Stratum corneum hydration remains an
important field of care research, mainly targeting the
improvement of the Stratum corneum barrier function.7
Thus, the necessity to measure and objectively quantify cuta-
neous hydration aroused in order to better understand and
improve the Stratum corneum hydration. The hydration
mechanism is not completely understood and several hypoth-
eses have been issued concerning the occlusive property of
the hydrating agent, restoration of barrier function, or inter-
action with Stratum corneum components (NMF, lipids). The
most famous and widespread moisturizer in the field of cos-
metics is glycerine, the mechanism of which is not yet per-
Several biophysical techniques have been developed dur-
ing the last decade in order to improve skin description
and knowledge, driven by medical, pharmaceutical, and
cosmetic researches.8–16Vibrational spectroscopic techni-
ques, such as infrared absorption and Raman scattering,
are among such approaches that have emerged in this
direction. The potential of Raman spectroscopy for moni-
toring skin hydration has been recognized. For this tech-
nique, no sample preparation is required and it can be
applied to specimens in any physical state, including gas,
liquid, gel, and amorphous phases. Because of its noninva-
sive and nondestructive nature, this technique can be oper-
ated without contact and shows a great potential for the
study of biological tissues. Early studies aimed at tissue
characterization and pathological tissue classification,
many of them targeting the skin.17,18The first in vivo stud-
ies of human skin using Raman spectroscopy were reported
by Williams et al., Schrader et al., and Shim et al. The
advent of in vitro and in vivo confocal devices allowed
enhancing in-depth measurements of the skin from the
surface to several tens of microns below the skin surface.
These techniques therefore provide a straightforward way
to get better insight into the chemical structure and physi-
cal behavior of the upper skin layers. Recently, we have suc-
cessfully demonstrated the suitability of the confocal
Raman microprobe to evaluate in vivo the hydration effect
induced by a glycerol treatment.19,20The improvement of
confocal Raman spectroscopy equally in an in vitro and in
vivo approach allowed us to study a new type of moisturiz-
ing agent in a continuous way.
Our study aimed at assessing a new biocompatible mois-
turizer, pMPC, the efficiency of the vectorization and the
synergistic effect of two moisturizing polymers, respectively
pMPC and Hyaluronic acid. We also focused on the pMPC
hydration mechanism after application on the skin.
A polymeric phospholipid,21–25poly [2-methacryoylox-
yethylphosphorylcholine] (pMPC), was synthesized as a
new type of biomaterial.24It is an analog of phospholipids,
which bears the bio-mimic phosphorylcholine moiety (as
the side group) and methacrylate unit. pMPC has been rec-
ognized for its unique water-holding capacity to give the
excellent nonthrombogenic surfaces as a result of its ability
of inhibiting the protein adsorption.24pMPC increases the
biocompatibility with the membrane or lipids,26and allows
to maintain the skin function barrier even after posthydra-
tion disturbances.22Mump, a phosphorylcholine polymer,
is the first biomimetic moisturizing ingredient that can
hold water efficiently, which enhances Stratum corneum
functions, especially the absorption/retention of water and
the prevention of water loss.21,24,25To improve cosmetic
properties of pMPC, like the skin softness effect, we com-
bined it with a well known hydrating agent, the Hyaluronic
acid, which can maintain also the hydration effect over the
Hyaluronan is a glycosaminoglycan that contains an
unbranched polysaccharide built up from repetitive units of
D-glucucronic acid and D-N-acetylglucosamine dissacharides.
This natural polymer is most abundant in many tissues and
demonstrates remarkable rheological, viscoelastic, and hygro-
To demonstrate the hydration efficiency of a new polymer,
pMPC, evaluated in Raman microscopy, we carried out a
Stratum corneum hydration study following two approaches,
both in vitro and in vivo. The in vitro experiment redraws
the different steps of the polymer improvement in skin
hydration. The in vivo work was carried out on healthy indi-
viduals using a newly developed in vivo confocal Raman
microprobe operating with an optical fiber and a 633 nm
laser. This probe permits acquisition of the spectrum over
the whole frequency range from 500 to 3600 cm?1. There-
fore, information about the fingerprint region (low frequen-
cies, 500–1500 cm?1) and the higher frequencies containing
water vibrations (2800–3600 cm?1) are collected simultane-
ously without using two different wavelengths as previously
Many applications in the field of cosmetics and dermatol-
ogy would significantly benefit from a combination of nonin-
vasive and original evaluation tool in vitro and in vivo for
the assessment of new moisturizing products. This is a prom-
ising advance in skin hydration and polymer research.
360Chrit et al.
Biopolymers DOI 10.1002/bip
MATERIALS AND METHODS
Moisturizing Polymers. pMPC1was provided by NOF Corpo-
ration (Tokyo, Japan) and Hyaluronic Acid (MW:1.100.000) by Sol-
iance (Colombes, France).
Microcapsules. We developed a new type of microcapsules that
are able to entrap aqueous solutions of polymers. These original
particles are based on a cellulose ester polymer and made by a mul-
tiple emulsion process. First, we dispersed an aqueous phase (water
and moisturizing polymers) in an organic phase composed of a
lipophilic solvent and the cellulose ester. Then, this emulsion is
dispersed in a second aqueous phase in order to obtain a stable mul-
tiple emulsion. The solvent is removed at low pressure and the cellu-
lose ester precipitated around the droplet of the primary emulsion
to form multi-wall particle (c.f. Figure 1A). The particle size lies
between 30 and 50 ?m and the pMPC release can be obtained by
the friction movements during its application on the skin. In this
way, a highly concentrated solution of moisturizing polymers is
In the first step, the microcapsules containing a 20% pMPC
aqueous solution were tested. In the second, the microcapsules con-
taining an aqueous solution of 10% of pMPC and 0.5% of Hyal-
uronic acid were evaluated.
In Vitro Analysis
Biological Samples. Plantar Stratum corneum was extracted by a
dermatologist, desiccated with silica gel, sterilized with Gamma
radiation, and was finally pressed in a disc form by an infrared press
ning electron microscopy. The microspheres size varies between 30 and 50 ?m. After a platinum
metallization, the sample was observed by a SEM 300 Hz JSM 6300 F. The first picture depicts dif-
ferent microspheres and their different sizes; whereas the second one represents a unique micro-
sphere. The last picture displays the microspheres cut in cryostat. B: Comparison of the water con-
tent brought by a pMPC in water of 2%, versus a solution containing microspheres of pMPC and
the control. Potentialization of the encapsulation of the pMPC indicated with the arrow (four times
higher). The three curves respectively describes the hydration induced by the microspheres of
pMPC (full line), pMPC in solution (dotted line) and the initial hydration level of the Stratum
corneum without treatment (dashed line).
A: Visualization of the microspheres containing pMPC and Hyaluronic acid by scan-
Hydration Effect: Efficiency of pMPC as Moisturizing Agent361
Biopolymers DOI 10.1002/bip
(10 T cm?2) (Eurolabo, Paris, France). The discs had a weight of
about 80–100 mg, an area of 1 cm2, and thickness of several hun-
dred micrometers. For the Raman measurements, the volume used
for hydration was calculated using a ratio of 0.5 ?g water/?g sample
dry sample and it was deposited in the middle of the sample and
then dispersed over the whole sample. Spectra were recorded after
15 min for equilibration and then in-depth hydration was probed
starting from the top surface. In these conditions, the hydration was
made homogeneous and no water pockets were noticed. For each
case, a minimum of three samples were analyzed.
Abdominal Stratum corneum sheets extracted from abdominal
parts were used in the mechanical studies. The samples of Stratum
corneum used in the mechanical and TEWL (Transepidermal water
loss) studies were isolated from human skin subjected to plastic sur-
gery (abdomen). The Stratum corneum was separated from the
whole skin according to the method of J. Ferguson, rinsed in dis-
tilled water to remove trypsin, and kept in ambient atmosphere. For
TEWL measurements, the samples were delipidized by immersion
for 2 h in a chloroform-methanol mixture (ratio 2v/1v) at room
Transmission Electron Microscopy.
Biological Samples. Stratum corneum extracts, obtained from
the legs of subjects exhibiting dry skin, were placed in a 1% pMPC
aqueous solution or in the vehicle alone, for 1 h, before being proc-
essed for transmission electron microscopy (TEM).29
Transmission Electron Microscopy. pMPC-treated samples
and control samples (untreated and water-treated dry flakes) were
fixed with 2% acrolein (Aldrich, France) in 0.1M sodium cacodylate
(Fluka Chemica) buffer, pH 6.8. They were postfixed in 0.25% ru-
thenium tetroxide (Polysciences) with 0.5% potassium ferrocyanide
(Aldrich) in the same buffer, dehydrated, and embedded in EPON
812 resin (Euromedex). Thin cross-sections (80 nm) were examined
after double staining with uranyl acetate (Aldrich) and lead citrate
(Aldrich) using a Zeiss CEM 902 electron microscope (Carl Zeiss,
Dermometer and Tewlmeter (TEWL)
Measurement of Elasticity.30The elasticity of abdominal Stra-
tum corneum (dynamic elastic modulus) was measured at 180 Hz by
a dynamic method described previously. Relative humidity and tem-
perature in the measurement chamber were regulated at 75% HR
and 308C. The equilibrium times employed prior to measurement
were 2 and 20 h. Accurate measurements of the same sample were
made before and after treatment.
Determination of TEWL. An evaporimeter (Dermalab, Lon-
dres, Royaume-Uni) was used to measure the TEWL (Trans Epider-
mal Water Loss) of delipidized abdominal Sratum corneum, before
and after treatment, in order to study the eventual occlusive effect
of the treatment. Relative humidity and temperature in the mea-
surement chamber were regulated at 40% HR and 308C. The equi-
librium times employed prior to the measurement were as men-
Micro-Raman Spectroscopy. Initial in vitro Raman experiments
were carried out on a LabRam microspectrometer (Jobin-Yvon-
Horiba, France). The instrument was equipped with a He:Ne laser
(633 nm wavelength, power 8.4 mW) and a CCD (Charge coupled
device) detector. The laser beam was focused on the skin by a
microscope objective (?50, Olympus) and the micro-Raman data
were collected from the sample surface to several microns deep
using the point-by-point mode. Raman scattering measurements
were obtained from micro volumes (<5 ?m3) of the sample because
of the high spatial resolution (1–2 ?m2in this case) and confocal
properties of the system. In this way, depth profiling from 0 to
20 ?m, using a step of a few microns was feasible. Other experimen-
tal conditions for profiles acquisition were as follows: confocal hole,
200 ?m; slit, 100 ?m; acquisition time, 30 s; and wavenumber range
2800–3700 cm?1for the hydration analysis. Nevertheless, to better
understand the interaction between the Stratum corneum compo-
nents and mMPC, the so-called fingerprint region (400–2000 cm?1)
was equally measured.
In Vivo Analysis
Test Subjects. The in vivo study was performed on 26 volunteers,
mainly healthy Caucasian types, all females between 24 and 59 years
of age, and without skin disease or allergy. They were recruited from
L’Oreal’s staff at the Chevilly research center (France) and the selec-
tion was based on the phototype criteria, which means including
phototype 1A to 3A and skin dryness. All participants had refrained
from using skin care products on the test areas for at least 3 days
prior to the start of the study. The measurements were carried out
during December and April.
Test Procedure. Three tests sites were selected and marked
(square of 4 ? 4 cm2) on the both volar forearms of each volunteer.
First, the test sites were wiped with a tissue paper moistened with
alcohol (ethanol 95%) in order to get rid of contaminants. Biophys-
ical readings start before any manipulation of the skin, as well as
after treatment with cosmetic agents. All measurements were per-
formed after a 20–30 min acclimatization period in an air-condi-
tioned room in order to equilibrate the skin in a controlled environ-
ment where the temperature was at 218C and the relative humidity
around 40–50%. After application of the product the skin is fric-
tionned to accelerate penetration in the stratum corneum. Excess
product is wiped off and we waited one hour before the Raman
measurements. The measurements were taken from each test area
and mean values were used for subsequent statistical analysis. More-
over, this test was randomized on the six areas in order to diminish
any possible influence related to the site. This procedure clearly
improves the comparison of the results.
Experimental Set-Up. An in vivo microspectrometer confocal
Raman was developed and the full details of our Raman equipment
is well described in our previous study,19,20which demonstrates the
feasibility of this microprobe in determining skin molecular and
structural composition. This device comprised a fiber-coupled
Raman spectrograph and a confocal Raman probe (HORIBA JOBIN
YVON, France), set up in an inverted configuration for this study.
Both the confocal set-up and fluorescence reduction improved
greatly the quality of the skin spectra. The objective used in this
experiment is a 50? long working distance objective (Olympus),
with a numerical aperture of 0.5, which operates in air. A piezo-elec-
tric device directly controlled by the data acquisition software (Lab-
Spec, HORIBA JOBIN YVON) allows controlling automated depth
362 Chrit et al.
Biopolymers DOI 10.1002/bip
profiling procedures with a high accuracy in z motion. The excita-
tion source was a 633 nm He–Ne laser, delivering about 8–10 mW
at the sample. The spectrograph was equipped with an air-cooled
CCD detector, with a chip size of 1024 ? 256 pixels and a 600 g
mm?1grating, which allowed to cover a large spectral window rang-
ing from 500 to 3600 cm?1in a single shot acquisition. The acquisi-
tion time was 20 s per measurement and at depths ranging from 0
to 20 ?m, with a 5 ?m step size enabling to perform rapid depth
All spectra were acquired and pre-processed following this scheme:
linear baseline subtraction (in order to get rid of the intrinsic skin
fluorescence), normalization using both bands, respectively, at
1450 cm–1(which corresponds to the common ?(CH2) band of lip-
ids and proteins in the Stratum corneum) for the 400–2000 cm–1
range and at 2940 cm–1for the 0.800–3600 cm–1range.19,20
Concerning the in-depth profiling, the ratio of the bands OH/
CH3(water content related to proteins) was calculated as previously
described.19,20,31–34For ratio calculation, the integrated areas under
each band were used. All these functions, together with the averag-
ing procedure, were performed using the data acquisition software
(LabSpec, HORIBA JOBIN YVON).
To assess natural skin hydration level, water hydration profiles were
observed at T0(experiment time before treatment) from the surface
down to 20 ?m deep. The effect of the microspheres of pMPC and
Hyaluronic acid has been tested on the difference DT ¼ T1h? T0
(T1hcorresponds to 1 h after cosmetic treatment) using a linear
mixed model35with the subject factor as random and the factors
such as treatment, depth, and their interaction, as fixed. The statisti-
cal significance for the two-sided tests was fixed at 5%. The analysis
was performed using SAS 8.2 (SAS Institute Inc., Cary, NC) for the
inferential part and SPSS 13.0 (SPSS Inc., Chicago, IL) for the de-
pMPC, a Synthetic Original Moisturizing Agent: An
In Vitro Approach
Hydrating Effect: In Vitro Approach. The water content is
calculated from the ratio of the respective integrated band
areas corresponding to water (3350–3550 cm?1)/protein
(2915–3030 cm?1) and allowed to classify the efficiency of
cosmetic agents in terms of hydrating effect.19,20,31–34
Figure 1B depicts the in vitro tests showing increase in
water content level after application of pMPC in water (2%)
in comparison with the control (untreated). We observed an
elevation of 20% of the water content in the Stratum corneum
in comparison with the untreated sample. This increase was
observed from the surface of the Stratum corneum down to
several microns below the skin.
Hydration Effect: The Efficiency of the pMPC Vectoriza-
tion. Using the method previously described, we have found
that the microencapsulation of pMPC improved by four
times the water content in the Stratum corneum in compari-
son with the pMPC in solution (Figure 1B). This increase of
water content was revealed from the surface of the skin to
several microns deep.
The Synergic Combination: pMPC and Hyaluronic Acid
(HA). Both polymers were encapsulated into an aqueous con-
centrated solution form following a concept so-called multiple
tion of both polymers is illustrated in the Figure 2A; the pMPC
is very efficient at the beginning (until 40 min) while the Hyal-
uronicacidismore hydrating and more remanentafter 40min.
polymer ‘‘in solution’’ application. The dotted line describes the
pMPC hydration; the dashed line corresponds to the Hyaluronic
acid. The full curve is the result of the combining polymers respec-
tively pMPC plus Hyaluronic acid. B: Synergy between two poly-
mers encapsulated: the Hyaluronic acid and the pMPC, a better
long lasting hydration after combination of the Hyaluronic acid and
the pMPC. The histogram plot presents the reference in blank, the
pMPC in solution in gray and the pMPC encapsulated in dark color.
All those results are extracted from a fixed depth namely at 10 ?m.
A: The Stratum corneum hydration induced after a
Hydration Effect: Efficiency of pMPC as Moisturizing Agent 363
Biopolymers DOI 10.1002/bip
Figure 2B displays the gain and the synergic combination of
both polymers for the hydration of the Stratum corneum at a
fixed depth of 10 ?m and at two different experimental times
(respectively 15 and 45 min after cosmetic treatment). In these
conditions, we noted a significant increase (þ47%) of the water
content in the sample treated by the microspheres (pMPC and
Hyaluronic acid) in comparison with a hydration induced by
microspheres of pMPC after 45 min.
To compare the hydration efficiency of the microspheres
containing pMPC and Hyaluronic acid and the cosmetic ref-
erence (glycerol 3%), we conducted an assessment by Raman
spectroscopy. The results obtained are summed up in Figure
3. As can be seen, the microspheres of both polymers enhance
by about two times the water content in the Stratum corneum
when compared with a reference solution of glycerol 3%.
Uptake of Water After 24 h in a Relative Humidity Con-
trolled Chamber. The question of the retention in water was
also treated by placing samples in a chamber with a con-
trolled hygrometry of 80% of relative humidity after the
application of different products and measurement after
about 24 h (Figure 4). The sample treated by the micro-
spheres containing the combination of polymers was more
efficient in reuptake of water by about 85% higher than the
reference, glycerol 3%.
In Vivo Approach for Measuring the Hydration
Effect of pMPC
The same tests were undertaken by in vivo Raman spectros-
copy on 26 volunteers using the application on the volar
forearm of the hydrating agent in comparison with the bare
skin (without treatment). Considering the water content evo-
lution, we can classify the products in relation with the Stra-
tum corneum hydration. Figure 5 shows a significant increase,
compared with the reference (bare skin), of the hydration
level after the application of the microspheres containing
both polymers. Such results clearly highlight the moisturiz-
ing efficiency of the microspheres (pMPC and Hyaluronic
acid) in consistency with the in vitro results.
The in vivo results evidently demonstrates a higher and
significant hydration level induced after application of pMPC
and HA microspheres in comparison with the control. The
mers (pMPC and Hyaluronic acid). A two-fold increase in the mois-
ture content of the Stratum corneum was observed compared with a
reference solution of glycerol 3%. The histogram plots presents the
reference (without treatment) in blank color, the pMPC plus Hyal-
uronic acid microspheres in dark color and the glycerol in solution
(cosmetic reference) in gray ones. All those results are extracted
from a fixed depth namely at 10 ?m.
Synergistic effect of microencapsulation of both poly-
relative humidity), the microcapsules of pMPC and of Hyaluronic
acid (full line) allowed an 85% higher uptake of water by the Stra-
tum corneum compared with a solution of glycerol 3% (dashed
line). The dotted curve depicts the natural water uptake of the Stra-
tum corneum without treatment.
In conditions of high relative humidity (24 h at 80%
induced 45 min after a cosmetic application, namely microspheres
of the pMPC plus Hyaluronic acid, measured by the water evolution
from T1h? T0. Data are expressed as mean value and their confi-
dence interval (CI). For the statistical significance test, ? ¼ 0.05.
Comparison with the bare skin of the global effect
364Chrit et al.
Biopolymers DOI 10.1002/bip
effect of pMPC plus hyaluronic acid has been tested on the
difference T1h? T0(T1h, 1 h after treatment and T0before
treatment) using a linear mixed model with the subject factor
as random and the other factors, such as treatment, depth,
and their interaction, kept fixed. We did not observe any sig-
nificant variation in the bare skin (P ¼ 0.6356) but a highly
significant effect of the microspheres entrapping pMPC and
HA (P ¼ 0.0062) (see Table I). Finally, it seems that the
microspheres produce a hydrating effect on the Stratum cor-
neum. The comparison between different treated groups of
the microspheres (pMPC plus HA) versus bare skin is highly
significant (P ¼ 0.0020) and homogeneous throughout the
skin (P ¼ 0.7954). This comparison is realized on the whole
depth and on the whole experiment time. Moreover we can
observe a significant increase of hydration with depth (P
¼ 0.0016) (see Table II), which is corresponding to the water
gradient within the Stratum corneum.
The In Vitro Effects Induced by Synthetic
Phospholipids Polymer on Stratum corneum Function
Penetration of the pMPC into the Stratum corneum. As
described previously, the pMPC is an interesting polymer
with both a characteristic polar head and a hydrophobic
part. The study of follow-up of penetration of the pMPC
and the hydration of Stratum corneum was conducted by
means of confocal Raman microspectroscopy. First of all,
the spectral signature of the pMPC was performed in order
to determine a characteristic vibration band different from
the Stratum corneum. The Raman spectra revealed a char-
acteristic band at 717 cm?1, which can be used as a marker
band to follow the pMPC within the Sratum corneum. This
band is mainly assigned to the in phase CH2rock vibration
belonging to the noncyclic CH2chains. Moreover, the pen-
etration of the pMPC was associated to an increase of the
intensity of the band at 1080 cm?1, corresponding to
?(CC) modes of the lamellar lipids. Figure 6 shows the
potential of the Raman microprobe to reveal presence of
this polymer down to a penetration depth of 10–12 ?m
into the Stratum corneum, in spite of its high molecular
weight. Because of this observation, we therefore decided
to evaluate the hydration induced by this polymer by prob-
ing with Raman spectroscopy, the Stratum corneum from
the surface to about 10 ?m below, namely the core of the
Plasticizing Effect. Variations in the dynamic modulus of
elasticity (Young’s modulus) after application of polymer so-
lution and after 2 and 20 h of equilibrium are shown in Fig-
ure 7. The results are expressed in terms of percentage of
reduction of the dynamic elastic modulus of the Stratum cor-
Difference T1? T0Using a Linear Mixed Model with the Subject
Factor as random and Other Factors such as Treatment, Depth,
and their Interaction Kept Fixed
The Effect of pMPC þ Hyaluronic acid Tested on the
Least Squares Means
Error DFt ValuePr > |t|
HNA þ LIP
DF: degree of freedom; t: Student test; Pr: probability.
plus Hyaluronic acid versus bare skin
The between treatment group comparison of pMPC
Tests of Fixed Effects
Num DFDen DFF Value Pr > F
Num: numerator; DF: degree of freedom; Den: denominator; F: Fischer
test; Pr: probability. The interaction treatment and depth has been also
pMPC into the Stratum corneum by the Raman microprobe. The
black lines represent the pMPC treated Stratum corneum from 0 to
12 ?m in depth, whereas the gray line the reference Stratum cor-
neum (without treatment). The first arrow designs the characteristic
pMPC vibration at 717 cm?1whereas the second one depicts the
increase of the 1080 cm?1vibration corresponding to the ? (CC)
The follow up the penetration (10–12 ?m) of the
Hydration Effect: Efficiency of pMPC as Moisturizing Agent 365
Biopolymers DOI 10.1002/bip
neum after application of the treatment (versus before treat-
ment). The results confirm the significant plasticizing effect
of the Stratum corneum by pMPC 1% and Sodium Hyaluro-
nate 1%. However, it must be noted that this effect was still
inferior to the plasticizing effect observed with the solution
of glycerol 3%.
TEM Observation and TEWL Analysis. We compared 1%
pMPC aqueous solution treated dry flakes with untreated or
water-treated dry flakes. In the control cases, thin cross-sec-
tions, following ruthenium tetroxide exposure, revealed (see
Figure 8A) densely packed corneocytes in the upper Stratum
corneum layers, abnormal retention of corneodesmosomes,
which are the major cohesive structures, and abnormally
arranged lipid lamellae within the extra-cellular spaces.
Scarce lipid lamellae are depicted in some areas and some of
them are squeezed against the corneocyte envelopes by an
amorphous material in intercellular dilatations. In the water-
treated samples, prominent swellings and disorganized lipid
lamellae could be clearly seen (data not shown). In the case
of application of pMPC (see Figure 8B), the upper extracellu-
lar spaces recovered lipid lamellar arrangement.
Accurate measurements of TEWL on the same delipid-
ited sample were made before and after application of the
polymer solution, after 2 and 20 h of equilibrium. The
after different cosmetic treatments on the Stratum corneum. Those
measurements have been done at two experimental times at 2 and
20 h. The cosmetics products tested are mentioned in the Figure 7.
The asterisks indicate the references. The lowest is the young modu-
lus, highest is the plasticizing effect.
Variation of the module of elasticity (Young’s modulus)
pMPC, following ruthenium tetroxide exposure.
The TEM observation of (A) an untreated Stratum corneum and (B) after treatment
366Chrit et al.
Biopolymers DOI 10.1002/bip
results are expressed in terms of percentage of reduction of
the water vapor diffusion gradient of the Stratum corneum
after the treatment (versus before treatment). These are
shown in Figure 9.
Thus, the application of pMPC tends to decrease the
water loss through the Stratum corneum. Yet, a vaseline-like
occlusive effect is not observed.
The pMPC is a polymer soluble in water and a by-product of
the 2-me ´thacryloyloxyethylphosphorylcholine (MPC). This
pMPC presents a structure similar to the polar group of the
phospholipids of cellular membranes.21–27The amphiphilic
nature of pMPC, a polar head and a hydrophobic part, is very
interesting and it was therefore relevant to assess its hydration
effect induced on the Stratum corneum after its application.
Moreover, both the issues of interaction and penetration of
pMPC within the Stratum corneum (ref NOF) were equally
approached, with the aim to better understand the hydration
mechanism. Thus, to estimate the moisturizing capacity of
pMPC, we followed the Stratum corneum hydration (more
precisely the follow up of water content) induced after its
application, using confocal Raman microspectroscopy.19,20
This study was divided into two stages; firstly the validation
concept during an in vitro approach and secondly during an
in vivo application. Clearly, at the same time, this original
work strongly emphasizes on the potentials of a new method,
confocal Raman microspectroscopy, which is amenable to
both in vitro and in vivo approach. This is obviously a helpful
support entailing for new other applications such as analyzing
and detecting new materials such as hydrating polymers. This
new advance, which takes into consideration the skincare
studies’ requirements, turns out to be very promising.
Accordingly, in vitro, we studied not only the impact of
pMPC vectorization but also the synergic effect after combi-
nation with another natural polymer, the hyaluronic acid.
The in vivo approach mainly targeted first, the potentials and
the robustness of an in vivo Raman optical microprobe for
the semiquantitative evaluation of natural skin hydration
and second, the classification of the moisturization effect
resulting from cosmetic agents.
The hydration rate was determined by calculating the water
to protein ratio demonstrated by Huizinga et al.31in their work
on eye lenses and recently by Bauer et al.32in their research on
the cornea. This method was used by Caspers et al.33,34on the
skin to evaluate and describe the water content profile in vivo.
Our previous works conducted on the skin in an in vivo con-
text19,20demonstrated successfully the suitability of the confocal
Raman microscopy to track the water content from the surface
to several micrometers below the skin before and after a cos-
metic treatment. The results obtained in vitro demonstrated
clearly a higher water content brought by an aqueous solution
of microspheres of pMPC in comparison with the pMPC in
water 2%. This efficiency could be explained by a better vectori-
zation, accurate targeting of the active ingredient within the
skin, more precisely certain components NMF or lipids),36,37
and a more concentrated ingredient release from the micro-
spheres. To improve the pMPC cosmetic formulation, the com-
bination of pMPC with Hyaluronic acid was studied. Thus, the
Hyaluronic acid is the most important constituent of the extra
cellular matrix.27,28It is a glycosaminoglycane composed of a
repetitive units of N-acetyl-D-Glucosamine and glucuronic acid.
Indeed, the Hyaluronic acid is known to preserve skin hydration
because of its high water retention capacity and increases the
skin softness sensation. This better hydration could be enlight-
ened by the original polymer structure, which is a biomimetic
phopholipid-like structure of cellular membrane. Besides, the
high capacity of both pMPC and Hyaluronic acid to capture
water would likely contribute to a higher retention of water
than pMPC alone because of a synergistic effect.
Currently, in the cosmetic field, the most famous and effi-
cient moisturizer is the glycerol.19,36–42In our study, we dem-
onstrated clearly a better skin hydration after application of
the microspheres containing both the polymers in compari-
son with the glycerol.
The in vivo study is a crucial step to validate the efficiency of
a new cosmetic product before its launching. In a second phase,
after the in vitro experiments, we have tested on several volun-
teers the hydration level ensuing from the application of micro-
spheres (pMPC and Hyaluronic acid) in comparison with refer-
ence skin (untreated). We undoubtedly highlight the higher
moisturizing effect produced by the microspheres. Nevertheless,
the experiment with the glycerol 3% has been tested in a previ-
metic treatment. Those measurements have been done at two exper-
imental times at 2 and 20 h. The most occlusive product is the Vase-
line. Obviously, pMPC is slightly occlusive, equivalent to the Hyal-
uronic acid effect at T20h, whereas at T2his more diffusive.
Variation of TEWL on the Stratum corneum after cos-
Hydration Effect: Efficiency of pMPC as Moisturizing Agent367
Biopolymers DOI 10.1002/bip
ous study,19in respect with the experimental conditions
employed here. Considering this trial, we could consider a more
efficient hydration enhanced by the microspheres thanwith glyc-
erol, which is in correlation with the in vitro results.
Those results could be explained by a different mechanism
of the pMPC with regard to glycerin. The pMPC and the Hy-
aluronic acid enhance both the water holding and availability
within the Stratum corneum. The capacity of water absorp-
tion by the polymers (pMPC and Hyaluronic acid) is prob-
ably higher than that of glycerol. At the same time, glycerin
must probably cause a stronger linkage with the water mole-
cules and makes them less available and thus less detectable
by the Raman microprobe. This is probably related to bound
to free water ratio. In view of elucidating the hydration
mechanism, we studied the penetration of the polymer
pMPC within the Stratum corneum. This synthetic polymer
is well described in the literature as biomimetic of the phos-
pholipids of cellular membranes because it has both hydro-
philic and hydrophobic groups.21–26This specificity allowed
us to predict the behavior of the polymer and its penetration
into the skin. As predicted, we have followed the pMPC from
the surface down to 10–12 ?m in depth, in spite of its high
molecular weight, thanks to a characteristic Raman vibration
of the pMPC.21This Raman observation is in accordance
with the Small-angle X-ray diffraction analysis conducted by
Shaku et al. Effectively, they discussed the penetration of
pMPC into the hydrophobic layer of lamellar structure
through Small-angle X-ray diffraction.43
nization whose main bands are present between 1030 and 1130
cm?1, corresponding to the lamellar lipid.20The 1080 cm?1
band appears more important in the treated Stratum corneum,
suggesting a higher disorganized lipid conformation, therefore
an increase of the flow of lipids. So the lipid phase could be
modified after the pMPC application. These lipid interactions
were confirmed by the ultrastructural analysis on the Stratum
corneum using transmission electron microscopy. Thereby,
these analyses demonstrate a reorganization of lamellar lipids
via the integration of the pMPC. This already confirms the
mode of action by biomimetism,23,43indicating that the pMPC
interacts with Stratum corneum lipids. Highly ordered lamellar
unit structures become localized in the upper interstices while
recovering their water holding properties. In fact, the resultant
barrier recovery is supposed to provide necessary hydration for
the normal Stratum corneum functioning. This could be an ex-
planation for the moisturizing mechanism of this phospholi-
pids-like polymer. Moreover, TEWL results confirm that
pMPC is able to restore the skin barrier function.22–24The
plasticizing effect was also studied because it is a strong hydra-
The result presented here could be related to the binding
state of water molecules brought by the polymers and their
interaction with Stratum corneum components. Despite the
important water content brought by the polymers, our hy-
pothesis is that the major part of these water molecules is not
tightly bound to the Stratum corneum. Nevertheless, pMPC
increases bound water of the Stratum corneum even after the
appearance of free water by acting on both intercellular lipids
and hygroscopic components, which could be partially
explained by the water holding experiment (Figure 4). More-
over, the differential scanning calorimetry experiment under-
taken on the freeze-dried hairless mouse Stratum corneum by
Shaku et al., described an increase of bound water in Stratum
corneum after addition of pMPC.
In conclusion, pMPC is an original polymer, which inter-
acts at different levels: First, on the hygroscopic properties by
the retention of water, second, on the intercellular lipids or-
ganization of Stratum corneum, inducing the restoration of
the barrier function, and third, on the biomimetism resulting
from the polymer penetration into the skin.
In vitro and in vivo confocal Raman spectroscopy represents
novel techniques for nondestructive spatially resolved deter-
mination of skin molecular composition and more specifi-
cally water concentration profiles. We have demonstrated
here that the water concentration profiles across the Stratum
corneum can be measured both in vitro and in vivo (at differ-
ent sites of the forearm) in a continuous approach, before
and after cosmetic treatment. Consequently, this follow-up
of water allowed to detect and to potentialize a new hydrat-
ing agent, pMPC, better than the widespread cosmetic refer-
ence, glycerin. The in vitro tests allowed us to improve the
pMPC efficiency after vectorization and combination with
another polymer, the hyaluronic acid. At the same time, the
in vivo study conducted us to confirm the microspheres
(pMPC plus HA) moisturization efficiency, which is a deter-
This research step is useful, a key in the development of
new cosmetic products, and can help to a better understand-
ing of moisturizer mechanism within the skin.
Furthermore, the classification of different hydrating prod-
ucts based on their moisturizing effect induced on the skin was
possible and appropriate. As this technique is suitable for fol-
lowing changes in molecular concentration or concentration
profiles induced by topical application, it may be extended to
other applications such as identification of other types of hyd-
rating agents, rapid screening, and improvement of new vector.
This seems to be a milestone in the cosmetic field.
368 Chrit et al.
Biopolymers DOI 10.1002/bip
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Hydration Effect: Efficiency of pMPC as Moisturizing Agent369
Biopolymers DOI 10.1002/bip