Article Not peer-reviewed version

Hyaluronic acid: elucidating its

penetration into, and effect on hair

fibers using confocal Raman

spectroscopy and biometric techniques

*
Cloe Boira , Mohammed Essendoubi , Marie Meunier , Carole Lambert , Daniel Auriol , Michel Manfait ,

Olivier Piot , Amandine Scandolera , Romain Reynaud

Posted Date: 17 January 2024

doi: 10.20944/preprints202401.1325.v1

Keywords: Hair penetration; Hyaluronic acid; Keratin conformation; Raman spectroscopy

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Article
Hyaluronic Acid: Elucidating Its Penetration Into,
and Effect on Hair Fibers Using Confocal Raman
Spectroscopy and Biometric Techniques
Cloe Boira 1,*, Mohammed Essendoubi 2,3, Marie Meunier 1, Carole Lambert 1, Daniel Auriol 1,
Michel Manfait 2, Olivier Piot 2, Amandine Scandolera 1 and Romain Reynaud 1
1 Givaudan Active Beauty, Science and Technology, France
2 EA 7506 Biospectroscopie Translationnelle (BioSpectT), Faculty of Pharmacy, University of Reims
Champagne-Ardenne, France
3 Biophysic Laboratory, Faculty of Medicine and Pharmacy of Tangier, AbdelMalek Essâdi University,

Tangier, Morocco
* Correspondence: [email protected]; Tel.: +33-6-22-24-06-41

Abstract: Background: Hyaluronic acid (HA) is well known in the cosmetic industry for its hydrating
properties linked to its interaction with keratin in the stratum corneum. Hair is also mainly composed of
keratin, but no evidence of penetration and interaction of HA in hair had yet been established. Herein, we
examined, with appropriate methods, this penetration and interaction. Methods: Confocal Raman
Spectroscopy was used to track HA penetration in hair fibers. Human hair was UV-irradiated, or not, before
washing with a number of different shampoo formulations. Low-molecular weight hyaluronic acid, high-
molecular weight HA, and an optimized blend of low- and high-molecular weight HA, were used. The benefits
of HA for cosmetic application were then evaluated at the ex vivo level. Results: Raman analysis of non-
irradiated samples revealed that the HA blend penetrated the hair cortex 5.9 times more than the placebo and
with irradiated samples, a 1.8-fold increase in penetration was observed. Whatever the irradiation status,
penetration of this optimal HA blend was significantly greater than that of the other two active substances.
Compared to the placebo, the HA blend significantly decreased spontaneous frizzing by 11%, repaired the hair
by increasing the elastic modulus and the break force, and increased the water content in the hair shafts.
Conclusion: Confocal Raman Spectroscopy can be considered as a powerful, non-invasive technique to
investigate the penetration of active ingredients into the hair.

Keywords: hair penetration; hyaluronic acid; keratin conformation; Raman spectroscopy

1. Introduction
Hair is a specialized derivative of the skin that is ideally organized to protect the human scalp.
The well-known structure of hair consists of an external cuticle composed of overlapping scales that
acts as a barrier to protect the inner structure. The central medulla is surrounded by the cortex, which
is mainly composed of the protein keratin [1]. This protein plays an important role in the physical
and mechanical properties of hair. At the molecular level, keratin is a helical protein composed of
two types of fibers: type I, with acidic amino acid residues, and type II, with basic amino acid
residues. One strand of each type combines to form a coiled-coil dimer. These dimers coil together in
antiparallel tetramers known as protofilaments. Finally, the protofilaments interact to form a single
intermediate filament organized into micro-fibrils and larger macro-fibrils [2]. The conformation of
the keratin chain is controlled by hydrogen bonds, ionic forces, Van Der Waals interactions, and
disulfide bonds. The disulfide bonds, originating from sulfur-containing cysteine residues, create
strong crosslinks between adjacent chains, giving each individual’s hair its unique shape. Indeed, the
more α-helical conformation the keratin has, the greater the curve in the protein chain, and the curlier
the hair fiber. Conversely, a β-sheet conformation in the keratin decreases the formation of disulfide
bonds and produces smoother hair fibers [3]. In the haircare industry, many products have been
developed to modify the disulfide bonds, with the aim of shaping or straightening the hair. These

© 2024 by the author(s). Distributed under a Creative Commons CC BY license.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

include chemical agents such as alkaline reducing agents, thioglycolates, or guanidine, which are
used to break and rearrange bonds. Unfortunately, all these products damage the hair and reduce its
strength [4]. As a consequence, alternative, safe, active ingredients that smooth without inducing
damage to the hair, the scalp, or the environment, are actively sought.
Hyaluronic acid (HA) is a key active ingredient in skincare products, but its benefits for hair
have been little described. To date, interactions between HA and keratin have been described in the
stratum corneum of the skin, where it is known to enhance skin hydration [5]. In previous work, we
first posed the hypothesis that HA can also interact with keratin in hair fiber, promoting keratin
interconversion from α-helix to β-sheet conformation. Our data showed an increase of β-sheet
conformation and a significant reduction in disulfide bonds after applying HA, thereby delivering
an anti-frizz property [3]. This interconversion was already observed during mechanical or chemical
hair straightening, which leads to hair smoothing and ultimately produces an anti-frizz effect [4].
Due to the cuticle on hair fibers, only low molecular weight molecules (˂ 10 kDa) have been found to
penetrate the cortex [6]. For molecules > 500 kDa, the penetration may only occur when the cuticle is
damaged, e.g., after bleaching [6]. In addition, no evidence of interaction of HA deep inside the hair
has yet been established; exploring the penetration of such molecules into hair fibers presents several
challenges. A range of techniques, including confocal fluorescence microscopy, have been used in
attempts to examine the penetration of tagged molecules, but autofluorescence consistently
compromises results [7]. Raman spectroscopy is a promising method for this type of evaluation. This
non-invasive technique detects the characteristic vibrational energy levels of molecules, and provides
structural information. In confocal mode, it can gather information at various depths inside a tissue,
and quantification is possible because inelastic Raman scattering correlates linearly with molecule
concentration [8]. In a previous study, we were able to track HA penetration ex vivo through the skin
using this technique [9]. Other studies performed on natural and intact human hair fibers
demonstrated that confocal Raman spectroscopy can be used to investigate the influence of cosmetic
ingredients on keratin structure. An example is the evaluation of a hair-smoothing or heat-protecting
effect by analyzing α-helix and β-sheet keratin conformation and the S-S disulfide bridges [10].
In the present study, we investigated whether Raman spectroscopy could be used to measure
HA penetration in hair, and to assess the benefits of a blended and optimized HA formulation for
use in haircare products.

2. Materials and Methods

2.1. Shampoo composition for all the tests involving a washing

The standard basic shampoo used contained water, sodium laurylether sulfate, sodium
cocoamidopropylbethaine, sodium chlorhyde, phenoxyethanol, guar hydroxypropyl trimonium, and
citric acid. This basic shampoo was blended with high molecular weight HA (HMW-HA; 1-1.4 MDa),
low molecular weight HA (LMW-HA; 20-50 kDa) or a blend of both (HA-Blend; confidential ratio
between LMW-HA and HMW-HA, commercially available under Resisthyal™ tradename supplied
by Givaudan Active Beauty).

2.2. Measuring HA penetration by Raman spectroscopy

Hair fiber preparation and treatment

One-gram samples (n = 10) of natural, undyed, light blond hair locks, measuring 10 cm in length,
from a white European donor, were used to limit fluorescence interference during Raman
spectroscopy measurements (INHIP, New York, USA). Five of the hair locks were irradiated at 20
J/cm² UVA and 0.6 J/cm² UVB in order to open the cuticle (Biosun, Vilber, Eberhardzell, Germany).
Hair locks were then washed for 2 min with 0.5 g shampoo containing 1% (w/v) of LMW, HMW, or
HA-Blend, rinsed 3 times with 200 mL distilled water, and dried for 3 min with a hair dryer kept 20
cm from the shaft. The washing, rinsing, drying, cycle was repeated 3 times before analysis. The same
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

shampoo without additive was used as control. Untreated hair locks were washed with distilled
water.

Raman spectroscopy and confocal measurements on hair fibers

Raman spectra were recorded using a near-infrared, confocal, Raman micro-spectrometer
(Labram, Horiba Jobin Yvon, Villeneuve d’Ascq, France). The unit comprised an optical microscope
(Olympus, BX41, France) coupled to a dispersive Raman spectrometer (Horiba Jobin Yvon,
Villeneuve d’Ascq, France) and a charge-coupled device (CCD) detector. Excitation was provided by
a 50-mW titanium-sapphire laser (Model 3900S, Spectra-Physics, France) generating a 785-nm beam
under the microscope objective. This irradiation is non-destructive for light blond hair samples and
causes no thermal or photochemical degradation. Confocal Raman measurements were recorded
using an optimized 100x infrared objective (Olympus, France) operating in air with a numerical
aperture of 0.8. To obtain axial profiles, the objective was mounted on a high-precision piezoelectric
device (Physik Instrumente, Germany) which allowed in-depth vertical scanning by focusing the
laser at various depths within the hair fibers. Spectra were recorded over the length of the hair fiber
with a 3-µm scanning step. For each spectrum, one 30-sec accumulation of laser exposure was
retained. Data were acquired using LabSpec 5 software (Horiba Jobin Yvon, Villeneuve d’Ascq,
France).

Spectral-data processing
Spectral data were pre-processed using LabSpec 5 software (Horiba Jobin Yvon, Villeneuve
d’Ascq, France). First, cosmic radiation was removed and all aberrant profiles were excluded from
the database. The remaining raw spectral profiles were corrected in order to clean the Raman signal.
The data were corrected for spectral shifts, noise was reduced using a 5-point-average Savitzky-Golay
smoothing filter, and baseline was adjusted using a fifth-degree polynomial function to remove
background fluorescence. Three independent axial profiles were recorded for each hair sample. The
processing of corrected data maps was performed by using original software based on Least Squares
fitting method that operates in the Matlab environment (The Math Works Inc., Natick, MA, USA). A
thorough description of this statistical analysis has been published previously [9]. Briefly, the method
involves mathematical modeling of HA reference spectra in the overall spectral axial (Z) profiles to
determine the contribution and distribution of these spectra within the measured profile.

2.3. Ex vivo analysis of anti-frizz properties

Hair locks with various curl levels - from wavy to curly - were used (n = 10/condition). At Time
0, hair locks were divided into 3 equal parts (2 treated parts and one untreated part) in order to have
an equal distribution of the different curl levels. Photographs were taken before washing. Then, hair
locks were washed for 10 sec with shampoos (1 g shampoo/2 g hair) containing 1.5% (w/v) or 3%
(w/v) HA-Blend, placebo shampoo, or water (for untreated locks). All locks were rinsed for 30 sec
with distilled water and then air-dried. Photographs were taken after treatment and drying.
For the frizz analysis, hair locks were smoothed with a thermal hair straightener and
photographed. Then they were placed in a room with extreme humidity (relative humidity 80% (SD
= 10% RH) for 8 h and photographed again. Images were analyzed using Photoshop® to study the
anti-frizz effect, by measuring hair-strand length before and after exposure to extreme humidity.

2.4. Ex vivo analysis of restorative properties by tensile test after bleaching

Natural Caucasian brown hair locks (1 g each) were bleached twice using a Platifiz bleaching
powder made of sodium persulfate, potassium persulfate, ammonium chloride, and sodium
metasilicate (SP Equation). Then, locks were washed for 60 sec with shampoos containing 3% (w/v)
HA-Blend, placebo shampoo, or plain water for non-treated hair locks, then rinsed for 20 sec. Treated
locks were dried for 30 min at 40° C in a helmet hairdryer.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

A Mobile Tensile Tester (MTT) was used to evaluate hair damage. The system is based on a
circular sample cassette, which allows for automatic measurement of up to 100 fiber samples. For this
study, 75 hair fiber samples were mounted using brass crimps and placed on a rotary cassette. A
Laser (Mitutoyo) measured the cross section of the hair fibers to normalize tensile-strength data. After
this measurement, a pneumatically operated sample gripper picked up the sample. The gripper was
mounted on a load cell which measures the force applied to the sample during elongation. The test
was conducted with 100% relative humidity and ambient room temperature.

2.5. Ex vivo analysis of keratin integrity following UV irradiation

Hair fiber preparation and treatment

One gram of natural, undyed, light-blond hair locks, 10 cm long, from a white European donor,
were used to limit fluorescence interference during measurement (INHIP, USA). Locks were washed
3 times for 2 min with 500 µL of shampoo containing 3% (w/v) HA-Blend, rinsed 3 times with 200
mL distilled water, and dried for 3 min with a hair dryer placed 20 cm from the shaft. The locks were
irradiated 7 times at 9 J/cm² UVA and 0.33 J/cm² UVB to open the cuticle (Biosun, Vilber, Germany).
The same shampoo, without the active substance, was used as a placebo. Non irradiated hair locks
were used as a negative control.

xPOLAR® analysis for keratin integrity

For each condition, 30 sections of 1-cm each, from different fibers, were mounted on a slide and
covered with a coverslip. We performed 1 xPolar measurement on each segment of hair, so there
were 30 measurement points per condition. In each pixel, a dimensionless value termed Kmax is
dependent on the keratin birefringence (intrinsic physical parameter of the material with a numerical
value that depends on the structural state of the keratin at the molecular level), and keratin thickness
traversed by light. Oscillations in the Kmax parameter are caused by the irregularities in the thickness
of the hair crossed.

2.6. Ex vivo analysis of hydration

Fifteen natural, brown hair locks of 10 g each, and measuring 10cm, were used. Locks were
washed using a neutral shampoo. After washing, the locks were slightly patted with a towel and then
dried with a hairdryer. After drying, hair locks underwent product application as described above.
Product (2 ml) was applied to wet hair locks, rubbed for 20 sec, and rinsed for 30 sec. After product
application, locks were dried with a hairdryer, and then measured. Hair-moisture content was
measured indirectly using a Tewameter TM 300 (Courage+Khazaka, electronic, GmbH). Tewameter
is a probe that measures the density gradient of water evaporation. The measuring head of the probe
is a narrow, hollow, cylinder (10-mm diameter, and 20-mm height), in order to minimize the influence
of air turbulence inside the probe. Water loss from the hair was continuously measured for 1 h. A
calibration curve was obtained using known amounts of water (0, 10, 50, 100, 200, 400, 600 µl).

2.7. Statistical analysis

Results that followed a normal distribution were analyzed statistically by applying a Student’s
t-test. Non-normally distributed data were analyzed nonparametrically using the Mann–Whitney
test. Significant p values are presented in figures as follows: #p value < 0.1; *p value < 0.05; **p value
< 0.01; *** p value < 0.001.3.

3. Results

3.1. HA penetration in hair fibers

We used Raman spectroscopy to evaluate the penetration of hyaluronic acid through the hair
fiber. Compared to the placebo shampoo (without any HA), we tracked the penetration of LMW,
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

HMW and HA-Blend formula shampoos in irradiated and non-irradiated hair fibers using Raman
spectroscopy. The cuticle was defined as a depth of 0 - 12 µm, and the cortex of 12 - 42 µm. With non-
irradiated hair locks (Figure 1), penetration of the active substances into the cuticle was significantly
higher than that of the control, increasing by 6.9x, 4.9x, and 6.9x for HMW, LMW, and HA-Blend
shampoos, respectively. Only the HA-Blend significantly penetrated the cortex (5.9-fold) more than
did the placebo and significantly in comparison to the other HA shampoos.

Non irradiated
of products penetration (% of placebo)

*
1000
Semi-quantificatification

x6.9** *
800 x6.9**
x5.9***
600 x4.9**

400 x2.6ns
x2.1ns

200

0
HMW 1%

LMW 1%

HMW 1%

LMW 1%
HA-Blend 1%

HA-Blend 1%
Placebo

Placebo
Untreated

Untreated

Cuticle Cortex

Figure 1. Semi-quantification of the penetration of LMW 1% (w/v), HMW 1% (w/v) and HA-Blend
1% (w/v) shampoos in non-irradiated hair fibers. Penetration was monitored in the cuticle (left) and
the cortex (right). Mann-Whitney test with *p < 0.05; **p<0.01; ***p < 0.001.

When hair fibers were irradiated with UVA and UVB, the difference in penetration into the
cuticle was significantly reduced. Indeed, the HA-Blend showed only 0.1-fold penetration compared
to the placebo. In contrast, the quantity penetrating the cortex was significantly increased to 1.8-fold
of the placebo level. Once again, the amount of 1% (w/v) HA-Blend detected in the cortex of the
irradiated hair fibers was significantly higher than the amount of 1% HMW (w/v) or 1% (w/v) LWM
(Figure 2).
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

UV irradiation

of products penetration (% of placebo)

**
# (p=0.0503)
Semi-quantificatification 250
x1.8*
200

150 x1 x0.9
ns x0.8 ns
ns
100
x0.3
ns
50
x0,1**

0
HMW 1%

LMW 1%

HMW 1%

LMW 1%
HA-Blend 1%

HA-Blend 1%
Placebo

Placebo
Untreated

Untreated
Cuticle Cortex

Figure 2. Semi-quantification of penetration of LMW 1%, HMW 1% and HA-Blend 1% shampoos in

hair fibers irradiated with UVA and UVB. Penetration was monitored in the cuticle (left) and the
cortex (right). Mann Whitney test with # p < 0.1; * p < 0.05; ** p < 0.01.

3.2. Anti-frizz study

To assess a straightening effect, we worked at the ex vivo level on hair shafts. The smoothing
effect was calculated based on variations in hair length in photos taken before and after exposure to
humidity (Table 1). The effect of HA-Blend at 3% (w/v) was significant, smoothing the hair 11% more
than the placebo shampoo, and producing a visible effect (Figure 3).

Table 1. Comparison of anti-frizz effect of shampoos after exposure to extreme humidity for 8 h.

Smoothing percentage mean

Condition and parameters (SD) and p value Δ(%) versus placebo
(Student t test)
Untreated 69 (3) (p<0.001)
Placebo 83 (4) (p<0.01)
HA-Blend 1.5% 87 (2) (p<0.01) 4% (ns)
HA-Blend 3% 94 (2) (p<0.01) 11% (p < 0.05)
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Figure 3. Macrophotographs illustrating the anti-frizz effect of HA-Blends and the placebo.

3.3. Reparation study

After bleaching with a chemical bleaching powder, the elastic modulus and the maximum force
needed to break a fiber significantly decreased by 20% and 11%, respectively. The placebo did not
shown any significant effect on the hair lock. After treatment with HA-Blend at 3% (w/v), the elastic
modulus and the break force significantly increased by 4% for both parameters in comparison to the
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

untreated damaged condition. The effect was also significant in comparison to the placebo (Figures
4 and 5).

140

Elastic Modulus MPa

+20%

(% of bleached)
****
120 **

-1% +4%*
ns
100

Bleached hair

HA-Blend 3%
Placebo
Untreated

+Bleaching

Figure 4. Elastic modulus evaluated after tensile-test analysis on hair locks damaged or not with
bleaching powder and washed with HA-Blend 3% (w/v) or placebo shampoos. Mann-Whitney test
with *p < 0.05; ** p < 0.01; **** p < 0.0001.

140
Break Force MPa
(%of bleached)

120 **
+11%
****
+4%
-1%
**
ns
100
Bleached hair

HA-Blend 3%
Placebo
Untreated

+Bleaching

Figure 5. Maximum force needed to break fibers of hair locks damaged with bleaching powder, or
not, and washed with HA-Blend 3% (w/v) or placebo shampoos. Mann-Whitney test with ** p < 0.01
and **** p < 0.0001.

3.4. Keratin integrity

The HA-Blend at 3% was able to repair damaged hair after bleaching. UV irradiation also
damaged the hair and affected keratin structure. After irradiation, the keratin integrity was
significantly reduced by 16%. The placebo did not produce any significant change. After treatment
with HA-Blend at 3% (w/v), the keratin integrity was improved by 31% in comparison to the
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

damaged condition. The effect was also significant in comparison to the placebo shampoo condition
(Figure 6).

relative to irradiated sample (A.U)

**

Kmax Birefringence parameter

100
+31%
-16%* ***
80 +6%ns

60
*
40

20

Irradiated

Placebo

HA-Blend 3%
Non-irradiated

+ 9J/cm² UVA
and 0.33J/cm² UVB

Figure 6. Keratin integrity measured using the Kmax parameter based on the keratin photonic
birefringence. The hair locks were washed with HA-Blend 3% (w/v) or placebo shampoos before
irradiation. Mann-Whitney test with *p < 0.05; ** p < 0.01; *** p < 0.001.

3.5. Hydration study

To evaluate whether HA is also able to moisturize hair, we measured the water evaporation
using a Tewameter®. Under the tested experimental conditions, HA-Blend 3% (w/v) increased the
water content when compared to the same formulation without the active ingredient (placebo) by
189-times (Figure 7). The increase in hair moisture content was described in the Table 2 below.

Figure 7. Illustration of the keratin integrity measured using the Kmax parameter based on the keratin
photonic birefringence. The hair locks were washed with HA-Blend 3% (w/v) or placebo shampoos
before irradiation.
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10

Table 2. Water content of hair locks after treatment with HA-Blend 3% (w/v) in comparison to
placebo.

Fold change and p-value

Water content (µL),
Condition versus placebo
mean (SD)
(Student t test)
Placebo 0.056 (0.01)
HA-Blend 3% 10.567 (1.18) x189 p < 0.05

(Fold change versus placebo)

15
Water content in µl
x189*
10

HA-Blend 3%
Placebo

Figure 8. Hair moisture content after treatment with HA-Blend 3% (w/v) or placebo shampoos.
Student’s t-test with * p < 0.05.

4. Discussion
The aim of this study was to develop a method to measure HA penetration in hair, and to assess
the benefits of a blended and optimized HA formulation for use in haircare products.
We established an innovative method to demonstrate the penetration of hyaluronic acid into the
hair fiber in rinse-off conditions. UV irradiation damage is a leading cause of hair protein loss and
color change [11]. Using UVA and UVB irradiation, we damaged the hair to open the cuticle and
enhance product penetration. The semi-quantification of the total Raman signal showed an increase
in the quantity of LMW, HMW, and placebo (shampoo without the active substance) detected in
damaged hair (over undamaged hair) (Data not shown). It is interesting to note that the total quantity
of HA-Blend detected in irradiated hair fibers is the same but the penetration profile is different, with
a lower quantity of HA-Blend in the cuticle and a higher amount in the cortex. That could suggest
that the penetration of HA-Blend is already optimal in undamaged condition. We have thus
compared the penetration of HA in normal or damaged hair locks to gain a greater understanding of
how the product works. When using non-irradiated hair locks, the three active shampoos (LMW,
HMW, and HA-Blend) had a similar ability to penetrate the cuticle; they were consistently better than
the placebo shampoo. In contrast, when examining the cortex, the HA-Blend outperformed all the
other formulations. With UV-damaged hair, penetration of the HA-Blend into the cuticle was
significantly reduced, but penetration into the cortex was still significantly higher than that of the
placebo or the two other active shampoos. This apparent discrepancy is due to the fact that when hair
has undergone UV-induced damage, the placebo more readily penetrates the cuticle, whereas
hydrophilic compounds such as HA bind more tightly to proteins such as keratin, which is in the
cortex [12]. In other words, once the HA-Blend penetrates the cuticle in damaged hair, this optimal
formulation may be attracted to the keratin in the cortex, leaving only small amounts on the cuticle
to repair the cortex. Even in the non-irradiated condition, only the HA-Blend significantly penetrated
the cortex, due to its optimized ratio of low- to high-molecular weight HA formulation, and its
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

11

capacity to bind to keratin. Besides, the optimal process behind the HA-Blend involved the addition
of lactic acid to reduce the pH of the formula. It has been found that pH plays a critical role in
molecule absorption and can have opposite effects depending on the initial ionization of the molecule
(a cationic or anionic compound). Indeed, change in pH could lead to structural changes in keratin
including changes in the binding affinity and the number of available binding sites [12]. At pH > 6.0,
hair keratin is negatively charged and attracts positively charged molecules. Inversely, at pH 2.0,
cationic species showed a low level of binding to keratin due to the electrostatic repulsion [12]. HA
is an anionic compound and we hypothesize that acidification of the pH induced by lactic acid
improved binding of HA-Blend to keratin.
By binding to the keratin, this optimal HA-Blend modified the keratin conformation, decreasing
α-helices and promoting the formation of β-sheets, leading to smoothing of the hair. These data
corroborated results from our previous study, in which analyses showed that this HA-Blend
decreases the number of disulfide bonds in keratin and induces a change in the α-helix/β-sheet ratio
[3]. The results presented here establish that the change in keratin conformation is correlated to the
effective penetration of the HA-Blend, allowing a direct interaction between HA and keratin. This
anti-frizz potential was confirmed in ex vivo experiments, with the HA-Blend producing a significant
smoothing effect 11% better than the placebo, thereby resulting in a visible benefit.
Current straightening methods, involving the thermal method (with an iron) or chemical agents,
have been found to damage hair by reducing its physico-mechanical properties [13]. Those treatments
break and rearrange disulfide bonds, change the structure of keratin in favor of a β-sheet
conformation, and disrupted cuticle [14,15]. We have shown that HA can straighten the hair and
change the keratin conformation without damaging the fiber, as shown by the tensile-strength
analysis. Indeed, we have demonstrated that HA treatment smoothed the hair while improving the
elastic modulus and the maximum strength needed to break a fiber by filling damaged hair from the
inside. We have demonstrated that the HA-Blend is also able to repair keratin integrity after UV
irradiation.
Moreover, thermal straightening has been found to lead to a reduction of water content into the
hair fiber [16]. The authors of that paper explained that the changes in protein conformation and a
reduction of the α-helix conformation may change the water accessibility. We have shown that even
if HA interacts with keratin and changes its conformation, it is able to significantly increase water
content in the fiber. This interaction between HA and keratin has already been described in the skin
to provide hydration and maintain skin-barrier integrity [5]. We hypothesize that the damage caused
at the cuticle level by thermal straightening is mainly responsible for water loss, but using high
molecular weight HA in the HA-Blend coats the fiber and improves hair hydration while smoothing
the fiber.

5. Conclusions
Our results demonstrated that Confocal Raman Spectroscopy is a powerful, non-invasive
technique to investigate the penetration of cosmetic ingredients into human hair fibers. To the best of
our knowledge, this study is the first evidence of HA penetration into hair fibers, and the first
comparison of the penetration of different molecular weights of HA. Our results show that an
optimized formulation containing different molecular weight HA plus lactic acid penetrates deeply
into the hair cortex to interact with keratin and exert a smoothing, hydrating, and reparative effect.
This insight opens new doors for the haircare industry to understand the mechanisms of action of
active molecules for applications in haircare.

6. Patents
This optimal blend is protected by the international patent application WO 2018/162604.

Author Contributions: Conceptualization, Cloe Boira, Marie Meunier, Amandine Scandolera and Romain
Reynaud; methodology, Cloe Boira, Mohammed Essendoubi, Marie Meunier, Carole Lambert, Daniel Auriol,
Michel Manfait, Olivier Piot, Amandine Scandolera and Romain Reynaud; software, Mohammed Essendoubi,
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 January 2024 doi:10.20944/preprints202401.1325.v1

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Michel Manfait and Olivier Piot; validation, Cloe Boira, Mohammed Essendoubi, Marie Meunier, Carole
Lambert, Daniel Auriol, Michel Manfait, Olivier Piot, Amandine Scandolera and Romain Reynaud; formal
analysis, Cloe Boira, Mohammed Essendoubi, Marie Meunier and Carole Lambert; investigation, Cloe Boira,
Mohammed Essendoubi, Marie Meunier and Carole Lambert; writing—original draft preparation, Cloe Boira;
writing—review and editing, Cloe Boira, Mohammed Essendoubi, Marie Meunier, Carole Lambert, Daniel
Auriol, Michel Manfait, Olivier Piot, Amandine Scandolera and Romain Reynaud. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Acknowledgments: The authors would like to thank Dermscan, Farcoderm, Kamax Innovative System and SP
Equation for their participation in the studies presented.
Conflicts of Interest: The authors declare no conflict of interest.

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