Abstract

Background: Hyaluronan (HA) is a gold standard of the cosmetic industry for skin moisturization. Although HA can penetrate stratum corneum, structural properties of the polysaccharide confer electronegative charges at physiological pH, limiting adhesion on skin surface that is negatively charged too. Using HA in rinsed formula, meaning shorter time of contact with skin, decrease its moisturizing effectiveness.
Aims: A cationic rather low molecular weight HA (c-HA), with innovative high degree of cationization, was developed and its hydrating properties were evaluated.
Methods: c-HA was formulated in shower gel, tested in a clinical trial, then ex vivo studies were conducted to figure out its efficacy. Skin adhesion was visualized and quantified through HA-binding protein labelling. Hydrating properties of c-HA were compared to unmodified low (LMW-HA) and high (HMW-HA) molecular weight HA on skin explants, after 5 min to 30 s application, using Raman spectroscopy. Aquaporin-3 and Filaggrin expression was quantified.
Results: In vivo, c-HA increased skin moisturization by +11.1% vs placebo, on very dry skin, 6 h after only 1 min application and rinse. c-HA showed better adhesion to skin explants surface compared to the LMW-HA (+107%) and HMW- HA (+23%). It provided higher water content in the stratum corneum than both reference compound (+67% vs LMW-HA, +50% vs HMW- HA), even after 30 s application. Filaggrin and Aquaporin-3 expressions increased by +35% and +16% respectively, indicating that improvement of epidermal water transport and barrier function could support the hydrating properties of c-HA.
Conclusions: Cationization promotes HA adhesion to skin and improves HA hydrating properties after short-time application, allowing new use in rinse-off products.

Keywords:Cationization; Hyaluronan; Epidermis; Moisturization; Aquaporin; Filaggrin; Raman

Introduction

Hyaluronan or hyaluronic acid (HA) is a non-sulfated linear glycosaminoglycan made of repeating units composed of D-glucuronic acid and N-acetyl glucosamine (Figure 1) [1]. HA is highly hydrophilic due to the presence of hydroxyl and acid groups. At physiological pH, carboxyl groups are negatively charged and form hyaluronate polyanion. HA is highly hygroscopic, and this capacity to bind water depends on the polymer concentration, its molecular weight and the aqueous environment of HA [2,3]. Indeed, molecular weight determines structural, physicochemical and biological properties of HA [4,5]. HA is abundant in human skin, where it plays a role in water homeostasis and extracellular matrix structure but also in inflammation, cell proliferation and healing [6,7]. Skin moisturization mostly depends on epidermal water regulation: stratum corneum composed of fully differentiated cornified keratinocytes embedded in lipid ceramide-rich matrix, ensures barrier function and limits transepidermal water loss; natural moisturizing factor, notably filaggrin peptides and glycerol, retain water in cornified envelop; corneodesmosomes and tight junctions enable cohesion and continuity of barrier; aquaporin-3 (AQP3) realizes water and glycerol transport within the tissue [8]. In the epidermis, HA hygroscopic properties contribute to deep layers hydration but its functions go beyond by regulating keratinocytes proliferation and differentiation and contributing to the establishment of a competent epidermal barrier [9]. Moisturizing products are considered as a basic beauty routin [8,10]. Due to its hydration property, biocompatibility and obtention by mastered biotechnological process, HA has become a reference moisturizer in the cosmetic industry [11,12]. As demonstrated by a search performed on Mintel’s Global New Products Database (GNPD, Mintel Group Ltd., London, UK, accessed on March 8th, 2024), 40% of new launches in the Beauty and Personal Care industry over the past year claimed “moisturizing” or “hydrating”, and among these products, 24% contained sodium hyaluronate as active ingredient.

Topically applied HA of low and high molecular weight have been clinically proven to increase skin water content and decrease transepidermal water loss [13-17]. The mechanisms by which exogenous hyaluronic acid improve skin hydration are not fully elucidated. High molecular weight HA (HMW-HA), over 1000 kDa, is not able to penetrate skin and stays at the stratum corneum level, while low molecular weight HA (LMW-HA), under 50 kDa, accumulates within stratum corneum as well, but is able to reach the viable epidermis [18-22]. HMW-HA is described as a film-former, limiting water loss, and LMW-HA attract much more attention for its biological effects. Topical application of 50 kDa HA on reconstructed epidermis modulates the expression of 120 genes involved in keratinocytes differentiation and cohesion, including gene encoding aquaporin-3 and tight junctions [21]. In a similar model, a 10 kDa HA increases filaggrin (FLG) and its degrading enzyme caspase-14, suggesting an increase in natural moisturizing factor and promotion of stratum corneum moisturization [23]. The modification of HA represents a strategy to increase its efficacy and extend the possible applications of the molecule [24,25]. As previously mentioned, HA is anionic, and the surface of stratum corneum also carries a negative charge, limiting the interaction between HA and the skin. Positively charged polymers are known for years to better diffuse within stratum corneum and this was recently demonstrated again with nanoemulsion [26,27]. Cationization of HA was achieved in previous studies and is reported to improve ocular drug delivery as well as moisture absorption and retention capacities of the polysaccharide [28,29]. So, the development of cationic HA appeared an opportunity to boost hydration properties of LMWHA that already displays a certain ability to penetrate skin and triggers biological response within epidermis. A rather LMW-HA was highly cationized through quaternary ammonium grafting and this work presents the clinical evaluation of the cationic HA (c-HA) followed by ex vivo studies to quantify skin adhesion and hydration compared to “native” LMW-HA and HMW-HA. Aquaporin and filaggrin expression were measured to assess the potential biological function of c-HA.

Materials and Methods

Hyaluronic acid

LMW- HA (INCI: hydrolyzed sodium hyaluronate), HMW- HA (INCI: sodium hyaluronate) and c-HA (INCI: hydroxypropyltrimonium hyaluronate) were manufactured by Givaudan.

c-HA cationization

The starting material for this study was hyaluronic acid (HA) with a molecular weight specification between 20 and 80 kDa. Under basic conditions, HA was reacted with 2,3-epoxypropyltrimonium chloride to produce a cationized form of HA (hydroxypropyltrimonium hyaluronate) with a cationization degree ranging from 1.4 to 3.0. The detailed procedure is described in patent WO/2023/285663A1.

Clinical evaluation on rinse-off application

All the subjects participating in the study gave their informed consent signed at the beginning of the study. The study was conducted according to the guidelines of the Declaration of Helsinki. This study performed on cosmetic products was within the definition of article L. 5131–1v of the French Public Health Code and is in accordance with decree n2017-884 of 9 May 2017, modifying some regulatory requirements concerning research involving human subjects.
INCI formula: AQUA/WATER, SODIUM LAURETH SULFATE, COCAMIDOPROPYLBETAINE, PHENOXYETHANOL, 1.2-HEXANEDIOL, CAPRYLYL GLYCOL, SODIUM CHLORIDE, FRAGRANCE ± HYDROXYPROPYLTRIMONIUM HYALURONATE

Panel description

A placebo-controlled double blind clinical study was performed on 29 volunteers with average age 33 ±5.9 years old divided in two groups; one group applied formula containing c-HA at 0.1% and the other one the placebo formula. Volunteers have been recruited by matching with the inclusion criteria which was having dry skin on calves’ area with corneometry values comprised between 10 to 40 a.u. Volunteers applied once the shower gel containing or not c-HA at 0.1%. They soaped for 30 s and wait 30 s more before generous rinse. Skin hydration was measured before application (T0) and then 6 h after application by using Corneometer®.

Craniometry measurements

The hydration of the stratum corneum causes changes in its electric characteristics. The stratum corneum behaves like a dielectric body, and any modification of its state of hydration results in a variation of its electric capacity measured by a condenser. Higher is the hydration, higher the capacity value is, because very strong dipolar nature of stratum corneum increases the electrical permittivity of the environment and its conductibility. Measurements were realized by Corneometer® CM825TM (Courage & Khazaka electronics). The probe linked to a condenser allows applying at all the time the same pressure on the technique in order to not disturb the measures and to obtain good experimental conditions reproducibility.

Skin source

All experiments were performed on primary cells and skin explants obtained from skin surgical residues following plastic surgery. Skin explants were obtained from donors who have sustained abdominoplasty and lifting (Polyclinique Courlancy, Reims) after reading, understanding and signing an “information and no objection” form for use, for dermocosmetic research purpose, of tissues, cells, and products of the human body collected during surgery (surgical residues), aligned with articles L. 1211-2 alinéa 2, and L. 1245-2, Code de la santé publique.

Skin adhesion versus LMW-HA or HMW-HA

Human fresh skin explants were topically treated for 1 h with LMW-HA, or HMW-HA, or c-HA at 1%, compared to untreated control. After 1 h of treatment, skin explants were rinsed twice with sterile water and liquid excess was softly absorbed with cleaning paper. Skin explants were embedded in OCT for HABP staining on cryoslices. For comparison study with LMW-HA, skin explants came from a 22-year-old woman donor having breast reduction, and comparison with HMW-HA was conducted on skin explants from a 53 years old woman donor, having abdominal surgery.

HABP staining

HA deposit was evidenced with a fluorescent staining using HA-binding protein on 8μm thickness cryoslices. Briefly, nonspecific sites were saturated with successive baths in avidin, biotin and 0.1% bovin serum albumin solutions. Biotinylated HAbinding protein was then incubated on cryoslices for 2 hours at room temperature, followed by rinses, and another incubation with Streptavidin coupled to Alexa fluor 568 for 30 min at room temperature in the darkness. Samples were rinsed and assembled with coverslips and mounting medium. Images were collected with Axio Observed Inverted fluorescence microscope (Zeiss). Fluorescent intensity specific of HA deposit on skin stratum corneum was quantified.

Skin hydration versus LMW-HA or HMW-HA

Human fresh skin explants were topically treated for 5 min with LMW-HA at 0.1% or HMW-HA with the c-HA at 0.1% or with water (untreated control). After 5 min of treatment, all skin explants were rinsed 5 times with sterile water. Topical treatment and rinse were repeated every day, for 2 days. Skin water content was analyzed on fresh skin explants by Raman spectroscopy after two days following repeated treatments. Analysis of Aquaporin-3 and filaggrin expression was conducted on c-HA treated explants and immunostaining was performed on formalin-fixed skin explants embedded in paraffin. For comparison study with LMWHA skin, explants came from a 22 years old woman donor, having breast reduction, and comparison with HMW-HA was conducted on skin explants from a 40-year-old woman donor, having abdominal surgery.

Flash rinse-off application – Hydration

Human fresh skin explants (41 years old woman donor having breast reduction) were topically treated for 30 s with LMW-HA at 0.1% or HMW-HA at 0.1% or with the c-HA at 0.1% compared to untreated control. After 30 s treatment, skin explants were rinsed 5 times with sterile water. These treatments were repeated every day for 2 days. Skin water content was analyzed on fresh skin explants by Raman spectroscopy after 2 days following repeated treatments.

Raman spectroscopy analysis - Hydration

The axial Z profiles were recorded directly on the skin samples. The Z profiles consist of an in-depth scanning through the skin. Raman spectra were collected at different focus points on skin surface, from Z = 0 μm to Z = 30 μm with a 3 μm step, corresponding to the upper layers of epidermis mainly the stratum corneum. A total of 40 Raman profiles were recorded (4 profiles per condition, n=4). For the assessment of skin water content, we calculated the integrated intensity of the OH vibration band on average stratum corneum spectra. This band represents the water content of the skin. The spectral range used for the calculation is νOH: 3100-3600 cm-1.

Aquaporin-3 and filaggrin immunostaining

Skin explants were cut in 4 μm thickness slices, dewaxed and antigenic retrieval was performed. Non-specific sites were saturated with bovine serum albumine and primary antibodies targeting FLG and AQP3 were then incubated on skin slices overnight at 4°C, following the dilutions recommended by the supplier. Next day, the excess antibody was washed with Tris buffer, and secondary antibodies coupled with Alexa fluor 488 were incubated for 1 h at room temperature with Hoechst 33342. Excess of antibody was washed with Tri’s buffer and mounting medium was added with coverslips. Pictures of the emitted fluorescent signal were taken with an inverted epifluorescent microscope (Axio Observer, Zeiss). Fluorescence intensity for each condition was measured using ImageJ software and results obtained with the treatments were compared to the untreated condition considered as the 100% control.

Statistical analysis

For ex vivo and clinical studies, data normality was first verified regarding the Gaussian law using Shapiro Wilk test. According to the results, parametric or nonparametric tests were used to compare the effect of c-HA versus the untreated condition or versus placebo or others HA, with # p < 0.1; * p < 0.05; ** p < 0.01 and *** p < 0.001.

Results

Clinical evaluation of c-HA hydrating properties

The study was conducted on calves’ area, previously demonstrated to be a dry zone of the body [30]. The Inclusion criteria was having very dry skin, characterized by Corneometer® value between 10 to 40 a.u. Placebo shower gel or formula containing c-HA 0.1% were applied for 1 min in total and generously rinsed. Figure 2 presents skin hydration values after 6 h vs T0 in placebo and active groups. Six hours after 1 min application, skin hydration increased by +6.6% vs T0 in volunteers applying shower gel with 0.1% c-HA. Placebo shower gel decreased skin hydration by -4.5%, meaning that active formula increased hydration by 11.1% vs placebo.

Ex vivo study of c-HA skin adhesion vs LMW-HA and HMWHA

Skin adhesion capacity of c-HA was evaluated in rinseoff application by HA-binding protein labelling of HA deposit, observed by fluorescence microscopy, and compared to LMW-HA (Figure 3) and HMW-HA (Figure 4) as references. Cationic charge increased skin adhesion of c-HA by 107% vs LMW-HA and by +23% vs HMW-HA.

Ex vivo study of c-HA skin hydration vs LMW-HA and HMWHA

The skin hydration properties of c-HA were compared to the ones of LMW-HA and HMW-HA using Raman spectroscopy. A 5 min application once a day for 2 days was realized on skin explants. Results are presented in Figure 5. While the three HA increased significantly skin hydration compared to untreated skin explants, c-HA demonstrated the highest efficacy with up to +108% vs untreated condition, and +58% and +37% vs LMW-HA and HMW-HA respectively.

Ex vivo study of Aquaporin-3 and Filaggrin expression after c-HA application

AQP3 and FLG protein expression were studied in presence of c-HA following the same conditions as used for hydration study. AQP3 is a water- and glycerol-transporting channel directly linked to skin moisture.31 Figure 6 presents fluorescence microscopy pictures of aquaporin-3 immunostaining (green fluorescence). AQP3 was localized in the plasma membrane and intracellular compartment of the keratinocytes from basal layer and stratum spinosum. An increase of aquaporin-3 by 16% was observed in explants treated with c-HA. Figure 7 presents fluorescence microscopy pictures of FLG immunostaining (green fluorescence). FLG is a major structural protein, binding keratin fiber in the epidermis, contributing to the terminal differentiation of epidermal cells and so to barrier function.32 FLG was mainly localized in the stratum corneum and fluorescence intensity increased by 35% in c-HA treated explants, suggesting a higher expression of FLG.

Ex vivo study of c-HA flash rinse-off application vs LMW-HA and HMW-HA

It was previously observed that c-HA increased hydration after 5 min application and rinse (Figure 4). Another study was conducted with only 30 s application following the same procedure. Again, c-HA was compared to LMW-HA and HMW-HA. Water content was measured and results are presented in Figure 8. LMW-HA and HMW-HA increased water content by +46% and +63% respectively compared to untreated skin explants, while the c-HA allowed to reach a +113%, meaning +67% compared to LMW-HA and +50% compared to HMW-HA. Despite the drastic shortening of time application, c-HA provided the highest increase in skin water content.

Discussion

Protecting the body from excessive water loss is a vital function of skin and appearance of normal skin is closely related to its hydration status. It is not surprising that “moisturizing” or “hydrating” were claimed on 40% of new cosmetic products over the past year and HA has become a key ingredient of moisturizers market, incorporated in 24% of such products (source GNPD, Mintel Group Ltd., London, UK, accessed on March 8th 2024).10,12. HA is electronegative, as the skin surface is, and cationization was selected to improve stratum corneum adhesion of c-HA with the perspective to develop rinse-off applications. Moreover, cationization of HA improves its water absorption capacity.29 The choice fell on LMW-HA due its ability to penetrate deeper in the skin and impact epidermal biological process.18–21,23 In the search of HA derivatives with augmented properties, several combinations of molecular weight and cationization degree were tested (data not shown) to design an optimal c-HA that was obtained with a rather low molecular weight and high cationization degree. A clinical study first assessed the hydration properties of 0.1% c-HA on very dry skin. The formulation, a shower gel, was applied for only 1 min and rinsed. Despite the short time of contact between c-HA and skin, hydration evaluated by corneometry, increased significantly by +6.6% vs T0 and +11.1% vs placebo as soon as 6 h after application. Previous findings observed LMW-HA (50 kDa) to increase hydration level (corneometry) after 30 days by +4.7% to +24% vs D0 following daily face cream application.13,22 In view of this values, immediate hydration provided by c-HA appears appreciable and confirm its effectiveness in rinse-off products. Ex vivo studies were conducted on skin explants to describe how c-HA behaves within the skin compared to nonmodified LMW-HA and HMW-HA. Firstly, skin adhesion capacity of the three HA was observed: more c-HA deposits were observed on the skin compared to LMW-HA and HMW-HA: +107% and +23% respectively. As expected from literature data, cationization improved the interaction with the skin surface [27,28].

Raman spectroscopy was then used to quantify water in skin explants treated for 2 days with c-HA 0.1% applied for 5 min once a day followed by rinse. Skin water content increased with all HA but c-HA-treated skin explants exhibited the highest values: +65% to +108%. Even when shortening the contact time to 30 s, c-HAtreated skin explants still displayed the highest water content, +113%, with similar efficacy in skin hydration improvement and the difference with HMW-HA and LMW-HA was even more pronounced. Hydration improvement is consistent with the better adhesion of c-HA to skin surface and the larger difference between c-HA and other HA after very short-time application, supports this conclusion. HA is highly hygroscopic and serve as a water reservoir in human skin. Cationization of hyaluronic acid doubles its moisture absorption capacity and improves its ability to retain water, compared to non-modified HA [29]. The water content increase in skin explants is probably related to water associated to c-HA. Though, the increase of AQP3 and FLG expression indicates that c-HA could also biologically act on skin hydration.

AQP3 is a transmembrane channel of water and glycerol expressed in keratinocytes. AQP3 contributes to maintain constant water content across the epidermis, and distributes glycerol, being part of the NMF, up to stratum corneum [31,33]. Increasing AQP3 expression with active ingredients could be a strategy to improve dry skin condition [34]. In this work, AQP3 expression was localized in the plasma membrane and intracellular compartment of keratinocytes from the basal layer and stratum spinosum in accordance with previous findings [31]. Expression increased by +16% in these compartments. The link between HA and AQP3 is not fully understood but was previously reported: LMWHA topically applied on reconstructed epidermis upregulates the gene encoding AQP3 and molecular dynamics simulations recently evidenced that HA is a regulator of AQP3 [21,35]. Further experiments would be of great interest to decipher the interaction of HA and AQP3.

FLG is a protein involved in keratinocytes differentiation into corneocytes, embedded in an insoluble matrix called cornified envelop [32]. FLG binds keratin fibers and the skeletal protein of stratum corneum, stabilizing the cornified envelop. In the terminal stage of differentiation, FLG is proteolyzed by caspase-14 into amino acids, contributing to the NMF. Indeed, FLG fluorescence was mainly detected in the stratum corneum, and c-HA treatment increased by +35% the FLG expression in the upper layer of epidermis. Although the role of HA in keratinocytes differentiation is still controversial, experiments evidenced a link between HA and FLG [9]. Expression of pro-filaggrin, the precursor of FLG, increases in cultured human keratinocytes treated with HA [36]. Recently Hashimoto et al., topically applied LMW-HA (<10 kDa) on 3D epidermidis model: pro-filaggrin gene expression increased with FLG protein, caspase-14 activity and HA content into epidermal basal layer [23]. Authors concluded that topical application of HA would result in an increase in NMF and promote moisturization of stratum corneum. AQP3 and FLG could both contribute to better hydration of skin by distributing water within epidermis, contributing to NMF and to the establishment of a competent barrier function. The data provided by the literature are consistent with a control of AQP3 and FLG by c-HA of which clinically proven hydration efficacy could be related to these proteins upregulation. This work evaluated the effect of HA cationization on the hydrating properties of the polymer. An innovative high degree of cationization combined to a rather low molecular weight, improved HA skin adhesion and hydrating capacities compared to “native” LMW-HA and HMW-HA. Up-regulation of AQP3 and FLG outlined the biological mechanisms that could support the clinical efficacy of c-HA. Finally, it demonstrates that augmented HA can be reached by combining the right molecular weight with the appropriate functionalization, opening a new use of HA in rinseoff applications.

Ethical Statements

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Contribution Statement

Conceptualisation, R.R. and A.S.; methodology, M.M., E.C. and N.J.; validation, A.S. and R.R.; skin sourcing, J.T.; investigation, M.M., M.B. and L.L.; writing-original draft preparation, M.M.; writingreview and editing, M.M. and A.S.; supervision, A.S. and R.R.; project administration, R.R.

All authors have read and agreed to the published version of the manuscript.

Acknowledgement

The authors would like to thank all team members for their participation on the conducting of the studies.

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