High molecular weight hyaluronic acid vectorised with clay provides long‐term hydration and reduces skin brightness

Abstract

Background

Hyaluronic acid (HA) is a widely used active cosmetic ingredient. Its multiple skin care benefits are modulated by its molecular weight. Low molecular weight (LMW) HA can penetrate the skin, but high molecular weight (HMW) HA remains at the surface. Here, we assessed how vectorization of HMW HA with bentonite clay—achieved with an innovative technology—enhances its cosmetic and hydrating properties.

Materials and Methods

The two HA forms were applied to skin explants; their penetration and smoothing effects were monitored by Raman spectroscopy and scanning electron microscopy. The two forms were biochemically characterised by chromatography, enzyme sensitivity assays, and analysis of Zeta potential. Cosmetics benefits such as, the smoothing effect of vectorised‐HA was assessed in ex vivo experiments on skin explants. A placebo‐controlled clinical study was finally conducted applying treatments for 28 days to analyse the final benefits in crow's feet area.

Results

Raman spectroscopy analysis revealed native HMW HA to accumulate at the surface of skin explants, whereas vectorised HMW HA was detected in deeper skin layers. This innovative vectorisation process changed the zeta potential of vectorised HMW HA, being then more anionic and negative without impacting the biochemical structure of native HA. In terms of cosmetic benefits, following application of vectorised HMW HA ex vivo, the skin's surface was visibly smoother. This smoothing was clinically confirmed, with a significant reduction in fine lines.

Conclusion

The development of innovative process vectorising HMW HA allowed HMW HA penetration in the skin. This enhanced penetration extends the clinical benefits of this iconic cosmetic ingredient.

1. INTRODUCTION

Hyaluronic acid (HA) is produced in the human body, and is ubiquitous in vertebrates, its use is thus extremely safe. It contributes to a number of biological processes, including cell differentiation, embryo development, inflammation and wound healing. ¹ , ² HA is increasingly used in cosmetic products for its hydrating and anti‐wrinkle effects. ² , ³ This polymeric compound exists at several MW, each providing benefits in the short or longer term, and at various depths in the skin. ² Thus, low‐MW variants (<50 kDa) penetrate deeper into the skin triggering biological pathways, whereas high‐MW presentations (>1000 kDa) remain at the surface, creating a hydrated layer. ⁴ The larger HMW HA can bind more water, providing enhanced hydration compared to its LMW counterpart. However, its size hinders its capacity to access deeper layers within the skin. As a consequence, the hydrating effect of HMW HA remains at the surface of the skin, where it is generally short‐lived—being rinsed or gradually sloughed off after application. Stronger interactions between this molecule and the skin could lead to greater benefits for cosmetics users.

Skin penetration routes are the focus of an abundant body of research, for both cosmetic and medical purposes. Depending on the nature of the molecule—hydrophilic or lipophilic, large or small, charged or neutral—the route will differ. Thus, lipophilic molecules penetrate the stratum corneum (SC) via the intercellular lipid bilayers separating the corneocytes, whereas more water‐soluble molecules take the transcellular route—passing through corneocytes and intercellular lipid bilayers—thanks to interactions with the polar head groups of the phospholipids making up the cellular membranes. ⁵ Beyond this general rule, empirical studies have shown that the permeability coefficient can be predicted for a given molecule based on four physicochemical parameters: molecular weight, octanol‐water partition coefficient (logD), number of H‐bond donors and H‐bond acceptors. ⁶ Based on these observations, LMW HA is expected to take the transcellular or intercellular pathway to penetrate the SC. This route is not accessible to HMW HA due to its larger size, but penetration of even large molecules can be enhanced by associating them with vector molecules.

Vehicle‐assisted penetration of active compounds has been extensively studied, in particular for medical applications. ⁷ Promising results have been obtained with vesicular systems, with the active molecule encapsulated in lipososmes. The skin's negative charge ⁸ , ⁹ has been exploited to enhance delivery, with charged liposomes showing stronger interactions with the skin. ¹⁰ , ¹¹ Thus, positive liposomes are associated with higher absorption through the stratum corneum than negatively charged vesicles ¹⁰ Alternatively, negative liposomes provide higher absorption through the viable epidermis and dermis than positively charged liposomes. ¹¹ This result was confirmed by other authors. ⁸ , ¹² Coating of the liposome surface with cationic polymers has also been found to effectively enhance absorption of its cargo. ¹³ All these studies related to drug delivery, but cosmetic applications could also benefit from the use of negatively‐charged active carriers. However, in such non‐essential applications, it is important to only potentiate molecules that have been proven to be completely safe. ¹⁴ For cosmetic purposes, the properties of the carriers themselves could enhance the characteristics of the final products.

Bentonite is a naturally negatively charged type of clay derived from sedimentary volcanic ash. It contains a significant fraction of smectite and is used both internally and externally as part of traditional remedies. ¹⁵ It has been included in dermal applications where it has soothing effects, for example on contact dermatitis ¹⁶ and nappy rash or diaper dermatitis. ¹⁷ , ¹⁸ It could thus be an interesting complement to HA in cosmetic products. Bentonite has a lamellar structure, with aggregated lamellar platelets held together by electrochemical forces. The structure contains interposition water and trapped exchangeable cations, such as calcium, magnesium or sodium. Bentonite can be activated to efficiently entrap molecules in a cation‐rich environment (WO 2023/135170). ¹⁹

For this study, we associated HMW HA with bentonite, to produce a clay‐HA complex using particular conditions. We hypothesised that the vectorised HMW HA molecules would retain their negative charge thanks to their contact with cations, and adopt a particular clay interaction mediated‐ compacted organization which potentially allowing them to better penetrate the skin's layers to provide hydration at a deeper level. In addition to chemically characterising the vectorised HMW HA to scientifically support the improvement of skin penetration, we performed a series of ex vivo and clinical studies. The results obtained demonstrated a number of cosmetic benefits following application of products containing the vectorised formulation.

2. MATERIAL AND METHODS

2.1. Vectorisation

HMW HA was obtained from Givaudan was vectorised according to the protocol described in the following patent (WO 2023/135170).

2.2. Skin penetration analysis by Raman spectroscopy

2.2.1. Skin explant culture and preparation for Raman spectroscopy and histological analysis

Human skin explants were prepared and kept in survival medium (MIL215001, Biopredic) for 24 h at 37°C under 5% CO₂. The next day, 10% (w/v) vectorised HMW HA (corresponding to 1% (w/v) HA) and 1% (w/v) HMW HA solutions in distilled water were topically applied to skin explants. Explants were incubated for 8 h at 37°C under 5% CO₂. The baseline control condition received no treatment. Following incubation, the skin surface was cleaned to eliminate any excess product. Skin explants were then frozen at −80°C and sectioned longitudinally using a cryotome to produce 20‐µm sections. For each explant, three tissue sections were selected and deposited on a CaF₂ support for Raman imaging analysis. A total of nine Raman images were recorded per condition. Three adjacent 7‐µm sections were prepared for Haematoxylin & Eosin staining to verify histological parameters.

2.2.2. Raman micro‐imaging

Raman images measuring Y: 10 µm/X: 100 µm were acquired with a 5‐µm step in both X and Y. Each Raman image is derived from 3Y spectra and 21X spectra (63 spectra per image). The parameters were as follows: Laser wavelength 600 nm, magnification 100 X, long focal length with 0.75 numerical aperture, acquisition time 25 s, accumulation 1X, spectral range 400–4000 cm, grating 950T, confocal aperture 300 µm, slit width 150 µm (spectral resolution 6.5 cm), X step 5 µm, Y step 5 µm.

The Raman spectrometer was calibrated with silicon before each experiment. The expected Raman peak of 520.7 cm was obtained, thus ensuring reproducible measurements. The laser power was continuously controlled at the sample level.

Raman images were pre‐processed to eliminate aberrant spectra (fluorescence, burning, saturation). Spectra were then baseline corrected, smoothed, despiked and normalised.

Corrected data maps were processed using in‐house software based on a least‐squares fitting method operating in MATLAB software. Reference spectra—average spectra for hyaluronic acid and clay—were mathematically mapped to the overall spectral image to determine the contribution of each spectrum to the image, and the distribution of each compound within the skin explant.

2.2.3. Biochemical characterisation

HMW HA and vectorised HMW HA were biochemically characterised to verify how the vectorisation procedure had affected the active molecule.

2.2.4. Physical characteristics

Size‐exclusion chromatography was used to verify that the size of the molecule had not been altered during the vectorisation process. The molecular weights (MWs) of HA were determined by size‐exclusion chromatography on two ViscoGEL A6000M columns (Malvern) used in series. Eluents were monitored by Viscotek GPCmax and Viscotek TDA 302 detectors (Malvern) to determine refractive index, light scattering (RALS and LALS) and viscosity. The eluent was PBS (Sigma–Aldrich) (pH = 7.0) at a flow rate of 0.35 mL/min. Columns and detectors were operated at 35°C. Calibration was done with a pullulan standard (180 kDa) (Malvern). The samples were dissolved in PBS at 45°C for 1 h and stored at 4°C–8°C overnight before analysis. The MWs were calculated using Malvern Omnisec software version 4.7.

2.2.5. Hyaluronidase sensitivity

Sensitivity to hyaluronidases was measured in tubo. Native and vectorised HMW HA were solvated in purified water (Milli Q+) at 12.5 mg/mL. An aliquot (300 µL) of each was placed in a 10‐mL Headspace vial (Agilent), 1.1 mL purified water (control condition) or Hyaluronidase (Sigma Aldrich) solution at 8 UI/mL (enzyme condition) was added. Tubes were incubated at 55°C for 16 h and cooled to room temperature before characterising the products by HPLC. Briefly, 100 µL of each sample was diluted in 780 µL mobile phase (Sodium chloride/Sodium phosphate buffer); 50 µL was injected into an OH‐Pak SB‐806 M column. Separation was performed in isocratic mode at 30°C at a flow rate of 0.5 mL/min over 40 min. Elution was detected at 200 nm.

2.2.6. Zeta potential

The zeta potential of the native and vectorised HMW HA was monitored over a pH range from 2 to 10 using a Zetameter (zetasizer Nano Z, Malvern Panalytical). Molecules were suspended in distilled water and transferred to a DTS 1070 cell (Malvern Panalytical). Results reflect the molecule's charge state based on its sensitivity to pH. The average from 10 measurements was calculated by the Zetasizer software, to determine each product's zeta potential.

2.3. Analysis of stratum corneum suppleness

2.3.1. Skin explant culture and preparation for atomic force microscopy

Human skin explants were prepared and maintained in a survival medium for 24 h at 37°C under 5% CO₂. The next day, 1% vectorised HMW HA (corresponding to 0.1% (w/v) HMW HA) and 0.1% (w/v) HMW HA solutions in distilled water were topically applied and incubated for either 8 or 24 h at 37°C under 5% CO₂. An untreated condition was included. At the end of each incubation period, skin explants were frozen in liquid nitrogen and stored at −20°C.

2.3.2. Atomic force microscopy for skin hydration assessment

Skin suppleness, correlated to skin hydration level, was measured by AFM (Bioscope Resolve, Bruker) combined with epifluorescence microscopy (Leica DMi8) to precisely position the AFM probe on the sample.

AFM was performed in QNM (Quantitative Nanomechanical Mapping) Peakforce mode. The AFM probe has a 0.35 N/m theoretical spring constant and a curvature radius 10 nm. Before each use, probe deflection sensitivity was measured on sapphire, and its spring constant was calibrated based on thermal noise.

Atomic force measurements were performed in liquid medium (PBS 1X), measuring the Force‐Volume (FV) on the stratum corneum over a scan size of 30 × 30 µm with 128 × 128 pixels, that is, a total of 16 284 measurement points. Each pixel in the image corresponds to a Force‐indentation curve from which the elastic modulus (Ea) can be extracted. Three independent skin samples were analysed per condition.

Some differences in the number of measurements may exist between areas, as some outlier data were automatically suppressed by the software.

The elastic modulus was determined from curves using BioMeca Analysis software. Approach curves obtained for each FV were processed to quantify the Ea (stiffness) of the stratum corneum. Sneddon's theoretical model was applied up to about 50% of the maximum force exerted. This value corresponds to an indentation ranging from 0 to 0.5 µm.

2.3.3. Ex vivo skin smoothing effect

Human skin explants were prepared and kept in survival medium for 24 h at 37°C under 5% CO₂. The next day, vectorised HMW HA at 1% (w/v) (corresponding to 0.1% HMW HA) and HMW HA at 0.1% (w/v) in distilled water was applied topically. Samples were incubated for 72 h at 37°C, under 5% CO₂ at reduced hygrometry (20% relative humidity). An untreated condition was included.

Following incubation, skin explants were cut into several parts for further study. Three independent skin samples were analysed per condition.

The smoothing effect was analysed by Scanning Electron Microscopy (SEM).

2.3.4. Evaluation of detoxification of the product on skin explants

Human skin explants were prepared and kept in survival medium for 24 h at 37°C under 5% CO₂. The next day, vectorised HMW HA diluted in sterile distilled water to a final concentration of 1% (w/v) was topically applied. Samples were incubated for 72 h at 37°C under 5% CO₂.

Skin explants were photographed using a Dermascope (DinoLite) before brushing with 3 mg of carbon particles to mimic exposure to pollution. The skin explants were photographed once again. Particles were rinsed off with a stream of sterile distilled water (3 × 10 mL for each zone). The particles remaining at the surface of the skin explants in each zone were then collected using a D‐squame before taking a final photograph. The percentage of the region of interest covered by particles was determined by image analysis using cellSens software.

2.4. Clinical evaluation

2.4.1. Study protocol and cohort

A double‐blind, placebo‐controlled clinical study was undertaken with 39 volunteers, aged from 35 to 55 years old (mean age 49 ± 5 years). Inclusion criteria included having dry skin on the cheek, and fine lines in the crow's feet area.

Volunteers were assigned to two groups. One group applied a cream containing 1% (w/v) vectorised HMW HA, the other group applied a placebo cream, identical but for the omission of the vectorised HMW HA. Volunteers applied the assigned cream twice daily to the whole face for 28 days.

Digital images of participants’ faces were recorded at different times using a 2D acquisition system to obtain standardised multimodal pictures at 24 mega‐pixel resolution. Pictures were taken on D0, before application, at 1 h after the first application, and 6 h after the first application to analyse immediate effects. Images acquired at the end of the study protocol—on day 28—provided information on the effects of long‐term use.

Images were analysed by ColorFace to measure the area covered by crow's feet wrinkles.

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 is in accordance with Decree n 2017–884 of 9 May 2017, modifying some regulatory requirements concerning research involving human subjects.

2.4.2. INCI FORMULA

AQUA/WATER, CETYL ALCOHOL, GLYCERYL STEARATE, PEG‐75 STEARATE, CETETH‐20, STEARETH‐20, ISODECYL NEOPENTANOATE, ± BENTONITE, SODIUM HYALURONATE, PHENOXYETHANOL, GLYCERIN, DIMETHICONE, FRAGRANCE

2.5. Statistical analysis

For in vitro and ex vivo studies, all results are presented as mean ± standard error of mean (SEM in vivo data are expressed as percent variation relative to D0.

A Shapiro Wilk test was used to verify whether the raw data was normally distributed. For normally‐distributed data, the mean values were compared using either an unpaired t test (≤2 groups) or one‐way ANOVA followed by post‐hoc analysis (≥2 groups). For non‐normally‐distributed data, a Kruskal–Wallis test was applied followed by a Mann–Whitney U test for unpaired values or a Wilcoxon test for paired values.

In all cases, results were considered significant with #p < 0.1, *p < 0.05, **p < 0.01 or ***p < 0.001.

3. RESULTS

3.1. Vectorisation of high molecular weight (HMW) hyaluronic acid (HA)

With a view to improving skin penetration of HMW HA and obtaining higher cosmetics benefits than native HMW HA, we applied a patented vectorisation technique to combine it with bentonite. Bentonite was mixed with HMW HA and exposed to pressure and shear forces to increase interactions between the inorganic matrix (bentonite) and its organic modifier (HA). Following treatment, the clay and the HA are both negatively charged and associated with cations which is stabilized into a lamellar sheet clay organization conferring particular organization to HA. This negative charge should enhance the interaction between the products and the skin.

3.2. Vectorization provides enhanced ex vivo skin penetration of high molecular weight hyaluronic acid

Vectorization process was developed to enhance skin penetration of HMW HA in the skin. The first step was then to verify this hypothesis. For this study, HMW HA or vectorised HMW HA was applied to skin explants at equivalent dosage. After 8 h in physiological conditions, Raman spectroscopy analysis confirmed that HMW HA was present on the skin's surface. In contrast, vectorised HMW HA was detectable deeper within the skin—down to a depth of 70 µm (Figure 1). The signal corresponding to bentonite remained at the surface of the skin.

FIGURE 1.

HMW HA penetrates significantly deeper into the skin after vectorisation. Skin penetration of native and vectorised HMW HA was measured on skin explants by Raman spectroscopy. Characteristic signals for HMW HA (upper panel) and clay (lower panel) were monitored.

This enhanced penetration could have been linked to increased sensitivity to hyaluronidase activity in the skin or degradation during manufacturing process itself, breaking the HMW compound into smaller entities that would penetrate more readily. To verify whether this was the case, we tested the sensitivity of both formulations to hyaluronidases in tubo. Native HMW HA and vectorised HMW HA showed similar sensitivities (Figure 2A). Indeed, the estimated size (in Da) was reduced by 99.3% and 99.9% for vectorised HMW HA and native HMW HA, evidencing a similar behaviour toward hyaluronidases, excluding a higher degradation of vectorised HMW HA that would have increased the penetration. Moreover, the size‐exclusion chromatography (SEC) indicated an identical MW for the two HMW HA forms, confirming that there were no alterations of the molecular structure during the manufacturing process (Figure 2B).

FIGURE 2.

Vectorisation of HMW HA does not alter its sensitivity to hyaluronidases. Native and vectorised HMW HA were solubilised in purified water before exposure (or not) to hyaluronidases 8 UI/mL. Products were detected by HPLC.

A last hypothesis was to have a modified electrostatic behavior of the HMW HA due to its interaction with clay which adapts particular organization following the process. To determine which properties of the vectorised molecule produced these effects, we performed a biochemical analysis. The vectorised HMW HA was confirmed to be more negatively charged than the native HMW HA based on its zeta potential. The vectorised HMW HA had a more negative profile than native HMW HA, making it more sensitive to pH variations promoting better electrostatic attraction into the skin (Figure 3).

FIGURE 3.

Vectorisation significantly alters the zeta potential of HMW HA without altering its molecular weight. (A) The zeta potential of native high molecular weight hyaluronic acid (HMW HA; green diamond) and vectorised HMW HA (green square) was measured over a pH range from 2 to 10 using Zetameter. (B) The molecular weights of native and vectorised HMW HA were measured by size exclusion chromatography.

3.3. Enhanced skin penetration is associated with greater hydration

Analysis of skin sections following application of HMW HA or vectorised HMW HA by atomic force microscopy (AFM) combined with epifluorescence microscopy indicated that both products significantly enhanced skin suppleness at 8 h by +47% and +98%, respectively, compared to no treatment or application of bentonite alone (+9%, ns) (Figure 4). However, the enhanced suppleness was longer‐lived with the vectorised HMW HA, persisting at 24 h with a significant increase of +108% in comparison with untreated condition. The skin suppleness is correlated to an increase of hydration.

FIGURE 4.

Durable enhanced skin suppleness associated with the application of vectorised HMW HA leads to an increase of hydration. The apparent elastic modulus was measured by atomic force microscopy on skin explants 8 and 24 h after applying HMW HA 0.1%, vectorised HMW HA 1% or bentonite 1%, solubilised in water. AFM readouts were normalised relative to the untreated control. Data are represented as mean ± SEM. Statistical significance was measured based on a Mann–Whitney test #p < 0.1, *p < 0.05 and **p < 0.01.

3.4. Enhanced ex vivo skin penetration and greater skin suppleness create a smoothing effect

The enhanced hydration and suppleness in the short‐ to medium‐term could lead to further effects after a longer treatment duration. To assess this, we observed skin explants by SEM after contact with HMW HA or vectorised HMW HA for 4 days (Figure 5).

FIGURE 5.

Durable enhanced skin suppleness associated with the application of vectorised HMW HA leads to a skin smoothing effect. Representative scanning electron micrographs of skin explants treated with HMW HA, vectorised‐HA, or bentonite for 4 days. The control sample was untreated.

Topical application of HMW HA led to slight smoothing, but corneocytes remained visible. In contrast, with vectorised HMW HA, the skin was significantly smoothed and the corneocytes were no longer distinguishable.

No smoothing was observed when skin explants were treated with bentonite alone.

3.5. Clinical evaluation

Having demonstrated improved penetration leading to enhanced suppleness and smoothing in our ex vivo experiments, we moved on to clinical evaluations. For this placebo‐controlled study, we recruited 39 volunteers aged. All volunteers had dry skin on the cheek, and fine lines in the crow's feet area. Volunteers were assigned to two groups—placebo or treatment group—and applied cream twice daily to the whole face for 28 days. The cream formulations were identical apart from the addition of 1% (w/v) vectorised HMW HA in the cream used by the treatment group.

3.6. Analysis of crow's feet

Just 1 h after a single application, the area occupied by crow's feet was significantly reduced in the treatment group, by 18.5% compared to before application. After 6 h, the reduction was slightly and significantly enhanced, reaching 21.5% (Figure 6). No such reduction was observed for the placebo group after a single application. This difference relative to the placebo was significant (p < 0.05).

FIGURE 6.

Application of vectorised HMW HA reduces area of crow's feet. The crow's feet area was imaged on volunteers’ faces before and at several timepoints after applying either a placebo cream or a cream containing vectorised HMW HA. (A) Representative images of a volunteer from the placebo group (upper panel) and a volunteer from the treatment group (lower panel). (B) Area occupied by crow's feet was quantified using ColorFace analysis. Immediate results (left panel)—at 1 and 6 h—and long‐term results (right panel)—at 28 days—are shown. Statistical analyses based on the Wilcoxon test and Mann–Whitney test depending on whether data were paired or unpaired. *p < 0.05, **p < 0.01, and ***p < 0.001.

Prolonged use of the 1% (w/v) vectorised HMW HA formulation for 28 days led to a similar level of smoothing, with a 27.3% significant reduction in the area occupied by crow's feet relative to the situation before treatment. Once again, no effect was observed with the placebo (Figure 6). This difference between the treatment and placebo groups was also significant (p < 0.001). Representative pictures are presented in Figure 7.

FIGURE 7.

Application of vectorised HMW HA reduces area of crow's feet. Representative images for a volunteer from the placebo group (upper panel) and a volunteer from the treatment group (lower panel).

4. DISCUSSION

The aim of this article was to demonstrate the innovative achievement behind the vectorisation process of HMW HA. The experiments described in this paper aimed to assess whether a vectorised form of HMW HA could provide better skin penetration than native HMW HA, and to examine its cosmetic effects. The vectorised form was produced by exposing a mixture of bentonite and HMW HA to shear forces and pressure. After the characterisation of the vectorised form, placebo‐controlled assays involving skin application were conducted both ex vivo and in clinical conditions.

Molecules penetrate the skin using a limited number of mechanisms: transappendageal (through appendages, such as the pilosebaceous unit), transcellular or intercellular. ⁵ , ⁷ , ²⁰ Only the latter pathway is considered realistic for ‘large’ molecules like HA where electrostatic interactions are involved.

For transdermal delivery of large drug molecules, research teams have attained some success using vehicles such as liposomes. ¹² With vehicle‐enhanced penetration, the drug must initially be released from the vehicle before it can partition into the stratum corneum (SC). From there, molecules can diffuse (as a result of the concentration gradient) before partitioning into the viable epidermis. Diffusion occurs once again through the viable epidermis until the molecule finally reaches the dermis, where it can gain access to the bloodstream. ⁷

In our ex vivo assays (Figure 1), skin penetration of HMW HA was improved after vectorisation with clay. Low MW HA is known to penetrate the skin better than HMW presentations. ⁴ We verified the MW profiles of the vectorised and native HMW HA forms to determine whether the vectorisation process had led to the breakdown of the HMW molecules. The MW profiles for the two formulations were found to be identical (Figure 3B). Therefore, the improved penetration observed was not due to altered MW before application.

Alternatively, the MW might have been altered after application. Indeed, products applied to the skin are exposed to enzymes and other factors present in this complex tissue. The vectorisation process could have altered the sensitivity of the HMW HA to metabolisation. Hyaluronidases present in the skin can break down HA applied to the surface, ²¹ thus producing smaller molecules that would penetrate more easily to reach the deeper layers of the dermis. Enzymatic tests demonstrated that the vectorisation process had no effect on the molecule's sensitivity to hyaluronidases compared to the native form (Figure 2).

The only chemical difference between the two forms of HMW HA that we identified was the altered charge state, detected based on the formulations’ zeta potentials (Figure 3A). The zeta potential of hydrogels is known to influence penetration. ²² Previous studies have examined the charge state of bentonite alone, and found that to equilibrate its natural negative charges, the lamellar structure can entrap exchangeable cations, such as calcium, magnesium or sodium. ²³ The vectorisation process associates these cations with HMW HA, allowing it to retain its hyaluronate (negatively charged) form within the lamellar structure. We hypothesise that in this form, the interaction between the product and the skin was strengthened, allowing the HMW HA to better penetrate the upper layers. Moreover, due to opposite charges between the product and pH gradient existing in the epidermis, the negatively charged vectorised HMW HA may have been attracted thanks to electrochemical attraction. ¹² , ²⁴

Other authors have also attempted to vectorise HMW HA (1200 kDa) to enhance its skin penetration, for example by nanoparticulation. ²⁴ Their negatively charged vectorised formulation allowed better skin penetration of HA than HA applied alone. Using fluorescently‐labelled HA molecules, they were able to demonstrate that the transdermal penetration route was altered by the vectorisation process, with native HA penetrating via a transcellular route whereas their nanoparticulate HA penetrated via an intracellular route. They attributed access to this pathway to stronger interactions with cellular membranes. A similar process could occur here with our vectorised HMW HA—the overall negative charge allows the product to bypass the keratinocytes at the surface of the skin to interact more strongly with cells in the upper skin layers, paving the way for enhanced absorption.

After elucidation of mode of skin penetration of our innovative process, we verified the skin benefits brought by a deeper penetration of HMW HA. We demonstrated on skin explants that the vectorised HMW HA was able to hydrate and smooth the skin in a visual manner thanks to SEM. The smoothing effect was then evidenced in a clinical study in which we demonstrated a significant reduction of crow's feet surface after application of the treatment. Indeed, the results were immediate (after 1 and 6 h) and prolonged after 28 days of application. The effect was significant in comparison to the placebo.

5. CONCLUSION

In conclusion, our data indicate that the use of innovative vectorisation process, a HMW HA penetrated the skin, extending the clinical benefits of this iconic cosmetic ingredient.

CONFLICT OF INTEREST STATEMENT

The authors declare not conflict of interest.

ETHICAL STATEMENT

The study was conducted according to the ethical guidelines of the Declaration of Helsinki. This study, performed on cosmetic products within the definition of article L. 5131‐1 of the French Public Health Code, is in accordance with Decree n 2017–884 of 9 May 2017, modifying some regulatory requirements concerning studies involving human participants. All participants provided written informed consent and a photo consent statement before starting the study. Written informed consent has been obtained from the patients to publish this paper.

De Tollenaere M, Meunier M, Lapierre L, et al. High molecular weight hyaluronic acid vectorised with clay provides long‐term hydration and reduces skin brightness. Skin Res Technol. 2024;30:e13672. 10.1111/srt.13672

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.