# Improvement of the rheological properties and performance of liquid crystal emulsions: influence of oil addition and/or high-frequency ultrasound treatment

Amanda Caritá, Hichem Kichou, François-Xavier Legrand, Alexis Verger, Xavier Perse, Laetitia Rapetti, Gislaine Ricci Leonardi, Guillaume Gillet, Emilie Munnier

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## ► To cite this version:

Amanda Caritá, Hichem Kichou, François-Xavier Legrand, Alexis Verger, Xavier Perse, et al. Improvement of the rheological properties and performance of liquid crystal emulsions: influence of oil addition and/or high-frequency ultrasound treatment. International Journal of Pharmaceutics, 2025, 682, pp.125934. 10.1016/j.ijpharm.2025.125934. hal-05146411

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Submitted on 13 Feb 2026

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# Improvement of the rheological properties and performance of liquid crystal emulsions: influence of oil addition and/or high-frequency ultrasound treatment

Amanda C. Caritá<sup>a</sup>, Hichem Kichou<sup>a</sup>, François-Xavier Legrand<sup>b, c</sup>, Alexis Verger<sup>a, c</sup>, Xavier Perse<sup>a</sup>, Laetitia Rapetti<sup>c</sup>, Gislaine Ricci Leonardi<sup>d, e</sup>, Guillaume Gillet<sup>f, d</sup>, Emilie Munnier<sup>a, e, f</sup>

## ARTICLE INFO

### Keywords:

Emulsion

Liquid crystals

High frequency ultrasounds

Rheology

SAXS/WAXS

Ex vivo penetration test

## ABSTRACT

This study investigates the impact of oil addition and processing methods on the physicochemical properties, stability, and skin delivery of vitamin C-loaded cosmetic emulsions containing liquid crystals. The research aims to assess how High-Frequency Ultrasound Treatment (HFUT) and oil incorporation influence particle size, rheology, and bioavailability. Control emulsions and emulsions enriched in vegetable oil were prepared with or without using high-frequency ultrasound treatment and analyzed for particle size distribution, microstructural organization (polarized light microscopy, X-ray scattering), rheological behavior, and stability over time. The ability of these emulsions to protect vitamin C and enhance its skin penetration was evaluated through ex vivo permeation studies using human skin and liquid chromatography analysis. HFUT-treated emulsions exhibited smaller particle sizes, increased viscosity, and improved stability compared to untreated emulsions. Structural analysis confirmed the presence of lamellar liquid crystals, contributing to enhanced formulation stability. Vitamin C retention in the emulsions remained high after prolonged storage. Permeation studies revealed that HFUT emulsions promoted vitamin C retention in the skin, while emulsions that have only been homogenized slightly facilitated deeper penetration into lower skin layers. HFUT treatment enhances the structural integrity and stability of cosmetic emulsions while influencing vitamin C delivery. These findings highlight the potential of process optimization to tailor cosmetic formulations for targeted skin benefits.

## 1. Introduction

Emulsions, recognized for their versatility, find extensive applications in the food, biomedical, and cosmetic fields (Kakran and Antipina, 2014). In cosmetology, emulsions must meet various requirements. Besides their primary role in delivering hydrophilic and lipophilic active ingredients to the skin while protecting them from degradation, emulsions are expected to maintain long-term stability during storage and use. Additionally, properties such as texture, ease of application, and skin feel are crucial considerations to ensure consumer acceptance

(Savic et al., 2011).

Essentially, emulsions are colloidal systems characterized by a liquid phase dispersed in another immiscible phase in the form of fine droplets. For example, the basic composition of a classical oil-in-water (O/W) emulsion includes water as the continuous phase and oil as the dispersed phase. The addition of emulsifiers, typically consisting of amphiphilic molecules (surfactants alone or combined with co-surfactants such as fatty alcohols, fatty acids, or monoglycerides), is usually required to reduce the interfacial tension between the liquids and to overcome the inherent instability of the system (Kouassi et al., 2022). Some specific

<footnote><sup>a</sup> Université de Tours, UPR CNRS 4301 CBM, Département NMNS « NanoMédicaments et NanoSondes », 31 Avenue Monge, 37200 Tours, France</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, 91400 Orsay, France</footnote>

<footnote><sup>c</sup> Alphenyx, 430 Avenue du Maréchal Lattre de Tassigny, 13009 Marseille, France</footnote>

<footnote><sup>d</sup> School of Medical Sciences, Universidade Estadual de Campinas (UNICAMP), SP, Brazil</footnote>

<footnote><sup>e</sup> School of Pharmaceutical Sciences, Universidade Estadual de Campinas (UNICAMP), SP, Brazil</footnote>

<footnote><sup>f</sup> GENIALIS, route d'Achères, 18250 Henrichemont, France</footnote>

<footnote>* Corresponding author.
E-mail address: emilie.munnier@univ-tours.fr (E. Munnier).</footnote>
surfactants and co-surfactant combinations, including mixtures of fatty alcohols and polysorbates, can interact with the continuous phase of the emulsion and spontaneously aggregate to form lyotropic liquid crystals (LCs) during cream manufacturing at temperatures between 50 and 80 °C and at high emulsifier concentrations (Ahmadi et al., 2020).

LCs are a unique state of matter that combines the properties of both the liquid and solid states. That is, these materials combine the structural behavior and rigidity characteristic of solids with the dynamic mobility, disorder, and fluidity observed in isotropic liquids (Müller-Goymann, 2004). The process of LC formation is driven by the hydrophobic effect and is concentration dependent. Besides, different liquid crystalline phases can be formed based on the geometry of the amphiphilic molecule and its critical packing parameter (Souza et al., 2017). For instance, a surfactant molecule with a well-balanced structure, where the cross-sectional area of the polar head group closely matches that of its tail, leads to the formation of a lamellar LC. This thermodynamically stable structure is characterized by parallel arrangements of lipid bilayers, where the hydrophilic head groups are in contact with water, and the hydrophobic tail groups point toward the center of the sheet (Suzuki, 2017). Furthermore, various types of lamellar phases can be encountered based on the state of the hydrophobic chains. The two most common are the Lα phase, where alkyl chains exhibit a liquid-like state, and the Lβ phase (also called lamellar gel phase), where stiff hydrocarbon chains are oriented perpendicular to the lamellar plane, forming axes organized in a hexagonal lattice (Datta et al., 2020).

Due to this unique structure and similarity to the natural lipidic arrangement of the *Stratum Corneum* (SC), lamellar LCs have found widespread application in cosmetic and dermatological formulations (Gosenca et al., 2013). Lamellar LCs can encapsulate a wide range of active ingredients, both hydrophilic and hydrophobic, within their distinct lipid and aqueous domains. This not only improves the chemical stability of the molecules (Caritá et al., 2021; Yang et al., 2023) but also allows for modulation of their delivery (Aytekin et al., 2013; Li et al., 2016). Furthermore, both Lα and Lβ phases entrap a significant amount of water in their interlamellar spaces, which impacts the moisturizing capacity of the system (Savic et al., 2011). For instance, Iwai and co-workers showed that emulsions containing LCs provide a superior moisturizing effect than conventional emulsions (Iwai et al., 1998). More recently, a clinical study demonstrated that emulsions containing lamellar LCs were able to reduce Transepidermal Water Loss (TEWL) and increase skin hydration immediately. This effect is maintained for at least 10 h (Teeranachaidekul et al., 2023).

Over the past few decades, considerable attention has been devoted to understanding how different formulation ingredients affect the formation of LCs. Several research groups have investigated the effects of factors such as surfactant type and concentration, surfactant/fatty acid ratio in the system, co-surfactant composition, emollient type and content, fatty alcohol chain length, presence of NaCl and 1,3-butanediol on LCs formation. Each of these parameters has the potential to influence the type and amount of liquid crystalline phase formed, as well as the thickness of the lamellae and the robustness of the water/lamellae interactions. Variations in the rheological properties of the formulations and the stability of the systems have also been reported (Ahmadi et al., 2020; Jia et al., 2018; Lukic et al., 2013; Savic et al., 2011; Teeranachaidekul et al., 2023; Terescenco et al., 2018a, 2018b).

However, it is well known that the manufacturing process can also affect emulsion characteristics. Key preparation parameters such as mixing energy, dispersion level, homogenization time, and temperature can influence the properties, structure, and stability of emulsions (Realdon et al., 2002). In LC formulations, the self-assembled system depends on a delicate balance of intermolecular forces, and even small changes have the potential to alter or disrupt the supramolecular arrangement of molecules (Souza et al., 2017). The effect of the process on the final properties of emulsions containing LCs has been discussed in the literature. Colafemmina and co-workers studied the effect of different cooling rates on the final structural and rheological properties

of formulations composed of cetyltrimethylammonium chloride, cetearyl alcohol and water. Their findings revealed that rapid cooling (40 °C/min) resulted in the formation of predominantly lamellar structures in the Lβ phase. In contrast, the slow cooling process (5 °C/min) facilitated the development of an alternative bilayer state coexisting with the Lβ phase, inducing changes in the mechanical properties of the samples (Colafemmina et al., 2020).

In addition, the influence of mixing on LCs formation has also been reported. Eccleston described that mixing affects the distribution of surfactants in the molten phase and in multilayers, which is linked to the mechanisms of lamellar arrangement of the system (Eccleston, 1990). Indeed, Wunsch and co-workers demonstrated that in the absence of shear, the lamellar Lβ phase cannot be formed. Instead, fatty alcohol molecules undergo recrystallization and are not incorporated into the lamellar structure (Wunsch et al., 2016). In a study by Cunningham and colleagues, various process parameters, including vane speed, were investigated for their impact on LCs formation. A ternary system composed of cetostearyl alcohol, behenyl trimethyl ammonium chloride and water was chosen. Samples were prepared and processed in a rheometer. It was observed that at the lowest vane speed (50 s⁻¹), the lamellar structure could not be formed, which affected the viscosity of the formulation. Increasing the vane speed to 600 s⁻¹ improved the internal structure of the systems (Cunningham et al., 2021). Conversely, Moraes et al. varied the mixing speed from 300 to 1500 rpm in the preparation of LCs-containing emulsions and reported that formulations prepared at the maximum speed did not exhibit LCs in their structure (Morais et al., 2014). Despite the valuable insights provided by these publications, the current understanding of the intricate relationship between system components and processing variables in the formation of LC structures and their subsequent impact on practical applications is limited. Further studies are needed to unravel these relationships and improve the general knowledge in this field.

In the recent years, the environmental consciousness and ecological awareness have significantly changed consumers' behavior, resulting in an increased demand for safer, more natural, and sustainable products (Pop et al., 2020). In the cosmetic sector in particular, the minimalist trend in formulation has been in the spotlight. This involves a deliberate reduction in the use of chemical additives, such as preservatives, colorants, surfactants, thickeners, and artificial fragrances (Lima et al., 2023). In this sense, the emulsification technique of High-Frequency Ultrasound Technology (HFUT) has attracted attention, especially for the possibility of producing emulsions without surfactants for topical application (Kichou et al., 2023; Kichou et al., 2022).

Fundamentally, ultrasound emulsification is based on the cavitation mechanism. In this process, piezoelectric transducers convert electrical pulses into acoustic energy waves, inducing cavitation – a phenomenon that leads to the formation of gas bubbles. These bubbles expand under the influence of the acoustic waves until they reach a critical size, resulting in their collapse. While this process is particularly violent in low-frequency ultrasound, causing significant temperature to rise in the system and structural destruction, the cavitation phenomenon is more restrained in high-frequency ultrasounds. The increased frequency used in these systems results in smaller bubbles, thus reducing the energy released (Perrin et al., 2022).

In the HFUT, patented by Genialis (N149668A1), the emulsion is premixed and then introduced into a thermostatic high-frequency ultrasound reactor equipped with twelve piezoelectric ceramics vibrating at high frequency under the influence of an electric field (Kaci et al., 2014). The emulsions produced by this method have been evaluated over the years. The studies demonstrate the narrow distribution and good stability of HFUT-treated emulsions (Kaci et al., 2017) as well as the biocompatibility of these systems with human mesenchymal cells (Kaci et al., 2016) and their efficacy in delivering active molecules to the skin (Kichou et al., 2023; Kichou et al., 2022).

If ultrasounds treatment on the organization and rheology of w/o systems has recently explored in the field of food (Mao et al., 2024), this
article presents, to the best of our knowledge, the first use of high-frequency ultrasound emulsification in the preparation of LC-containing O/W emulsions. For this purpose, O/W emulsions consisting of Cetyl alcohol, Polysorbate 60 and water were prepared for the topical delivery of Vitamin C (Vitamin C) – a well-established active ingredient in cosmetic and dermatological preparations (Boo, 2022). This emulsified vehicle, developed in a previous study by Carità and coworkers, exhibited a complex colloidal structure consisting of lamellar LCs in the Lα and Lβ phases and demonstrated high efficacy in protecting Vitamin C from degradation (Carità et al., 2021). Here, the same formulation was first prepared using conventional homogenizer emulsification and HFUT to systematically evaluate the effect of ultrasound on these systems. In addition, apricot kernel vegetable oil was included in the formulations at a high concentration to amplify the effect of the emollient on the system's organization. Although this article focuses on the effects of oil on the structure, rheology, and skin permeation of emulsions, it is important to mention that the addition of oil can have interesting effects on the general efficacy of emulsions. Apricot kernel oil is rich in unsaturated fatty acids, such as oleic and linoleic acid, reported to improve skin hydration and softness. Additionally, it is rich in polyphenols, which can improve the antioxidant capacity of the formulation (Ahmad and Ahsan, 2020; Makrygiannis et al., 2023). Several techniques, namely optical and polarized microscopy, particle size measurement, rheology, small- and wide-angle X-ray diffraction (SAXS and WAXS) were used to describe their properties. The stability of Vitamin C in the formulations was evaluated by high-performance liquid chromatography (HPLC). In addition, a skin permeation study was performed to verify the efficiency of the emulsions in delivering Vitamin C to human skin.

## 2. Materials and methods

### 2.1. Chemicals

Cetyl alcohol and Polysorbate 60 were kindly provided by from AMI Ingredients (Tauxigny, France). L-ascorbic acid was supplied by Cooper (Melun, France), and Apricot kernel (Prunus Armeniaca) vegetable oil was obtained from Herbarom Laboratoires (Aouste-sur-Sye, France). Methanol and Dulbecco's Phosphate Buffer Saline (D-PBS) were purchased from Fisher Scientific (Illkirch-Graffenstaden, France). Sodium metabisulfite was purchased from Alfa Aesar (Haverhill, MA). Ultrapure water Elix® (Merck – Darmstadt, Germany) was used throughout the whole work.

### 2.2. Human skin samples

Excised human skin was obtained from a healthy subject undergoing abdominal surgical resection. 20 mm dermatomed discs were prepared by Alphenyx (Marseille, France) in compliance with the French Public Health Code and European GDPR. All the authorizations were delivered by the French Ministry of Research, with the approval of the French Ethical Committee.

## 2.3. Methods

### 2.3.1. Preparation of emulsions

#### 2.3.1.1. Emulsions prepared by mechanical homogenization.
Control and AP-Turrax emulsions were developed as presented in Table 1. The oily phase of the Control emulsion contained 9 % of Cetyl alcohol and 6 % of Polysorbate 60, while the oily phase of AP-Turrax emulsion was composed of 9 % of Cetyl alcohol, 6 % of Polysorbate 60, and 20 % of Apricot kernel vegetable oil. The aqueous phase consisted of 5 % of Vitamin C and water for both formulations.

The emulsions were prepared by pre-dissolving the Vitamin C in a small amount of water (10 % of the total water content). The remaining aqueous and oily phases were heated separately at 75 ± 5 °C. The aqueous phase was added into the oily phase and the system was homogenized for 3 min at a stirring speed of 10000 rpm using a rotor-stator device Ultra-Turrax® T25 (IKA, Freiburg, Germany). Next, the mixtures were kept under agitation until cooling down to below 40 °C, and then, the Vitamin C was incorporated.

#### 2.3.1.2. Emulsions submitted to high frequency ultrasounds treatment (HFUT).
Control HUFT (C-HUFT) and AP-HFUT emulsions had the same composition as Control and AP-Turrax, respectively, and were prepared as described in the previous section. However, after homogenization by Ultra-Turrax®, the mixtures were transferred to the ultrasonic reactor operating at 1.7 MHz and treated for 30 min, following previous studies on emulsion stabilization with HFUT (Kaci et al., 2016; Kichou et al., 2023). Subsequently, the mixtures were agitated until they cooled to below 40 °C, and at that point, the Vitamin C solution was incorporated.

### 2.3.2. Particle size measurement

Particle size distributions were obtained by flow imaging microscopy using a particle counter, shape, and size analyzer FC200S + HR (Occhio, Angleur, Belgium). A 1:1000 dilution of emulsions prepared in deionized water was passed through the measuring cell while optical images were taken. A 50 µm spacer was used. Before each analysis, a background calibration was performed with ultrapure water. Information on the distribution of particle shape and size for each sample was extracted using Callisto software (Occhio, Angleur, Belgium). The Feret diameter, which is the distance between two parallel lines tangent to the particle shape, was used to determine the maximum diameter of the particles. The average diameter, D10, D50, and D90 were used for the characterization of the emulsions. The D10, D50, and D90 indicate the particle size corresponding to 10 %, 50 %, and 90 % of the analyzed particles, respectively (Kaci et al., 2017). The statistical significance was determined using Student's t-test with a level of significance set for $p \le 0.05$.

### 2.3.3. Optical and polarized microscopy

The microstructure of the emulsions was analyzed using an Axio-scope 5 optical microscope equipped with a camera CMOS Axiocam 305 (Zeiss, Oberkochen, Germany). The emulsions were observed firstly under a bright field and secondly under polarized light (using cross-polarizers) to evaluate the presence of anisotropic phases in the samples. ZEN LE Zeiss software was used for image treatment.

<table><caption>**Table 1**

Composition and method of preparation of the formulated systems.</caption><thead><tr><th>Component</th><th>AP-HFUT</th><th>AP-Turrax</th><th>C-HFUT</th><th>Control</th></tr></thead><tbody><tr><td>Cetyl alcohol</td><td>9</td><td>9</td><td>9</td><td>9</td></tr><tr><td>Polysorbate 60</td><td>6</td><td>6</td><td>6</td><td>6</td></tr><tr><td>Vitamin C</td><td>5</td><td>5</td><td>5</td><td>5</td></tr><tr><td>Apricot kernel oil</td><td>20</td><td>20</td><td>--</td><td>--</td></tr><tr><td>Ultrapure water</td><td>q.s. 100</td><td>q.s. 100</td><td>q.s. 100</td><td>q.s. 100</td></tr><tr><td>Preparation method</td><td>Homogenization + high frequency ultrasounds</td><td>Homogenization</td><td>Homogenization + high frequency ultrasounds</td><td>Homogenization</td></tr></tbody></table>
### 2.3.4. Small- and wide-angle X-ray scattering (SAXS)

X-ray diffraction patterns were acquired using a microfocus X-ray tube (IμS, Incoatec) and selecting the Cu Kα radiation. It was used with an intensity of 1000 μA and a voltage of 50 kV. The incident beam was focused on the detector with multilayer Montel optics and 2D Kratky block collimator. Small-angle (SAXS) and wide-angle (WAXS) X-ray scattering analyses were performed simultaneously using two position-sensitive linear detectors (Vantec-1, Bruker) set perpendicular to the incident beam direction, up to 7° (2θ) and at 19° to 28° (2θ) from it, respectively. The direct beam was stopped with a W-filter. The scattered intensity was reported as a function of the scattering vector $q = 4π sin θ/λ$ where θ is half the scattering angle. The repeat distances *d*, characteristic of the structural arrangements, were given by $q (Å⁻¹) = 2π/d (Å)$. Silver behenate, p-bromobenzoic acid and tristearin (β form) were used as standards to calibrate SAXS and WAXS detectors. All samples were introduced into thin-walled glass capillaries (GLAS, Müller, Berlin, Germany) of 1.5 mm external diameter which were then placed in a specially designed temperature-controlled sample holder (Microcalix, Setaram) maintained at 23 °C during the measurements.

### 2.3.5. Rheology measurements

Continuous and oscillatory measurements were performed with a Kinexus PRO + rotational rheometer (Netzsch-Gäretebau GmbH, Selb, Germany) equipped with a parallel plate geometry (PU40, roughened) with a diameter of 40 mm. All experiments were performed in triplicate at 25.0 ± 0.1 °C. A fixed gap between the geometries was set at 0.6 mm and the excess sample was removed. The rSpace software was used to process the data.

To obtain the flow properties of the emulsions, continuous ramp tests were performed by recording shear stress and viscosity at increasing shear rates ranging from 0.01 to 10000 s⁻¹ (logarithmic mode) for 860 s. The obtained data was fitted to the power law model.

The viscoelastic behavior of the samples was studied using oscillatory measurements. To determine the linear viscoelastic region, the amplitude sweep test was performed at a constant frequency of 1 Hz with increasing strain from 0.1 %-100 % of deformation (logarithmic mode). Once this region was established, a frequency sweep ramp was performed within the linear region from 0.1 to 10 Hz. The storage modulus (G') and loss modulus (G'') were then obtained.

### 2.3.6. Quantification of vitamin C by high-performance liquid chromatography (HPLC)

HPLC analysis was performed on an Ultimate 3000 (Thermo Fisher Scientific, Les Ulis, France) using Chromeleon 7.1 software. The system was equipped with a DAD UV Ultimate 3000 detector and a SUPELCO-SILTM LC-NH₂ column with a particle size of 5 µm and a length of 4.6 × 150 mm (Sigma Aldrich, Saint-Quentin-Fallavier, France). The column temperature was adjusted to 25 °C. Absorbance was recorded at 245 nm for Vitamin C detection. The analysis was performed in isocratic mode using an acetonitrile/water (70:30) mobile phase supplemented with 10 µM phosphoric acid. The injection volume was 10 µL at a flow rate of 1 mL/min for a total run time of 6 min.

To obtain the calibration curve, a stock solution of Vitamin C was prepared by dissolving 40 mg of Vitamin C in 40 g PBS. Serial dilutions were performed to obtain the following standard solutions: 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, and 0.0001 mg/mL. The solutions were then analyzed by HPLC. The observed retention time was 4.3 min for vitamin C. The area under the peak was calculated for each concentration and used for the calibration curve.

### 2.3.7. Stability assays

#### 2.3.7.1. Formulation stability.
To evaluate the stability of the emulsion, particle size distribution, and viscosity were measured three days after emulsion preparation and then after 1 month of storage at 4 °C. The

presence of phase separation on the samples was also visually inspected after 9 months of storage at 4 °C.

#### 2.3.7.2. Vitamin C stability.
The ability of the formulations to protect Vitamin C from degradation over time was evaluated by HPLC. For this purpose, the samples were stored in the dark at 4 °C for 9 months. To perform the experiments, an aliquot of the sample was weighed and diluted with water to the desired concentration for HPLC analysis. HPLC analysis was performed in triplicate. All results are expressed as mean ± SD (n = 3).

### 2.3.8. Skin permeation studies

Skin permeation studies were performed on excised and dermatomed human skin. Each disk of skin had a diameter of 20 mm and an average thickness of 501 ± 20 µm. The skin was stored at -20 °C until the day before the experiment. The tissues exposed to the formulations were obtained from the same donor (Caucasian female donor, 47 years old, phototype 3) to limit biological variability between replicates. The permeation test was conducted using static vertical glass Franz diffusion cells (9-cell setup) with a diffusion area of 0.64 cm² and a receptor volume of 5.0 mL (Permagear Inc, PA, USA).

#### 2.3.8.1. Experimental setup.
The evening before the experiments, human skin samples were removed from the freezer and thawed overnight in a cold room (4 °C). On the day of the assay, they were mounted on Franz diffusion cells. The receptor compartment of each cell was filled with degassed PBS (pH 7.4) containing 0.3 % sodium metabisulfite (added to prevent oxidation of Vitamin C during the experiments). The Receptor Fluid (RF) was maintained at 37 °C with continuous synchronous stirring (500 rpm) using a magnetic stirrer. The skin was equilibrated with the RF for 30 min and the Transepidermal Water Loss (TEWL) was checked with a VapoMeter (Delfin Technologies, Kuopio, Finland). Finally, approximately 0.7 g of formulation was evenly applied to the skin surface in the donor compartment. The experiments were carried out for 24 h. The cells were kept in the dark to avoid degradation of Vitamin C under light.

After this period, the diffusion cells were disassembled and the remaining emulsion in the donor compartment was collected. The skin surface was carefully swabbed with ethanol-soaked cotton swabs to remove any excess formulation. RF was collected and separated for analysis. The Stratum Corneum (SC) was then removed by the tape-stripping method (Kichou et al., 2023). The viable epidermis and dermis were separated by heat treatment in water at 62 °C for 1 min. The dermis was cut into small pieces using a scalpel. Vitamin C was extracted from the SC, epidermis, and dermis with 600 µL of water: methanol 1:1 mixed solvent by means of vortexing for 1 min. All collected samples were analyzed by HPLC. The experiments with C-HFUT and AP-HFUT emulsions were replicated *n* = 3 and the assays with control and AP-Turrax formulations were performed *n* = 2. The experiments were validated by the calculation of the mass balance. All results are reported as mean ± SD. The statistical significance was determined using Student's t-test with a level of significance set for $p \le 0.05$.

## 3. Results and discussion

### 3.1. Emulsion characterization

Particle size measurements were performed using flow image microscopy. The average particle size, D10, D50, and D90 (corresponding to 10, 50, and 90 % of the size of the particles analyzed, respectively) for the Control, C-HUFT, AP-Turrax, and AP-HUFT emulsions are shown in Table 2.

It was observed that the C-HFUT emulsion had a slightly smaller average size (1.183 ± 0.781 µm) compared to the Control sample (1.364 ± 0.755 µm). The same trend was observed for AP-Turrax (average
<caption>**Table 2**</caption>

Average particle size, D10, D25, D50, D75, and D90 of AP-HUF, AP-Turrax, C-HUF and Control formulations, 3 days and 30 days after preparation.

<table><thead><tr><th></th><th></th><th>AP-Turrax</th><th>AP-HFUT</th><th>C-HFUT</th><th>Control</th></tr></thead><tbody><tr><td rowspan="3">Mean[ µm]</td><td>D3</td><td>1.680 ± 1.102</td><td>1.605 ± 1.101</td><td>1.183 ± 0.781</td><td>1.364 ± 0.755</td></tr><tr><td>D30</td><td>1.579 ± 1.037</td><td>1.713 ± 1.178</td><td>1.254 ± 0.781</td><td>1.392 ± 0.814</td></tr><tr><td>D10 [µm]</td><td>0.561</td><td>0.519</td><td>0.519</td><td>0.533</td></tr><tr><td rowspan="3">D25 [µm]</td><td>D3</td><td>0.561</td><td>0.561</td><td>0.562</td><td>0.561</td></tr><tr><td>D30</td><td>0.793</td><td>0.744</td><td>0.636</td><td>0.734</td></tr><tr><td>D30</td><td>0.764</td><td>0.783</td><td>0.76</td><td>0.734</td></tr><tr><td rowspan="3">D50 [µm]</td><td>D3</td><td>1.429</td><td>1.328</td><td>0.939</td><td>1.234</td></tr><tr><td>D30</td><td>1.341</td><td>1.405</td><td>1.31</td><td>1.261</td></tr><tr><td>D75[µm]</td><td>2.225</td><td>2.108</td><td>1.503</td><td>1.807</td></tr><tr><td rowspan="3">D90 [µm]</td><td>D3</td><td>2.057</td><td>2.264</td><td>1.842</td><td>1.838</td></tr><tr><td>D30</td><td>3.244</td><td>3.14</td><td>2.174</td><td>2.39</td></tr><tr><td>D30</td><td>3.011</td><td>3.386</td><td>2.55</td><td>2.39</td></tr></tbody></table>

particle size of 1.68 ± 1.102 µm) and AP-HFUT (1.605 ± 1.101 µm) emulsions. The results were found to be statistically different with $p < 0.05$. These observations, confirmed by the D90 values, indicate that the ultrasonic treatment caused a slight decrease in particle size due to the deformation and breakage of the oil droplets during the propagation of the acoustic waves (Kaci et al., 2014). On the other hand, the addition of oil to the systems led to an increase in particle size for both AP-Turrax and AP-HFUT compared to the Control and AP-HFUT formulations, respectively. The results exhibit differences that are statistically significant with $p < 0.05$.

At the overall level, it can be observed that all emulsions exhibit an adequate particle size for cosmetic applications (Calvo et al., 2020). Besides, the small particle size reported here is also usually correlated with good colloidal stability. To validate this hypothesis and to gain a deeper understanding of the influence of formulation and process on these systems, additional measurements were performed. The results will be presented in the following section.

## 3.2. Influence of formulation and process on the organization of liquid crystals

### 3.2.1. Optical and polarized microscopy

The results of optical and polarized microscopy are displayed in Fig. 1. Optical microscopy of all the developed emulsions shows a classical pattern of oil droplets dispersed in the continuous aqueous phase. However, as can be seen in Fig. 1 micrographs (c) and (a) corresponding to the AP-Turrax and AP-HFUT samples, respectively, the addition of apricot kernel oil resulted in larger droplets compared to the Control (g) and C-HFUT (e) emulsions. This observation reinforces the results obtained by particle size measurements. In addition, the presence of LCs in all samples was confirmed by polarized light microscopy.

The technique of polarized light microscopy records specific birefringence because of the formation of anisotropic structures in a sample. The types of LCs are assigned by observing their structural defects. Typically, the observation of Maltese crosses indicates the presence of lamellar LCs (Müller-Goymann, 2004). These structures are visible in the micrographs (h) and (f) corresponding to Control and C-HFUT emulsions respectively. The addition of oil to the formulations transforms it into a texture commonly referred to as "onion rings" characterized as anisotropic droplets. They can be observed in the respective micrographs of both AP-Turrax (d) and AP-HFUT (b) emulsions. The reminiscent anisotropy found in the surrounding oil droplets also points to a lamellar organization (Pantelic et al., 2015). This fact indicates that the oil incorporation can modify the structure of the formulation but does not destroy the main lamellar pattern of the samples. Similar results have already been reported in the literature (Terescenco et al., 2018a).

Regarding the effect of HFUT on LCs, several points can be highlighted. HFUT did not affect the LCs formation, and the lamellar phase is

preserved in both formulations. However, despite the qualitative information, it is possible to observe that the Maltese crosses are larger in the C-HFUT emulsion than in the Control sample. Considering that the structural defects present in the lamellar phase are very sensitive to shear, it can be suggested that the ultrasonic homogenization created inhomogeneities in the system, resulting in larger Maltese crosses in the C-HFUT formulation. In theory, a larger Maltese cross corresponds to a larger multivesicular structure.

In addition, the AP-HFUT emulsion shows a significant increase in anisotropic structures compared to AP-Turrax. In the literature, these structural changes are usually correlated with an increase in emulsifier/oil concentration in the sample (Lukic et al., 2013; Pantelic et al., 2015). In 2018, Terescenco and colleagues investigated the influence of the alkyl polyglucoside/cetostearyl alcohol ratio on LCs formation. They conducted experiments with a series of solutions that varied in both the alkyl polyglucoside/fatty alcohol ratio and its concentration in water. The authors found a correlation between the presence of Maltese crosses, which correspond to the lamellar phase, and the concentration of the surfactant mixture in the system. Solutions containing more than 2.5 % of the surfactant mixture formed more Maltese crosses and showed better stability (Terescenco et al., 2018c). Hence, it can be postulated that the HFUT treatment led to an expansion of the lamellar LC matrix in the sample, especially in the AP-HFUT emulsion, which could improve the stability of the formulation.

### 3.2.2. SAXS/WAXS measurements

The structure of the emulsions was analyzed at 23 °C using SAXS and WAXS measurements. Fig. 2a shows the plot of q in the small angle region as a function of scattering intensity. All the prepared emulsions present a complex structure that could be described as the superposition of two lamellar patterns. The first one corresponds to the formation of a multilamellar structure described by the presence of Bragg peaks with order 1:2:3, indicated in Fig. 2a as small colored vertical lines. The second arrangement, indicated by the presence of long dotted lines in Fig. 2a, corresponds to the four diffraction orders of the orthorhombic form of hexadecanol (see Fig. S1 for the SAXS pattern of pure cetyl alcohol), as previously reported (Wunsch et al., 2016). The presence of a crystalline fraction of cetyl alcohol may enhance the stability of the prepared emulsions, particularly if some crystals become located at the oil/water interface during recrystallisation. Although this is not a Pickering emulsion in the strict sense of the term, the particles formed during recrystallization upon cooling may contribute to the stabilization of the emulsions, or at the very least, do not compromise their stability. The lamellar repeat distance is of the same order of magnitude for the four emulsions and is comparable to the value previously obtained at 25 °C by Carità et al. (Carità et al., 2021).

High-frequency ultrasound treatment resulted in a slight decrease in the lamellar repeat distance, as the lamellar repeat distance after HFUT decreased from 124 ± 1 Å to 120 ± 4 Å for conventional emulsions and from 125 ± 1 Å to 122 ± 3 Å for emulsions prepared with apricot kernel oil. It should be noted that the addition of apricot kernel oil does not alter the lamellar organization of the system, since the lamellar repeat distances are the same for each type of emulsion (124 ± 1 Å for Control emulsion and 125 ± 1 Å for AP-Turrax; 120 ± 4 Å for C-HFUT and 122 ± 3 Å for AP-HFUT).

The WAXS profiles shown in Fig. 2b revealed the presence of a sharp main peak which position ($q = 1.51 Å^{-1}$ corresponding to a distance of 4.16 Å) is the same regardless of the emulsion studied. This position indicates a tightly packed hexagonal arrangement of hydrocarbon chains and has been previously reported in the literature in systems containing fatty acids (Carità et al., 2021; Savic et al., 2011; Terescenco et al., 2018b; Wunsch et al., 2016). This phase has been referred to as α-gel. Overall, the results show that the use of two different emulsification processes or the incorporation of apricot kernel oil does not alter the WAXS profiles. A minor additional peak was observed at $1.70 Å^{-1}$ in all samples, which could be attributed to the crystalline fraction of cetyl
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Optical micrograph under bright field (a,c,e,g) and polarized light (b,d,f,h) of emulsion: AP-HFUT (a,b), AP-Turrax: (c,d), C-HFUT: (e,f), and Control (g,h).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 2. SAXS (a) and WAXS (b) profiles of the different formulations. AP-HFUT emulsion is represented in grey, AP-Turrax emulsion in yellow, C-HFUT emulsion in green, and Control emulsion in blue. The symbol * corresponds to a signal relating to the setup.</figcaption></figure>

alcohol present (compare with the emulsions prepared with 4.5 % of cetyl alcohol instead of 9 %, using a rotor-stator device; see Fig. S2).

### 3.2.3. Rheology measurements

#### 3.2.3.1. Continuous measurement – Flow behavior.
The rheological behavior of the developed emulsions was first investigated by flow test. The results showed that all formulations exhibited shear thinning pseudoplastic behavior, i.e. a decrease in viscosity with increasing shear rate (Fig. 3). Generally, shear thinning is considered a favorable rheological attribute for topical products since it indicates that the formulation can be spread smoothly on the skin (Marto et al., 2020).

The curves obtained through the flow test can be well described by a

power-law relationship between viscosity ($\eta$) and shear rate ($\gamma$) as defined by the Ostwald equation (Dabbaghi et al., 2021):

$$ \eta = K\dot{\gamma}^{n-1} $$

where K = ‘flow coefficient’ or ‘consistency index’ (Pa.s) and n = ‘power law’ or ‘fluidity index’. The K and n parameters obtained for the four emulsions measured, along with their correlation coefficients, are shown in Table 3.

Table 4 presents the obtained data for apparent viscosity collected at different ranges of shear: 0.01, 0.1, 1, 10, 100, and 1000 s⁻¹ three days after preparation. Notably, these distinct shear rate ranges correspond to typical physical processes employed when utilizing cosmetic emulsions.

<figure><img src="image_6.png" /><figcaption>Fig. 3. Viscosity as a function of shear rate for the different formulations. AP-HFUT emulsion is represented in grey, AP-Turrax emulsion in yellow, C-HFUT emulsion in green, and Control emulsion in blue.</figcaption></figure>
<table><caption>**Table 3**
The rheological power-law parameters obtained from the different formulations.</caption><thead><tr><th>Emulsion</th><th>n</th><th>K (Pa.s)</th><th>Correlation coefficient</th></tr></thead><tbody><tr><td>AP-HFUT</td><td>0.2962</td><td>54.99</td><td>0.9865</td></tr><tr><td>AP-Turrax</td><td>0.2811</td><td>32.97</td><td>0.9934</td></tr><tr><td>C-HFUT</td><td>0.3366</td><td>47.00</td><td>0.9926</td></tr><tr><td>Control</td><td>0.3059</td><td>25.03</td><td>0.9929</td></tr></tbody></table>

<table><caption>**Table 4**
Comparison of viscosity values (in Pa·s) obtained at different shear rates (in s⁻¹) during the flow test of the four emulsions studied on day 3 (D3) and day 30 (D30).</caption><thead><tr><th></th><th></th><th>AP-Turrax</th><th>AP-HFUT</th><th>C-HFUT</th><th>Control</th></tr></thead><tbody><tr><td rowspan="2">η (0.01)</td><td>T0</td><td>5,702</td><td>5,912</td><td>5,583</td><td>3,194</td></tr><tr><td>T30</td><td>1,283</td><td>3,316</td><td>3,631</td><td>1,360</td></tr><tr><td rowspan="2">η (0.1)</td><td>T0</td><td>198.3</td><td>323.7</td><td>279.5</td><td>182.9</td></tr><tr><td>T30</td><td>137.8</td><td>293.3</td><td>170.2</td><td>78.7</td></tr><tr><td rowspan="2">η (1)</td><td>T0</td><td>37.1</td><td>49.5</td><td>42.0</td><td>22.6</td></tr><tr><td>T30</td><td>16.1</td><td>31.5</td><td>24.8</td><td>13.0</td></tr><tr><td rowspan="2">η (10)</td><td>T0</td><td>7.2</td><td>9.8</td><td>9.7</td><td>4.8</td></tr><tr><td>T30</td><td>1.7</td><td>3.6</td><td>4.7</td><td>1.7</td></tr><tr><td rowspan="2">η (100)</td><td>T0</td><td>1.1</td><td>2.4</td><td>2.4</td><td>1.0</td></tr><tr><td>T30</td><td>0.2</td><td>0.3</td><td>0.5</td><td>0.2</td></tr><tr><td rowspan="2">η (1,000)</td><td>T0</td><td>0.3</td><td>0.4</td><td>0.5</td><td>0.2</td></tr><tr><td>T30</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td></tr></tbody></table>

For example, the range of 0.01 to 10 s⁻¹ relates to draining under gravity for emulsions on the skin. The interval of 10 to 100 s⁻¹ corresponds to dipping and pouring procedures, while shear rates around 1000 s⁻¹ are typical for spreading or rubbing applications (Barnes, 1994; Gilbert et al., 2013).

It can be observed that all the formulations exhibit high viscosity at “zero shear” (0.01 s⁻¹), which corresponds to the viscosity of the material at rest. This high viscosity is mainly related to the presence of Cetyl alcohol in the emulsions. Cetyl alcohol, or 1-hexadecanol, is a straight-chain fatty acid with 16 carbon atoms. Its most characteristic property is its ability to increase the viscosity and stability of oil-in-water (O/W) emulsions. As such, this molecule is widely used in the cosmetic and pharmaceutical industries to produce cream-like formulations. This increase in viscosity/stability can be related to the formation of an interfacial film due to interactions with a surfactant or to the formation of the liquid crystalline phase itself (Fukushima and Yamaguchi, 2001). When liquid crystalline lamellar phases are formed in the bulk aqueous continuous phase, these structures tend to increase its viscosity and reduce the mobility of the dispersed phase droplets. This reduction in mobility decreases the potential for droplet interactions and coalescence, reducing the possibility of creaming/sedimentation in the system (Kelm and Wickett, 2017). The effect of the liquid crystalline phase on system viscosity was also demonstrated by Terescenco and colleagues using different ratios of alkyl polyglucoside/cetostearyl alcohol dispersed in aqueous solutions. Increasing the amount of cetostearyl alcohol (which is a mixture of C16/C18 alkyl chains) in the system resulted in the evolution of micellar solutions into LCs, with a significant gain in viscosity (Terescenco et al., 2018b).

Another interesting point observed at “zero shear” is that the addition of oil to the AP-Turrax emulsion resulted in increased viscosity when compared to the Control formulation, despite a slight increase in droplet size (η increased from 3194 Pa·s in the Control to 5702 Pa·s in AP-Turrax). Remarkably, this oil-induced effect is absent in the ultrasound-treated formulations: both C-HFUT and AP-HFUT emulsions exhibit very similar apparent viscosity values (η of 5583 Pa·s for C-HFUT and 5912 Pa·s for AP-HFUT), with both values surpassing the viscosity of the Control emulsion.

At “low shear” (0.1 s⁻¹) it can be observed that both HFUT-treated emulsions have higher viscosity values than the formulations homogenized only with Ultra-Turrax, and the oil effect previously observed on

the viscosity of AP-Turrax is mitigated. Finally, at higher shear rates (100 to 1000 s⁻¹), only the HFUT treatment is effective in increasing viscosity.

In summary, the results highlight two distinct approaches to enhance emulsion viscosity at “zero shear”: the addition of oil through Ultra-Turrax® homogenization and High-Frequency Ultrasound Treatment (HFUT). However, when subjected to higher shear rates, only HFUT treatment contributes to an increase in viscosity. This observation can be attributed to the additional energy input into the system during HFUT treatment. Although further investigations are required to elucidate the impact of high-frequency ultrasound on LCs networks, it has already been reported that increased kinetic energy improves mixing and results in an enhanced internal structure with higher viscosity (Cunningham et al., 2021).

The capacity of HFUT treatment to enhance the viscosity of emulsions without altering the formulation ingredients is particularly interesting. This observation underscores the process-dependent nature of viscosity enhancement, showcasing HFUT treatment as a versatile strategy for increasing emulsion viscosity without the need for additional ingredients such as thickening agents. Such versatility aligns well with the minimalist trend in the cosmetic industry today.

### 3.2.3.2. Oscillatory measurements

#### 3.2.3.2.1. Strain sweep test.
To investigate the viscoelastic behavior of the emulsions, the systems were studied by means of dynamic oscillatory measurements. Fig. 4 shows the evolution of the elastic (G') and viscous (G'') moduli as a function of the oscillation strain. The results show that all the formulations exhibit a viscoelastic solid behavior, with G' higher than G''. This behavior is compatible with the thick texture of the emulsions (Franzol et al., 2021). It is noteworthy that the HFUT treatment significantly increased the G' and G'' moduli by at least three times for both C-HFUT and AP-HFUT compared to the Control and AP-Turrax emulsion, indicating greater resistance to deformation under shear stress and a more elastic-like behavior in these samples.

The strain test also provides information about the resistance to deformation. Fig. 3 shows that the two moduli G' and G'' are nearly parallel up to a certain frequency point (indicated by the lines in the graph). This region is called the Linear Viscoelastic Region (LVR). Beyond this point, the elastic modulus begins to decrease, and the network structure of the sample starts to break down (Tadros, 2013). The results indicate that all formulations have very similar LVR regions. HFUT increased the LVR only slightly (strain limit of 0.15 % for untreated emulsions and 0.25 % for HFTU-treated samples).

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Evolution of G' (represented by solid symbols) and G'' (hollow symbols) as a function of the oscillatory strain of the four emulsions studied. The solid line represents the end of the LVR of the HFUT-treated samples. The dashed line represents the end of the LVR of the emulsions homogenized by Ultra-Turrax only.</figcaption></figure>
Despite having a slightly longer Linear Viscoelastic Region (LVR) and more elastic characteristics, the HFUT-treated samples exhibit a more critical response to deformation, as evidenced by the crossover between the G' and G'' moduli, indicating system destruction. In contrast, the Ultra-Turrax treated samples exhibit greater resistance to flow. Notably, the AP-Turrax emulsion displays no crossover between G' and G''.

When considering the effect of oil on the samples, the most pro-
nounced change is observed in the samples homogenized with Ultra-
Turrax only. The addition of oil does not change G', but there is a sig-
nificant increase in G'' in the AP-Turrax emulsion compared to the
Control formulation, indicating an increase in the viscous component in
this system.

3.2.3.2.2. *Frequency sweep test.* Once the LVR has been defined by a strain sweep, the next step to further evaluate the samples is to perform a frequency sweep test. This analysis provides complementary information about the effect of colloidal forces operating in the system (Franzol et al., 2021). The results obtained are displayed in Fig. 5. Once again, all formulations exhibited a G' higher than the G'' implying a more pro- nounced elastic behavior over the viscous component. No crossover between the moduli was observed over the entire frequency range measured.

In addition, the frequency sweep test can also provide information
about the type of LCs present in the analyzed system. The literature
supports that lamellar LCs show G' parallel to G'', while hexagonal LCs
exhibit a cross point of the two moduli (Terescenco et al., 2018b).
Therefore, the response of all formulations to the frequency sweep test
indicates the presence of lamellar liquid crystals in the system, which is
consistent with the observations made by polarized light microscopy
and X-ray scattering techniques.

3.3. Formulation stability

To determine the stability of the formulations, particle size distri-
bution, and viscosity were measured three days after emulsion prepa-
ration (D3) and then after 1 month of storage (D30) at 4 °C. The stability
of the emulsions was also checked visually, and no phase separation
between the aqueous and oily phases was observed during the entire
duration of the experiments (9 months).

The values of particle size distributions are displayed in Table 2. It is
noteworthy that all formulations had very little variation in particle size,
an indication of good stability.

Regarding the viscosity data, some differences were found in the flow
behavior of the emulsions measured on the days D3 and D30, as pre-
sented in Table 4. The results showed a decrease in the apparent vis-
cosity during the storage time of all emulsions for the whole range of

<figure><img src="image_8.png" /><figcaption>Fig. 5. Frequency sweep test. Evolution of G' (represented by solid symbols) and G'' (hollow symbols) moduli as a function of frequency for the different emulsions (Log2 scale on the x-axis).</figcaption></figure>

shear rates applied. However, it is important to note that this decrease
was more pronounced in the formulations emulsified only by the Ultra-
Turrax® device, i.e. AP-Turrax and Control emulsions. For instance,
considering the viscosities at low shear rate (0.01 s⁻¹), the Control
emulsion presented a viscosity value of 1360 Pa.s at D30, contrary to the
initial value of 3194 Pa.s reported at D3, which represents a loss of 57 %
of its viscosity. On the other hand, the C-HFUT emulsion showed an
apparent viscosity value of 5583 Pa.s at D3 and 3631 Pa.s at D30, cor-
responding to a reduction of 34.97 %. This effect is even more evident
when comparing the formulations containing apricot oil. The AP-Turrax
emulsion showed a 77 % decrease in viscosity (apparent viscosity of
5702 Pa.s at D3 and 1283 Pa.s at D30), while the AP-HFUT emulsion
showed a 44 % viscosity reduction (apparent viscosity of 5912 Pa.s at D3
and 3316 Pa.s at D30). A decrease in viscosity in an emulsion is usually
associated with droplet coalescence and a consequent destabilization
process (Tadros, 2013). Nevertheless, the data presented in this study
did not indicate an increase in droplet size for any of the emulsions
(Table 4). Consequently, the results suggest that the reduction in vis-
cosity could be attributed to weaker interdroplet interactions, and the
HFUT treatment contributed to a more robust network structure,
thereby limiting this process.

3.4. Formulation efficacy

Once the emulsion stability has been assessed, the next crucial step in
evaluating the systems is to study the parameters related to their efficacy
on the skin. In this context, it is essential to ensure good stability of the
active molecule, vitamin C, in the formulations and to check the ability
of the emulsions to promote its delivery to the human skin. Both aspects
are addressed in this section.

3.4.1. Vitamin C stability

Cosmetic products are typically used under aerobic and moisture
conditions for relatively long periods of time. Therefore, to obtain an
effective product, it is essential to ensure good stability of the active
ingredient in the formulation under these conditions (Boo, 2022). In the
case of Vitamin C, this task can be challenging due to its unstable nature.
Vitamin C is easily oxidized in aqueous media in the presence of light,
oxygen, and metal ions. In addition, the rate of decomposition of
Vitamin C is pH-dependent and accelerated by temperature (Caritá
et al., 2020; Pullar et al., 2017; Stamford, 2012). There are several ex-
amples of these limitations in the literature. In 2001, Margolis and Park
published a technical report on the degradation of Vitamin C in auto-
sampler vials over the course of experiments (Margolis and Park, 2001).
Similarly, Golubitskii and co-workers observed that the peak area of a
Vitamin C chromatogram (in an aqueous solution pH 2.0, 0.1 % phos-
phoric acid) decreased by 27 % in 50 min (Golubitskii et al., 2007).
Regarding topical preparations, the half-life of Vitamin C in classical
water-in-oil (W/O) emulsions was estimated to be 20 days and 10 days
at 25 °C and 45 °C, respectively (Kim and Lee, 2018).

In the last decades, several strategies have been employed to mitigate
the degradation of Vitamin C. These approaches include controlling the
presence of oxygen and exposure to light during formulation and stor-
age, utilization of acidic pH (higher stability is obtained at pH ≤ 4),
reducing the water content by using anhydrous/non-aqueous formula-
tions, and the development of encapsulated delivery systems (Martínez-
Valverde et al., 2022; Sheraz et al., 2011; Yang et al., 2023; Zhang et al.,
2020). Additionally, the synergistic use of other antioxidant molecules
in the formulation, such as ferulic acid and vitamin E, has also been used
to prevent the degradation of Vitamin C (Lin et al., 2005).

In a previous work by Caritá and co-workers, the ability of O/W
emulsions containing LCs of mixed cetyl alcohol and polysorbate 60 to
protect Vitamin C against degradation was evaluated by HPLC at
different conditions of temperature (Caritá et al., 2021). The results
showed that the emulsions were stable for at least 4 months at 25 °C
(approximately 90 % of the initial Vitamin C content remained in the
formulations).

In the present study, the samples were stored in the dark at 4 °C for 9 months and the Vitamin C content was measured by HPLC. The remaining Vitamin C in the formulations after this storage period is shown in Fig. 6.

In the AP-HFUT formulation, 92.82 % of the initial Vitamin C was recovered after 9 months of storage, while 78.26 %, 96.72 % and 108.82 % of Vitamin C were obtained from the formulations AP-Turrax, C-HFUT and Control, respectively. In general, these results show that all formulations were able to protect Vitamin C from degradation and that neither the addition of oil nor the emulsification process had a significant effect on Vitamin C stability. These results are consistent with the ones reported by Caritá and co-workers and suggest that the molecular organization of the system plays a key role in Vitamin C protection (Caritá et al., 2021).

One theory to explain this protective effect is the partitioning and diffusion of oxygen between fluid and crystalline bilayers. In 2016, Möller and colleagues reported in a study on hydrated phospholipid bilayers that the oxygen concentration was nearly three times higher in liquid phospholipid bilayers than in the interlamellar aqueous solution. Conversely, a reverse trend was observed for crystalline bilayers below the chain melting temperature. Despite these considerable variations in oxygen partition coefficients, there was no significant impact on the passive diffusion of oxygen (Möller et al., 2016). Based on these findings from the literature, it is proposed that Vitamin C entrapped in the formulation is shielded from oxygen contact due to the presence of crystalline α-gel bilayers in the system, as confirmed by the WAXS measurements.

### 3.4.2. Skin permeation studies

Vitamin C is a small molecule (molecular weight of 176.12 g/mol), yet its skin permeation is very limited by its dissociation constant ($pK_a = 4.25$) and its high hydrophilic character ($\log P_{(o/w)} = -1.85$) (Iliopoulos et al., 2019; Liston et al., 2022). It is well reported in the literature that hydrophilic compounds suffer inefficient skin penetration due to the barrier function exerted by the SC, the outermost layer of the epidermis (Elmowafy, 2021). The SC consists mainly of dead, flattened

corneocytes, a tightly organized extracellular lipid matrix with a low water content of approximately 15–30 %. This intricate structure prevents most hydrophilic and large molecular weight (>500 Da) compounds from penetrating the intact skin layer (Ho et al., 2023; Zhang et al., 2023).

Vitamin C has many biological functions when topically applied. Vitamin C is expected to attenuate and prevent skin aging by reducing oxidative stress caused by internal and external factors such as solar radiation, smoke, and pollution. Studies have also demonstrated the effectiveness of Vitamin C in promoting collagen by directly stimulating collagen gene expression and by acting as a cofactor for proline and lysine hydroxylases, which stabilize the tertiary structure of collagen molecules. Vitamin C is also used to treat hyperpigmentation, melasma, and sunspots. This activity is related to its ability to interfere with the active site of tyrosinase, the enzyme responsible for melanogenesis. In addition, Vitamin C promotes cell differentiation of keratinocytes, improves dermal-epidermal cohesion, and acts on epigenetic mechanisms capable of preventing the onset of malignancies such as melanoma (Boo, 2022; Caritá et al., 2020; Pullar et al., 2017). However, these activities are directly dependent on the proper delivery of Vitamin C to the distinct layers of the skin.

To evaluate the capacity of the developed formulations to promote an adequate delivery of Vitamin C to the skin and to assess the impact of oil addition and preparation methods on the Vitamin C release, an *ex vivo* skin permeation study was conducted using Franz diffusion cells in conjunction with the tape-stripping method. Human skin from a single donor was chosen as the membrane model to limit the biological variability. Results were obtained after 24 h of exposure to the different formulations. The distribution of Vitamin C in the SC, Epidermis (Ep) + Dermis (D), and Receptor Fluid (RF) is shown in Fig. 7, along with the total amount of Vitamin C that permeated or was retained in the skin (Qabs). Results are presented in percentages relative to the applied quantity of Vitamin C.

The overall results indicate that all emulsions are effective in delivering Vitamin C to different layers of the skin, with no statistically significant differences between formulations. However, upon closer examination, some trends emerge. Emulsions containing oil, namely AP-Turrax and AP-HFUT, have smaller error bars compared to the Control and C-HFUT emulsions. This suggests that the inclusion of oil in the formulations contributes to more uniform permeation, which may be related to improved wetting properties. Although contact angle measurements were not performed to evaluate this parameter, the literature reports that changes in the oil phase of emulsions can indeed affect wetting parameters and consequently spreadability (Douguet et al., 2017; Eudier et al., 2019).

Among the formulations tested, the Control emulsion appears to be the most effective in delivering vitamin C to all skin layers and the RF. C-HFUT provides similar results to the Control emulsion on the SC, D + Ep, but a lower amount of vitamin C is retrieved from the RF. Therefore, C-HFUT emulsion should be preferred when a more superficial action of Vitamin C is desired, such as antioxidant and photoprotective activities, while the Control emulsion may be a more interesting alternative to deliver Vitamin C to the deepest layers of the skin, for example, to promote collagen synthesis (Caritá et al., 2020; Pullar et al., 2017). It is also worth noting that the human skin used in this study underwent dermatomization, resulting in the removal of the lower region of the dermis and the absence of subcutaneous tissue. Consequently, it is reasonable to infer that a significant portion of the recovered Vitamin C in the RF could be retained in the dermis under real-life conditions.

Considering oil-containing formulations, both AP-Turrax and AP-HFUT emulsions exhibit comparable permeation profiles at the SC, D + Ep, with Vitamin C permeation values lower than those observed for the Control and C-HFUT formulations. However, the AP-Turrax emulsion promoted greater vitamin C permeation to the RF compared to AP-HFUT, consistent with the results observed for the Control and C-HFUT formulations. In summary, the data suggest that HFUT treatment

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Amount of Vitamin C (%) recovered from AP-HFUT, AP-Turrax, C-HFUT and Control emulsions after 9 months of storage at 4 °C.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 7. Vit C permeation and retention in human skin after 24 h of experiment, expressed as the percentage of the applied dose. AP-HFUT emulsion is represented in grey, AP-Turrax emulsion in yellow, C-HFUT emulsion in green, and Control emulsion in blue. Abbreviations: (SC) *stratum corneum*, (Ep) Epidermis, (D) Dermis, (RF) receptor fluid. "Total" corresponds to the cumulated amount of Vitamin C recovered in SC + Ep + D + RF.</figcaption></figure>

promotes increased vitamin C retention in the skin, while Turrax emulsions enhance vitamin C delivery to deeper tissue layers.

## 4. Conclusion

The study highlights the distinct effects of oil integration and High-Frequency Ultrasound Treatment (HFUT) on the structure, stability, and rheological properties of a liquid crystal emulsion containing vitamin C. The incorporation of apricot kernel oil resulted in an increase in particle size, as confirmed by both flow image microscopy and optical microscopy. This increase was accompanied by a transformation of the lamellar liquid crystal structures into anisotropic droplets, commonly known as "onion rings," which indicate a preserved but modified lamellar arrangement. The addition of oil enhanced the viscosity of untreated emulsions at low shear rates, suggesting improved thickening effects. However, during storage, emulsions containing oil exhibited greater viscosity loss compared to their non-oil counterparts, which could imply weaker interdroplet interactions.

High frequency ultrasound treatment induced a slight reduction in particle size, likely due to the breakage of oil droplets under ultrasonic energy. Importantly, it contributed to a more structured lamellar liquid crystal matrix, as seen in the increased presence of Maltese crosses in polarized microscopy images. This structural reinforcement led to enhanced viscosity and viscoelastic properties, particularly under high shear conditions, making HFUT emulsions more resistant to flow. Additionally, HFUT improved the long-term viscosity retention of emulsions, reducing the extent of viscosity loss over time and indicating a more robust network structure.

When combined, oil integration and HFUT resulted in emulsions with improved structural integrity and stability. The AP-HFUT emulsion exhibited a more pronounced anisotropic liquid crystals organization, suggesting an expansion of the lamellar matrix, which could enhance formulation stability. While oil increased viscosity at low shear in untreated emulsions, HFUT emulsions maintained higher viscosity across all shear rates. Furthermore, the HFUT formulations promoted greater retention of Vitamin C within the skin, while oil-containing emulsions led to more permeation. This indicates that HFUT enhances emulsion stability and skin retention of active ingredients, while oil modifies structural organization without compromising LC integrity. In conclusion, while oil integration alters the structural and rheological

properties of emulsions, HFUT treatment provides significant advantages in terms of particle size reduction, viscosity enhancement, and long-term stability. The combination of both factors results in optimized emulsions with potential applications in cosmetic formulations, offering improved textural properties and controlled delivery of active compounds.

### CRediT authorship contribution statement

**Amanda C. Carità:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Hichem Kichou:** Writing – original draft, Methodology, Investigation, Formal analysis. **François-Xavier Legrand:** Writing – review & editing, Investigation, Formal analysis. **Alexis Verger:** Writing – original draft, Investigation, Formal analysis, Data curation. **Xavier Perse:** Investigation. **Laetitia Rapetti:** Writing – review & editing, Methodology.
**Gislaine Ricci Leonardi:** Writing – review & editing, Conceptualization. **Guillaume Gillet:** Writing – review & editing, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.
**Emilie Munnier:** Writing – review & editing, Validation, Supervision, Methodology, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Emilie Munnier reports financial support was provided by Centre-Val de Loire Region. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

This research was funded by the Conseil Régional Centre-Val de Loire (Project ARD CVL MINIONs 2020-00141275 and UFO 2020-00138875).

The authors thank Pr Mohammed Boussafir from UR 6293 GéHCO, Université de Tours, for the technical assistance for polarized microscopy.
# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2025.125934.

# Data availability

Data will be made available on request.

# References

Ahmad, A., Ahsan, H., 2020. Lipid-based formulations in cosmeceuticals and biopharmaceuticals. Biomed. Dermatol. 4, 1–10. https://doi.org/10.1186/s41702-020-00062-9.

Ahmadi, D., Mahmoudi, N., Heenan, R.K., Barlow, D.J., Lawrence, M.J., 2020. The influence of co-surfactants on lamellar liquid crystal structures formed in creams. Pharmaceutics 12, 864. https://doi.org/10.3390/pharmaceutics12090864.

Aytekin, M., Gursoy, R.N., Ide, S., Soylu, E.H., Hekimoglu, S., 2013. Formulation and characterization of liquid crystal systems containing azelaic acid for topical delivery. Drug Dev. Ind. Pharm. 39, 228–239. https://doi.org/10.3109/03639045.2012.671829.

Barnes, H.A., 1994. Rheology of emulsions—a review. Colloids Surf. A: Physicochem. Eng 91, 89–95. https://doi.org/10.1016/0927-7757(93)02719-0.

Boo, Y.C., 2002. Ascorbic acid (vitamin C) as a cosmeceutical to increase dermal collagen for skin antiaging purposes: emerging combination therapies. Antioxidants 11, 1663. https://doi.org/10.3390/antiox11091663.

Calvo, F., Gomez, J.M., Ricardez-Sandoval, L., Alvarez, O., 2020. Integrated design of emulsified cosmetic products: a review. Chem. Eng. Res. Des. 161, 279–303. https://doi.org/10.1016/j.chedr.2020.07.014.

Caritá, A.C., Azevedo, J.R., Buri, M.V., Bolzinger, M.A., Chevalier, Y., Riske, K.A., Leonardi, G.R., 2021. Stabilization of vitamin C in emulsions of liquid crystalline structures. Int. J. Pharm. 592, 120092. https://doi.org/10.1016/j.ijpharm.2020.120092.

Caritá, A.C., Fonseca-Santos, B., Shultz, J.D., Michniak-Kohn, B., Chorilli, M., Leonardi, G.R., 2020. Vitamin C: one compound, several uses. Advances for delivery, efficiency and stability. Nanomedicine. https://doi.org/10.1016/j.nano.2019.102117.

Colafemmina, G., Palazzo, G., Mateos, H., Amin, S., Fameau, A.L., Olsson, U., Gentile, L., 2020. The cooling process effect on the bilayer phase state of the CTAC/cetearyl alcohol/water surfactant gel. Colloids Surf. A: Physicochem. Eng 597, 124821. https://doi.org/10.1016/j.colsurfa.2020.124821.

Cunningham, G.E., Alberini, F., Simmons, M.J., O'sullivan, J.J., 2021. Understanding the effects of processing conditions on the formation of lamellar gel networks using a rheological approach. Chem. Eng. Sci. 242, 116752. https://doi.org/10.1016/j.ces.2021.116752.

Dabbaghi, M., Namjoshi, S., Panchal, B., Grice, J.E., Prakash, S., Roberts, M.S., Mohammed, Y., 2021. Viscoelastic and deformation characteristics of structurally different commercial topical systems. Pharmaceutics 13, 1351. https://doi.org/10.3390/pharmaceutics13091351.

Datta, A., Tanmay, V.S., Tan, G.X., Reynolds, G.W., Jamadagni, S.N., Larson, R.G., 2020. Characterizing the rheology, slip, and velocity profiles of lamellar gel networks. J. Rheol. 64, 851–862. https://doi.org/10.1122/8.0000011.

Douguet, M., Picard, C., Savary, G., Merlaud, F., Loubat-Bouleuc, N., Grisel, M., 2017. Spreading properties of cosmetic emollients: use of synthetic skin surface to elucidate structural effect. Colloids Surf. B.154, 307–314. https://doi.org/10.1016/j.colsurf.2017.03.028.

Eccleston, G.M., 1990. Multiple-phase oil-in-water emulsions. J. Soc. Cosmet. Chem. 41, 1–22.

Elmowafy, M., 2021. Skin penetration/permeation success determinants of nanocarriers: pursuit of a perfect formulation. Colloids Surf. B Biointerfaces 203, 111748. https://doi.org/10.1016/j.colsurf.2021.111748.

Eudier, F., Savary, G., Grisel, M., Picard, C., 2019. Skin surface physico-chemistry: characteristics, methods of measurement, influencing factors and future developments. Adv. Colloid Interface Sci. 264, 11–27. https://doi.org/10.1016/j.cis.2018.12.002.

Franzol, A., Banin, T.M., Brazil, T.R., Rezende, M.C., 2021. Assessment of kinetic stability of cosmetic emulsions formulated with different emulsifiers using rheological and sensory analyses. J Solgel Sci Technol 99, 469–481. https://doi.org/10.1007/s10971-021-05587-x.

Fukushima, S., Yamaguchi, M., 2001. Physical chemistry of cetyl alcohol: occurrence and function of liquid crystals in o/w creams. In: Matijević, E. (Ed.), Surface and Colloid Science. Springer, Boston, pp. 1–98. https://doi.org/10.1021/ja015272w.

Gilbert, L., Picard, C., Savary, G., Grisel, M., 2013. Rheological and textural characterization of cosmetic emulsions containing natural and synthetic polymers: relationships between both data. Colloids Surf A Physicochem Eng Asp 421, 150–163. https://doi.org/10.1016/j.colsurfa.2013.01.003.

Golubitskii, G.B., Budko, E.V., Basova, E.M., Kostarnoi, A.V., Ivanov, V.M., 2007. Stability of ascorbic acid in aqueous and aqueous-organic solutions for quantitative determination. J. Anal. Chem. 62, 742–747. https://doi.org/10.1134/s1061934807080096.

Gosencà, M., Bester-Rogać, M., Gašperlin, M., 2013. Lecithin based lamellar liquid crystals as a physiologically acceptable dermal delivery system for ascorbyl

palmitate. Eur. J. Pharm. Sci. 50, 114–122. https://doi.org/10.1016/j.ejps.2013.04.029.

Ho, M.J., Park, D.W., Kang, M.J., 2023. Design of novel tricaprylin-incorporated multi-layered liposomal system for skin delivery of ascorbic acid with improved chemical stability. Pharmaceuticals 16, 121. https://doi.org/10.3390/ph16101012.

Iliopoulos, F., Sil, B.C., Moore, D.J., Lucas, R.A., Lane, M.E., 2019. 3-O-ethyl-l-ascorbic acid: characterisation and investigation of single solvent systems for delivery to the skin. Int. J. Pharm. X 1, 100025. https://doi.org/10.1016/j.ijpx.2019.100025.

Iwai, H., Fukasawa, J., Suzuki, T., 1998. A liquid crystal application in skin care cosmetics. Int. J. Cosmet. Sci. 20, 87–102. https://doi.org/10.1046/j.1467-2494.1998.171741.x.

Jia, B., Zhang, Q., Zhang, Z., Chen, M., Zhang, W., 2018. Preparation of liquid crystal emulsion and its application performance study. J. Dispers. Sci. Technol. 39, 100–105. https://doi.org/10.1080/01932691.2017.1297721.

Kaci, M., Arab-Tehrany, E., Desjardins, I., Banon-Desobry, S., Desobry, S., 2017. Emulsifier free emulsion: comparative study between a new high frequency ultrasound process and standard emulsification processes. J. Food Eng. 194, 109–118. https://doi.org/10.1016/j.jfoodeng.2016.09.006.

Kaci, M., Arab-Tehrany, E., Dostert, G., Desjardins, I., Velot, E., Desobry, S., 2016. Efficiency of emulsifier-free emulsions and emulsions containing rapeseed lecithin as delivery systems for vectorization and release of coenzyme Q10: physico-chemical properties and in vitro evaluation. Colloids Surf B. 147, 142–150. https://doi.org/10.1016/j.colsurfB.2016.07.036.

Kaci, M., Meziani, S., Arab-Tehrany, E., Gillet, G., Desjardins-Lavisse, I., Desobry, S., 2014. Emulsification by high frequency ultrasound using piezoelectric transducer: formation and stability of emulsifier free emulsion. Ultrason. Sonochem. 21, 1010–1017. https://doi.org/10.1016/j.ultonsch.2013.11.006.

Kakran, M., Antipina, M.N., 2014. Emulsion-based techniques for encapsulation in biomedicine, food and personal care. Curr. Opin. Pharmacol. 18, 47–55. https://doi.org/10.1016/j.coph.2014.09.003.

Kelm, G.R., Wickett, R.R., 2017. The role of fatty acids in cosmetic technology. In: Ahmad, M.U. (Ed.), Fatty Acids. AOCs Press, pp. 385–404. https://doi.org/10.1016/B978-0-12-809521-8.00012-X.

Kichou, H., Caritá, A.C., Gillet, G., Bougassaa, L., Perse, X., Soucé, M., Gressin, L., Chourpa, I., Bonnier, F., Munnier, E., 2023. Efficiency of emulsifier-free emulsions in delivering caffeine and α-tocopherol to human skin. Int. J. Cosmet. Sci. 45, 329–344. https://doi.org/10.1111/ics.12837.

Kichou, H., Dancik, Y., Eklouh-Molinier, C., Huang, N., Soucé, M., Gressin, L., Gillet, G., Chourpa, I., Munnier, E., Bonnier, F., 2022. Highlighting the efficiency of ultrasound-based emulsifier-free emulsions to penetrate reconstructed human skin. Int. J. Cosmet. Sci. 44, 262–270. https://doi.org/10.1111/ics.12772.

Kim, S., Lee, T.G., 2018. Stabilization of L-ascorbic acid in cosmetic emulsions. J. Ind. Eng. Chem. 57, 193–198. https://doi.org/10.1016/j.jiec.2017.08.023.

Kouassi, M.C., Grisel, M., Gore, E., 2022. Multifunctional active ingredient-based delivery systems for skincare formulations: a review. Colloids Surf. B, 112676. https://doi.org/10.1016/j.colsurfB.2022.112676.

Li, Y., Dong, C., Cun, D., Liu, J., Xiang, R., Fang, L., 2016. Lamellar liquid crystal improves the skin retention of 3-O-ethyl-ascorbic acid and potassium 4-methoxysalicylate in vitro and in vivo for topical preparation. AAPS PharmSciTech 17, 767–777. https://doi.org/10.1208/s12249-015-0353-6.

Lima, L.L., Bispo-dos-Santos, K., Trevisan, I.M.C., Rapóso, C., Velho, P.E.N.F., Bagatin, E., Rezende, R.A., Silva, J.V.L., I, G.R., 2023. Developing botanical formulations for sustainable cosmetics. Cosmetics 10, 159. https://doi.org/10.3390/cosmetics10060159.

Lin, F.H., Lin, J.Y., Gupta, R.D., Tournas, J.A., Burch, J.A., Selim, M.A., Monteiro-Riviere, N.A., Grichnik, J.M., Zielinski, J., Pinnell, S.R., 2005. Ferulic acid stabilizes a solution of vitamins C and E and doubles its photoprotection of skin. J. Invest. Dermatol. 125, 826–832. https://doi.org/10.1111/j.0022-202X.2005.23768.x.

Liton, L.S., Rivas, P.L., Sakdiset, P., See, G.L., Arce, F., 2022. Chemical permeation enhancers for topically-applied vitamin C and its derivatives: a systematic review. Cosmetics 9, 85. https://doi.org/10.3390/cosmetics9040085.

Lukic, M., Pantelic, I., Daniels, R., Müller-Goymann, C., Savic, M., Savic, S., 2013. Moisturizing emulsion systems based on the novel long-chain alkyl polyglucoside emulsifier: the contribution of thermoanalytical methods to the formulation development. J. Therm. Anal. Calorim. 111, 2045–2057. https://doi.org/10.1007/s10973-012-2263-0.

Makrygiannis, I., Athanasiadis, V., Chatzimitakos, T., Bozinou, E., Mantzourani, C., Chatzilazarou, A., Makris, D.P., Lalas, S.I., 2023. Exploring the chemical composition and antioxidant properties of apricot kernel oil. Separations 10, 332. https://doi.org/10.3390/separations10060332.

Mao, J., Gao, Y., Meng, Z., 2024. Regulation of fat crystals in water-in-oil emulsions by high-intensity ultrasound: crystal size and tracing of droplet distribution. Food Res. Int. 188, 114493. https://doi.org/10.1016/j.foodres.2024.114493.

Margolis, S.A., Park, E., 2001. Stability of ascorbic acid in solutions stored in autosampler vials. Clin. Chem. 47, 1463–1464. https://doi.org/10.1093/clinchem/47.8.1463.

Martínez-Valverde, T., Crespo, N., Suner, E., 2022. Open-Label study to evaluate the efficacy of a topical anhydrous formulation with 15% pure ascorbic acid and ginger as a potent antioxidant. Cosmetics 9, 74. https://doi.org/10.3390/cosmetics9040074.

Martó, J., Nunes, A., Martins, A.M., Carvalheira, J., Prazeres, P., Gonçalves, L., Marques, A., Lucas, A., Ribeiro, H.M., 2020. Pickering emulsions stabilized by calcium carbonate particles: a new topical formulation. Cosmetics 7, 62. https://doi.org/10.3390/cosmetics7030062.

Möller, M.N., Li, Q., Chinnaraj, M., Cheung, H.C., Lancaster, J.R., Denicola, A., 2016. Solubility and diffusion of oxygen in phospholipid membranes. Biochimica et
Biophys. Acta (BBA) – Biomembr. 1858, 2923–2930. https://doi.org/10.1016/j.bbamem.2016.09.003.

Morais, G.G., Oliveira, W.P., Rocha-Filho, P.A., 2014. Influence of mixing speed in liquid crystal formation and rheology of O/W emulsions containing vegetable oils. J. Dispers. Sci. Technol. 35, 1551–1556. https://doi.org/10.1080/01932691.2013.868315.

Müller-Goymann, C.C., 2004. Physicochemical characterization of colloidal drug delivery systems such as reverse micelles, vesicles, liquid crystals and nanoparticles for topical administration. Eur. J. Pharm. Biopharm. 58. https://doi.org/10.1016/j.ejbp.2004.03.028.

Pantelic, I., Milic, J., Vuleta, G., Dragicevic, N., Savic, S., 2015. Natural emulsifiers of the alkyl polyglucoside type and their influence on the permeation of drugs. In: Dragicevic, N., Maibach, H.I. (Eds.), Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement: Modification of the Stratum Corneum. Springer-Verlag, Berlin, pp. 231–250. https://doi.org/10.1007/978-3-662-47039-8_14.

Perrin, L., Desobry-Banon, S., Gillet, G., Desobry, S., 2022. Review of high-frequency ultrasounds emulsification methods and oil/water interfacial organization in absence of any kind of stabilizer. Foods 11, 2194. https://doi.org/10.3390/foods11152194.

Pop, R.A., Säpläcan, Z., Alt, M.A., 2020. Social media goes green—The impact of social media on green cosmetics purchase motivation and intention. Information 11, 447. https://doi.org/10.3390/info11090447.

Pullar, J.M., Carr, A.C., Vissers, M., 2017. The roles of vitamin C in skin health. Nutrients 9, 866. https://doi.org/10.3390/nu9080866.

Realdon, N., Perin, F., Morpurgo, M., Ragazzi, E., 2002. Influence of processing conditions in the manufacture of O/W creams: I. Effect on dispersion grade and rheological characteristics. Il Farmaco 57, 341–347. https://doi.org/10.1016/S0014-827X(02)01213-2.

Savic, S., Lukic, M., Jaksic, I., Reichl, S., Tamburic, S., Müller-Goymann, C., 2011. An alkyl polyglucoside-mixed emulsifier as stabilizer of emulsion systems: the influence of colloidal structure on emulsions skin hydration potential. J. Colloid Interface Sci. 358, 182–191. https://doi.org/10.1016/j.jcis.2011.02.049.

Sheraz, M.A., Ahmed, S., Ahmad, I., Shaikh, R.H., Vaid, F.H.M., Iqbal, K., 2011. Formulation and stability of ascorbic acid in topical preparations. Surg. Neurol. Int. 2. https://doi.org/10.4103/0975-8453.86296.

Souza, J.F., Pontes, K.D.S., Alves, T.F., Amaral, V.A., Rebelo, M.D.A., Hausen, M.A., Chaud, M.V., 2017. Spotlight on biomimetic systems based on lyotropic liquid crystal. Molecules 22, 419. https://doi.org/10.3390/molecules22030419.

Stamford, N.P., 2012. Stability, transdermal penetration, and cutaneous effects of ascorbic acid and its derivatives. J. Cosmet. Dermatol. 11, 310–317. https://doi.org/10.1111/jocd.12006.

Suzuki, T., 2017. Effect of molecular assembly for emulsion and gel formulations. In: Sakamoto, K., Lochhead, R.Y., Maibach, H.I., Yamashita, Y. (Eds.), Cosmetic Science and Technology: Theoretical Principles and Applications. Elsevier, Amsterdam, Oxford, Cambridge, pp. 519–538. https://doi.org/10.1016/B978-0-12-802005-0.00030-6.

Tadros, T.F., 2013. Use of Rheological Measurements for Assessment and Prediction of the Long-Term Assessment of Creaming and Sedimentation. In: Bröckel, I.U., Meier, W., Wagner, I.G. (Eds.), Product Design and Engineering: Formulation of Gels and Pastes. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 95–120. https://doi.org/10.1002/9783527654741.ch4.

Teeranachaidekul, V., Soontaranon, S., Sukhasem, S., Chantasart, D., Wongrakpanich, A., 2023. Influence of the emulsifier on nanostructure and clinical application of liquid crystalline emulsions. Sci. Rep. 13, 4185. https://doi.org/10.1038/s41598-023-31329-w.

Terescenco, D., Picard, C., Clemenceau, F., Grisel, M., Savary, G., 2018a. A. Influence of the emollient structure on the properties of cosmetic emulsion containing lamellar liquid crystals. Colloids Surf. A: Physicochem. Eng 536, 10–19. https://doi.org/10.1016/j.colsurfa.2017.08.017.

Terescenco, D., Savary, G., Clemenceau, F., Merat, E., Duchemin, B., Grisel, M., Picard, C., 2018b. The alkyl polyglucoside/fatty alcohol ratio effect on the formation of liquid crystal phases in binary systems. J. Mol. Liq. 253, 45–52. https://doi.org/10.1016/j.molliq.2017.12.149.

Terescenco, D., Savary, G., Picard, C., Clemenceau, F., Merat, E., Grisel, M., 2018c. Influence of the emollient on emulsions containing lamellar liquid crystals: from molecular organization towards applicative properties. Int. J. Cosmet. Sci. 40, 565–574. https://doi.org/10.1111/ics.12498.

Wunsch, K., Relkin, P., Cuvelier, G., Clement, F., Nicolas-Morgantini, L., Benkhelifa, H., Flick, D., 2016. Effect of surfactant on structure thermal behavior of cetyl stearyl alcohols. J. Therm. Anal. Calorim. 123, 1411–1417. https://doi.org/10.1007/s10973-015-5074-2.

Yang, Y., Yan, S., Yu, B., Gao, C., Chang, K., Wang, J., 2023. Lamellar gel containing emulsions as an effective carrier for stabilization and transdermal delivery of retinyl propionate. Colloids Surf. A: Physicochem. Eng. 131834. https://doi.org/10.1016/j.colsurfa.2023.131834.

Zhang, M., Sun, R., Xia, Q., 2020. An ascorbic acid delivery system based on (W1/O/W2) double emulsions encapsulated by Ca-alginate hydrogel beads. J Drug Deliv Sci Technol 60, 101929. https://doi.org/10.1016/j.jddst.2020.101929.

Zhang, Y., Pan, W., Wang, D., Wang, H., Hou, Y., Zou, M., Piao, H., 2023. Solid-in-oil nanodispersion as a novel topical transdermal delivery to enhance stability and skin permeation and retention of hydrophilic drugs l-ascorbic acid. Eur. J. Pharm. Biopharm. 185, 82–93. https://doi.org/10.1016/j.ejpb.2023.02.004.