Confocal Raman spectroscopy coupled with in vitro permeation testing to study the effects of formalin fixation on the skin barrier function of reconstructed human epidermis.

Confocal Raman Spectroscopy is recognised as a potent tool for molecular characterisation of biological specimens. There is a growing demand for In Vitro Permeation Tests (IVPT) in the pharmaceutical and cosmetic areas, increasingly conducted using Reconstructed Human Epidermis (RHE) skin models. In this study, chemical fixation of RHE in 10 % Neutral Buffered Formalin for 24 h has been examined for storing RHE samples at 4 °C for up to 21 days. Confocal Raman Spectroscopy (CRS), combined with Principal Components Analysis, revealed the molecular-level effects of fixation, notably in protein and lipid conformation within the stratum corneum and viable epidermis. IVPT by means of high-performance liquid chromatography, using caffeine as a model compound, showed minimal impact of formalin fixation on the cumulative amount, flux, and permeability coefficient after 12 h. While the biochemical architecture is altered, the function of the model as a barrier to maintain rate-limiting diffusion of active molecules within skin layers remains intact. This study opens avenues for enhanced flexibility and utility in skin model research, promising insights into mitigating the limited shelf life of RHE models by preserving performance in fixed samples for up to 21 days.

Strat-M® positioning for skin permeation studies: A comparative study including EpiSkin® RHE, and human skin.

In the development and optimization of dermatological products, In Vitro Permeation Testing (IVPT) is pivotal for controlled study of skin penetration. To enhance standardization and replicate human skin properties reconstructed human skin and synthetic membranes are explored as alternatives. Strat-M® is a membrane designed to mimic the multi-layered structure of human skin for IVPT. For instance, in Strat-M®, the steady-state fluxes (JSS) of resorcinol in formulations free of permeation enhancers were found to be 41 ± 5 µg/cm2·h for the aqueous solution, 42 ± 6 µg/cm2·h for the hydrogel, and 40 ± 6 µg/cm2·h for the oil-in-water emulsion. These results were closer to excised human skin (5 ± 3, 9 ± 2, 13 ± 6 µg/cm2·h) and surpassed the performance of EpiSkin® RHE (138 ± 5, 142 ± 6, and 162 ± 11 µg/cm2·h). While mass spectrometry and Raman microscopy demonstrated the qualitative molecular similarity of EpiSkin® RHE to human skin, it was the porous and hydrophobic polymer nature of Strat-M® that more faithfully reproduced the skin's diffusion-limiting barrier. Further validation through similarity factor analysis (∼80-85%) underscored Strat-M®'s significance as a reliable substitute for human skin, offering a promising approach to enhance realism and reproducibility in dermatological product development.

Solubilization of biomimetic lipid mixtures by some commonly used non-ionic detergents.

Detergents are amphiphilic molecules often used to solubilize biological membranes and separate their components. Here we investigate the solubilization of lipid vesicles by the commonly used non-ionic detergents polyoxyethylene (20) oleyl ether (Brij 98), n-octyl-ß-D-glucoside (OG), and n-dodecyl ß-D maltoside (DDM) and compare the results with the standard detergent Triton X-100 (TX-100). The vesicles were composed of palmitoyl oleoyl phosphatidylcholine (POPC) or of a biomimetic ternary mixture of POPC, egg sphingomyelin (SM) and cholesterol (2:1:2 molar ratio). To follow the solubilization profile of large unilamellar vesicles (LUVs), 90° light scattering measurements were done along the titration of LUVs with the detergents. Then, giant unilamellar vesicles (GUVs) were observed with optical microscopy during exposure to the detergents, to allow direct visualization of the solubilization process. Isothermal titration calorimetry (ITC) was used to assess the binding constant of the detergents in POPC bilayers. The results show that the incorporation of TX-100, Brij 98 and, to a lesser extent, OG in the pure POPC liposomes leads to an increase in the vesicle area, which indicates their ability to redistribute between the two leaflets of the membrane in a short scale of time. On the other hand, DDM incorporates mainly in the external leaflet causing an increase in vesicle curvature/tension leading ultimately to vesicle burst. Only TX-100 and OG were able to completely solubilize the POPC vesicles, whereas the biomimetic ternary mixture was partially insoluble in all detergents tested. TX-100 and OG were able to incorporate in the bilayer of the ternary mixture and induce macroscopic phase separation of liquid-ordered (Lo) and liquid-disordered (Ld) domains, with selective solubilization of the latter. Combination of ITC data with turbidity results showed that TX-100 and OG can be incorporated up to almost 0.3 detergent/lipid, significantly more than Brij 98 and DDM. This fact seems to be directly related to their higher capacity to solubilize POPC membranes and their ability to induce macroscopic phase separation in the biomimetic lipid mixture.

Elastic cationic liposomes for vitamin C delivery: Development, characterization and skin absorption study.

The influence of hydrophilic surfactants acting on the membrane elasticity of liposomes on the skin absorption of vitamin C is investigated. The purpose of encapsulation inside cationic liposomes is to improve the skin delivery of vitamin C. The properties of elastic liposomes (ELs) are compared to that of conventional liposomes (CLs). ELs are formed by the addition of the "edge activator" Polysorbate 80 to the CLs composed of soybean lecithin, cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammoniopropane chloride), and cholesterol. The liposomes are characterized by dynamic light scattering and electron microscopy. No toxicity is detected in human keratinocyte cells. Evidences of Polysorbate 80 incorporation into liposome bilayers and of the higher flexibility of ELs are given by isothermal titration calorimetry and pore edge tension measurements in giant unilamellar vesicles. The presence of a positive charge in the liposomal membrane increases the encapsulation efficacy by approximately 30% for both CLs and ELs. Skin absorption of vitamin C from CLs, ELs and a control aqueous solution measured in Franz cells shows a high delivery of vitamin C into each skin layer and the acceptor fluid from both liposome types. These results suggest that another mechanism drives skin diffusion, involving interactions between cationic lipids and vitamin C depending on the skin pH.

Efficiency of emulsifier-free emulsions in delivering caffeine and α-tocopherol to human skin.

OBJECTIVE: Increasing consumer demand for natural and environmentally friendly products is driving the cosmetic industry to seek greener and safer processes. High-frequency ultrasound technology (HFUT) stabilizes emulsions without adding emulsifying surfactants (ES). In this work, the formulation characteristics of an HFUT-treated emulsion and a Reference emulsion were compared for both caffeine and α-tocopherol. METHODS: A comparison was made between ES-free emulsions and the Reference emulsions based on droplet size, viscosity, pH and rheology behaviour for both active cosmetic ingredients. The permeation of caffeine and the skin retention of α -tocopherol were studied in vitro using Franz diffusion cells on human skin biopsies, considered the gold standard for permeation assays. RESULTS: The formulations developed were stable and showed suitable droplet size distribution. In the case of ES-free emulsions, the average droplet size was inferior to 1.5 µm regardless of the polarity of the active. All formulations presented a shear-thinning pseudoplastic behaviour, an attribute usually desired for cosmetic products. The skin permeation studies showed that in the case of caffeine (model hydrophilic molecule), the ES-free emulsion presented a delivery capacity similar to that of the Reference emulsion. However, for α-tocopherol (highly lipophilic model molecule), differences were observed in the distribution of the active in the stratum corneum with an advantage for the Reference emulsion, probably due to the impact of surfactants on the SC lipids. CONCLUSION: This work demonstrates that HFUT is a reliable tool that is able to prepare stable ES-free emulsions loaded with hydrophilic or lipophilic active ingredients. Skin permeation studies confirm that the emulsions produced by HFUT promote the delivery of the actives to the human skin. In the case of α-tocopherol, the delivery efficiency was lower than with the Reference emulsion, especially in the SC layers, due to the absence of surfactants. Nevertheless, the ES-free emulsion still represents a good compromise between efficacy and the need for green cosmetics in the market.

OBJECTIF: La demande croissante des consommateurs pour des produits naturels et respectueux de l'environnement encourage l'industrie cosmétique à développer des procédés plus écologiques et plus sûrs. La technologie des ultrasons à haute fréquence (HFUT) permet de stabilizer les émulsions sans ajouter de tensioactifs émulsionnants (ES). Dans ce travail, les caractéristiques d'une émulsion traitée par HFUT et d'une émulsion de référence ont été comparées. La caféine et l'α-tocophérol ont été utilisés comme actifs modèles. MÉTHODES: Les émulsions sans ES et les émulsions de référence on été comparées en termes de taille des gouttelettes, de viscosité, de pH et de comportement rhéologique pour les deux actifs. La perméation de la caféine et la rétention cutanée de l'α-tocophérol ont été étudiées in vitro sur des biopsies de peau humaine, en utilisant des cellules de diffusion de Franz, le 'gold standard' des tests de perméation. RÉSULTATS: Les formulations développées sont stables et présentent une distribution appropriée de la taille des gouttelettes. La taille moyenne des gouttelettes des émulsions sans ES est inférieure à 1.5 µm, quelle que soit la polarité de l'actif. Toutes les formulations présentent un comportement rhéofluidifiant adapté à un usage cosmétique. Les études de perméation cutanée montrent que l'émulsion sans ES contenant de la caféine (molécule modèle hydrophile) présente une capacité de délivrance similaire à celle de l'émulsion de référence. Dans le cas de l'α-tocophérol (molécule modèle lipophile), des différences ont été observées dans la distribution de l'actif dans le stratum corneum (SC) avec un avantage pour l'émulsion de référence, probablement lié à l'interaction entre les tensioactifs et les lipides du SC. CONCLUSION: Ce travail démontre que le traitement par HFUT permet de préparer des émulsions stables sans ES, quelle que soit la polarité des actifs cosmétiques à véhiculer. Les études de perméation cutanée confirment que les émulsions produites par HFUT permettent la diffusion cutanée des actifs dans la peau humaine. Même si dans le cas de l'α-tocophérol la quantité accumulée était plus faible, l'émulsion traitée par HFUT propose un bon compromis entre efficacité et éco-responsabilité.

Stabilization of vitamin C in emulsions of liquid crystalline structures.

Emulsified systems are widely used for topical delivery with the aim of optimizing cutaneous absorption and offering a pleasant sensory. They also may provide a protection of the active molecule against oxidation and/or degradation. The oil phase of o/w emulsions may consist of liquid crystalline structures, especially lamellar structures which are similar to those found in the stratum corneum lipids. In the present work, o/w emulsions containing liquid crystals of mixed cetyl alcohol and Polysorbate 60 were developed for topical delivery of vitamin C, a potent antioxidant with several applications in the cosmetic and pharmaceutical fields. In addition to the well-documented lipid supplementation of the stratum corneum, the liquid crystal emulsions provide a significant chemical stabilization of vitamin C against its degradation. Emulsions were characterized by X-ray diffraction, polarized optical microscopy, and transmission electron microscopy. The stability of vitamin C in the formulations was evaluated upon storage in different conditions of temperature. The emulsions contain a complex colloidal structure, consisting of lamellar liquid crystalline (Lα) and crystalline lamellar gel (Lß) phases, that provide a very efficient protection of vitamin C against its degradation.

Recent Advances and Perspectives in Liposomes for Cutaneous Drug Delivery.

The cutaneous route is attractive for the delivery of drugs in the treatment of a wide variety of diseases. However the stratum corneum (SC) is an effective barrier that hampers skin penetration. Within this context, liposomes emerge as a potential carrier for improving topical delivery of therapeutic agents. In this review, we aimed to discuss key aspects for the topical delivery by drug-loaded liposomes. Phospholipid type and phase transition temperature have been shown to affect liposomal topical delivery. The effect of surface charge is subject to considerable variation depending on drug and composition. In addition, modified vesicles with the presence of components for permeation enhancement, such as surfactants and solvents, have been shown to have a considerable effect. These liposomes include: Transfersomes, Niosomes, Ethosomes, Transethosomes, Invasomes, coated liposomes, penetration enhancer containing vesicles (PEVs), fatty acids vesicles, Archaeosomes and Marinosomes. Furthermore, adding polymeric coating onto liposome surface could influence cutaneous delivery. Mechanisms of delivery include intact vesicular skin penetration, free drug diffusion, permeation enhancement, vesicle adsorption to and/or fusion with the SC, trans-appendageal penetration, among others. Finally, several skin conditions, including acne, melasma, skin aging, fungal infections and skin cancer, have benefited from liposomal topical delivery of drugs, with promising in vitro and in vivo results. However, despite the existence of some clinical trials, more studies are needed to be conducted in order to explore the potential of liposomes in the dermatological field.

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity.

Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose Resat-the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C12E8 and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.

Effect of Triton X-100 on Raft-Like Lipid Mixtures: Phase Separation and Selective Solubilization.

Under certain conditions, biological membranes exhibit resistance to solubilization, even at high detergent concentration. These insoluble fragments are enriched in sphingolipids, cholesterol, and certain proteins having a preference for more organized environments. Here we investigated the effect of detergent Triton X-100 (TX-100) on raft-like lipid mixtures composed of POPC (palmitoyl oleoyl phosphatidylcholine, an unsaturated lipid), SM (sphingomyelin, a saturated lipid), and cholesterol, focusing on the detergent-induced phase separation at subsolubilizing concentration and the extent of solubilization at higher concentration. Giant unilamellar vesicles (GUVs) of POPC/SM/chol containing a fluorescent probe known to prefer the liquid-disordered phase were prepared and observed with fluorescence microscopy. A phase diagram constructed in the presence and absence of 0.1 mM TX-100 showed that the detergent induces macroscopic liquid-ordered/liquid-disordered (Lo/Ld) phase separation over a wide range of membrane composition, indicating that TX-100 has the ability to rearrange the lateral heterogeneity of the lipid mixture. The extent of solubilization of the POPC/SM/chol GUVs was quantified by measuring the vesicle size before and after the injection of a high concentration of TX-100. In parallel, the solubilization extent of large unilamellar vesicles (LUVs) was assessed by turbidity measurements. The extent of solubilization decreases significantly as the fractions of SM and cholesterol in the mixture increase. The origin of the detergent resistance is the low partitioning of TX-100 in cholesterol-rich membranes, especially in SM-containing ones, as evidenced by isothermal titration calorimetry experiments on LUVs. Our results provide a guide to future research on the effects of TX-100 on raft-like lipid mixtures.