Members of the vitamin A family, include retinoic acid, retinol esters and retinol, are widely used in cosmetics and pharmaceuticals. Especially retinol is relatively milder, so it is more commonly used in cosmetics. External retinol preparations are a major class of anti-ageing agents, and their safety and efficacy has been a focus of investigation. In 2022, the EU SCCS revised the safety assessment for vitamin A derivatives (retinol, retinol acetate and retinol palmitate) for their use in cosmetics. The effects of cosmetics are directly related to absorption rate and amount of the active ingredients. In cosmetics research, it is important to understand the speed and range of the skin penetration of ingredients28. Therefore, appropriate detection methods are needed to evaluate and determine the absorption of active ingredients in cosmetics to ensure safety and effectiveness. Moreover, in vitro methods are used in conjunction with ex vivo and / or in vivo studies to more quickly translate newly developed topical and transdermal drug delivery systems from the laboratory to the clinic21.IVPT is an effective method that replaces equivalent clinical trials for skin drug delivery preparations and investigates the therapeutic equivalence of experimental drugs compared with reference drugs by evaluating their in vitro absorption kinetics through isolated human skin. IVPT can be used to screen drug preparations, evaluate the properties and mechanism of action of the preparation carrier system to promote skin penetration, and evaluate the systemic risk of skin exposure to chemicals by predicting the skin transport of drug molecules29. In vitro diffusion modelling is an important tool for screening the penetration ability of active ingredients in various formulations30.Therefore, in the current study, we used a modified vertical diffusion cell model, according to the relevant guidelines and literature, to perform in vitro diffusion cell experiments that simulate real skin absorption. In the evaluation of cosmetics, the experimental time of the in vitro Franz diffusion cell is related to the actual use of cosmetics. Yourick JJ et al.18. reported that the single best estimate of systemic retinol absorption from in vitro human skin studies is the 24-h receptor fluid value. Therefore, we carried out a 24 h Franz diffusion cell experiment on external retinol preparations.The results of the 24 h Franz diffusion cell experiment showed that retinol could not be detected in the receiving solution. As reported in the literature, retinol accumulates in the skin31,32, possibly because the concentration of retinol is too low to reach the detection limit or because it has not penetrated into the receiving solution. Mice and rats are easy feeding and be widely used in pilot study. Literatures showed that the permeability of rats and mice were greater than that of pigskin. By comparing the amount of retinol penetration into the three skin types (mouse, rat and pig), we found that the absorption of rat in full-thickness skin was the highest, followed by mice, and finally pig skin. The amount of penetration in rats and mice was about 2 times of pig skin, which corresponds with the existing scientific literature33. Minipigs are a promising animal model for predicting percutaneous drug absorption in humans34. Moreover, Ranamukhaarachchi et al.35 showed that porcine stratum corneum became a closer model for human stratum corneum after freezing by measuring Young’s (elastic) modulus. In summary, for the in vitro diffusion cell, the results of pig skin are closer to the actual skin absorption.In addition, through three in vitro animal models (pig, mouse, and rat skin), we also found that ROL can penetrate the stratum corneum and accumulate in skin, but the accumulated amount of retinol in the stratum corneum is greater than that in the skin. According to amount of retinol released into the skin over time, the percutaneous permeation of retinol is time dependent, but the absorption rate of retinol in the skin decreases over time, and the amount of retinol in the skin reaches its absorption peak at 2–4 h after administration. The rapid absorption and retention of retinol in the skin is conducive to its biological activity. What is more, by calculating the slope of the curve of the experimental results for the relative amount of retention in the full-thickness skin, we found that the absorption rate of the full-thickness skin in the ROL-C1 group was faster than that in the ROL-C2 group. Therefore, it can be preliminarily explained that the in vitro percutaneous penetration effect of retinol in the ROL-C1 group was better than that in the ROL-C2 group. However, in our study, the Franz diffusion cell experiment was static, which is not the actual application of the product. To overcome this obstacle, it was been reported that a modified Franz diffusion cell method achieved significant visual improvement in both the epidermis and dermis after 4 h by applying a rolling massage to skin treated with retinol emulsion36. Therefore, the static Franz diffusion cell has many limitations and it cannot substitute for the live animal evaluation.Raman is a label-free technique, and the main advantage of confocal Raman spectroscopy is the ability to detect many labels or probes simultaneously. When labelling small bioactive compounds, fluorescent dyes often affect biological activity; however, small Raman labels can be introduced without affecting biological activity, and bioactive compounds in living cells can be visualised37, therefore we utilised this noninvasive property of Raman spectroscopy for the analysis of isolated pig skin, which allows for the detection of topical cosmetics that cannot be used for localisation detection due to the inability to add labels. As a commonly used active ingredient in the cosmetic industry, the dermal delivery of nicotinamide (NIA) was examined by Iliopoulos F et al.38 using an in vitro 24-h finite-dose Franz diffusion cell method and in vivo confocal Raman spectrometry after 60 min of treatment. They analysed the results of the two methods and found that the in vitro cumulative transmittance of NIA was correlated with the in vivo dermal uptake of NIA at 2 μm. According to the results of Raman spectroscopy, we found that at the Raman shift of 1597 cm−1, the absorption peaks of ROL-C1 and ROL-C2 can reach a depth of more than 10 μm in the skin, showing transdermal penetration ability. In addition, the peak intensity in the ROL-C1 group gradually decayed with increasing skin depth, up to 16 μm. The intensity was higher than in the ROL-C2 group, consistent with the Franz diffusion cell experiment results. Raman mapping was not totally effective because of interference with other ingredients, especially when the concentration of the target compound in the mixture is relatively low39, but they were consistent with the Franz diffusion cell results.In vivo and in vitro correlation studies are also important components of transdermal permeation studies of topical formulations. Fan Y40 explored the differences in transdermal permeation properties between ex vivo dermal permeation experiments using Franz diffusion cells and in vivo dermal / haematological pharmacokinetic experiments facilitated by dual-site microdialysis sampling. In this study, for the homologous of mice and rat, we only employed the KM mouse model to quantitate the concentration of ROL in whole skin and plasma. In full-thickness skin (Fig. 4B), after treatment with ROLs, intradermal absorption rate reached a plateau after 4 h and decreased after that. Although ROL was detected in skin after 24 h, exogenous ROL was not detected in plasma (Fig. 4D). ROL was not detected in SD rat plasma either (Fig. 4C).It is interesting that ROL-C1 with lower ROL concentration has more penetration than in ROL-C2. The appearance of ROL-C1, a lecithin organogel, was clear. This preparaion enhances permeation and skin deposition41,42. A lecithin organogel may improve permeation at least 10 times43. Particles of ROL-C1 were smoother and spherical from the images of SEM and Polarizing microscope. In contrast, the surface of particles in the ROL-C2 were flaky, which prevented mobility (see more details in supplement). The preparation protocol for these samples maybe also important.In addition, by comparing the in vitro and in vivo data of mice, we found that the absorption of the full-thickness skin in vivo was about three times of the ex vivo skin. We speculated this might be related to some transport genes in the in vivo skin, which would be removed or deactivated during the process of ex vivo skin, because the skin would be removed the cells and the cell debris by some extraordinary methods. So we performed transcriptome sequencing to study the transport and absorption mechanism of retinol.The transcriptome changed after treatment with ROLs. The cellular and molecular changes were found in epidermis and dermis similarly as previous studies of retinol topical application44. Since the transcutaneous absorption and transport of retinol involves pathway activation, we focused on up-regulating differential genes. In our study, the development of the cuticle, epidermis and dermis in the skin was significantly activated after treatment with the ROLs, reflecting the biological function of the external retinol preparations. KEGG pathway enrichment analysis revealed that the ABC transporter and the retinol metabolism pathways being activated. The relationship between retinol transport and activation of the ABC transporter pathway is an interesting topic for future research. Among the 126 DEGs identified, we found genes related to retinol metabolism and transport. One of these, Stra6, encodes a transmembrane protein with unique structural characteristics that can catalyse the two-way transport of retinol and isolate retinol from target cells45. In addition, it has been reported that Stra6 acts as both an RBP receptor and a transporter for vitamin A uptake46. These findings suggest that retinol may be transported into cells through the Stra6 receptor after skin treatment with retinol topical preparations. However, the skin acts as a microretinol reservoir, and retinol as a fat-soluble molecule may also enter the skin cells through passive diffusion. Therefore, the next study on the process of retinol transport into cells in topical preparations will be interesting. After transported into the cell, the metabolic pathway of retinol is activated, resulting in metabolic processes, such as the Lrat gene, which catalyzes the esterification of all-trans retinol to all-trans retinol ester; the Rdh12 and Aldh1a3 genes catalyze the metabolism of retinol to retinoic acid, which in turn exerts biological functions.In summary, after ROLs treatment, the metabolism and transport pathways of retinol in the skin are activated, and there is an absorption and transport process that occurs in the skin. Retinol may enter skin cells through upregulated expression of the Stra6 gene and then be retained in the skin and play a role in skin development. However, RNA-seq analysis has several limitations; it is only a reference and needs quantitative verification. Thus, further experimental verification is needed.
