What vitamin makes skin thicker?

Vitamin C–squalene bioconjugate promotes epidermal thickening and collagen production in human skin.

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Synthesis and chemical characterization of Vit C–SQ

Vitamin C (ascorbic acid) is a charged and hydrophilic natural antioxidant with skin antiaging and photoprotective effects. Because it penetrates poorly into the skin, lipidic derivatives more able to enter stratum corneum have been designed as antioxidant excipient for topical formulations. Many fatty acids esterified forms of the C-6 primary hydroxyl group of vitamin C were thus introduced in pharmaceutical or cosmetic compositions. For example, 6-O-palmitoyl-L-ascorbic acid was shown to reduce the appearance of wrinkles, cracks, crevices and puffiness around the eyes23 and 6-O-stearoyl-L-ascorbic acid was found to retard skin aging24 and to be a potent inhibitor of hyaluronidase enzymes25. Many other derivatives including esters of oleic acid26, linoleic27, myristic acid28, retinoic acid29, docosahexaenoic acid30, etc. have been evaluated for potential application in cosmetic and as antioxidant in food industry. The anchoring of the lipidic moiety on the lateral chain of ascorbic acid preserves the redox properties of the tetronic core. Furthermore, this design considerably simplifies the synthesis since the primary hydroxyl group is the most nucleophilic function. We thus envisaged to access 6-squalenoyl ascorbic acid (Vit C–SQ) by direct condensation of 1,1′,2-trisnor-squalenic acid, easily available from squalene31,32, without protection of the tetronic ring. Several synthesis strategies were described to obtained SQ derivatives33,34,35. However, in the case studied here, most conventional methods failed to give the expected material. We finally found that when ascorbic acid was treated with squalenyl chloride in HCl saturated N-methyl-pyrrolidinone (NMP) according to the method of Yazawa et al.36, the desired 6-O-squalenoyl-L-ascorbic acid could be obtained in 30–35% yield after chromatographic purification. Although this method had the merit of giving the desired product, it suffered from extensive degradation during the removal of the high boiling solvent NMP. To bypass this hurdle, we examined the enzymatic acylation of ascorbic acid with stabilized lipase. Biocatalysts offered mild conditions and improved regioselectivity for the acylation of ascorbic acid, particularly when sensitive unsaturated fatty acids were involved37,38,39. The 6-O-squalenoyl-L-ascorbic acid was thus synthetized by treatment of a 2:1 mixture of ascorbic acid and squalenic acid with Novozyme 435, a lipase acrylic resin from Candida antarctica in t-AmOH at 50 °C for 2 days, using molecular sieves as drying agent. In these conditions the Vit C–SQ was obtained in 74% yield as a thick oil after chromatographic purification (Fig. 1). The Vit C–SQ derivative was stable upon 2 years storage in a freezer at − 20 °C. Figure 1 Synthesis of Vit C–SQ through chemical esterification (Method A) or enzymatic acylation (Method B). Full size image

Formulation of Vit C–SQ

Vit C–SQ and Vit C–Palmitate (used as a control) were formulated as creams to be directly applied on human explants. These oily compounds were dispersed in SQ at equivalent concentrations of Vit C (3 or 5 wt%) and homogenized by magnetic stirring. The Vit C–SQ and Vit C–Palmitate formulations were viscous, with an oily texture and slightly yellow color. Free Vit C could not be dispersed in SQ, so it was incorporated in carboxymethyl cellulose at equivalent contents.

Skin morphology was improved by Vit C–SQ bioconjugate

Formulations containing free or conjugated Vit C or SQ alone, were applied repeatedly for 10 days upon stratum corneum of human skin explants collected from two 30-year females and maintained in a proprietary survival medium. Untreated control explants from each donor were kept in the same conditions for the same time. The explants from both donors received distinct treatment panels, i.e. donor 1 received for 10 days Vit C either free or coupled to SQ or to Palmitate, at 1 or 3 wt% final Vit C concentration, while donor 2 received for 10 days free Vit C 5 wt% or Vit C–SQ at 3 or 5 wt%, or SQ alone. Thus, the experiment with Vit C–SQ 3 wt% was repeated in both donors, allowing comparison. In fixed and colored tissue sections from each explant, the skin morphology as well as the epidermis and dermis thickness were investigated. A significant increase of epidermis thickness was observed with Vit C–SQ 3 wt% in donor 1 (Fig. 2A, C), i.e. + 60% compared to the untreated control (69 ± 12 µm versus 43 ± 7 µm, respectively; p < 0.001). The thickening effect was more limited with the same concentration of free Vit C (+ 28%; p < 0.05) or of Vit C–Palmitate (+ 31%; p < 0.05). Consistently, in donor 2 a 22% increase in epidermis thickness was observed with Vit C–SQ 3 wt% (p < 0.05), as well as, with Vit C–SQ 5 wt% (p < 0.05), while only a non significant 14% increase was observed with free Vit C 5 wt% and no variation with SQ alone (Supplementary Fig. S1A, C), thus confirming the beneficial effect of Vit C–SQ on skin thickness. Epidermal thickness before treatment (40 ± 4 µm) was not significantly different from the value after 10 days without treatment (43 ± 7 µm; T0) in donor 1 (Fig. 2A). On the other hand, dermis thickness (128 ± 14 µm in control from donor 1; 125 ± 4 µm in control from donor 2) was not modified whatever the Vit C formulation used in both donors nor with SQ in donor 2 (Fig. 2D, Supplementary Fig. S1D). Finally, of all Vit C formulations studied, Vit C–SQ resulted in the highest gain of epidermis thickness. Figure 2 Topical treatment of human skin explants (donor 1) for 10 days with three formulations containing 3 wt% Vit C, increased epidermis thickness and collagen type III labeling in dermis. (A) In fixed and stained (Masson trichrome) whole skin sections, epidermis (pink) and dermis (blue) layers were observed before (T0) and after the treatment period. The control explant was kept untreated for 10 days. (B) Fixed skin sections labeled with goat anti-human collagen III antibody and revealed by DAB are shown. Pictures were realized with a tri CCD DXC 390P camera (Sony) and stored with Leica IM1000 software, version 1.10 (www.leica-microsystems.com). (C) The epidermis thickness and (D) dermis thickness were measured in each explant (n = 7 to 9 images; mean of 3 measures per image). (E) The intensity of collagen III immuno-staining signal in dermis was measured as the percentage of analyzed surface (n = 9 images; mean of 3 measures per image). One-way ANOVA with Tukey’s post-test (***p < 0.001, *p < 0.05) was used to compare treatments. Graphs were done with GraphPad Prism version 5.0.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com). Scale bar = 100 µm (panel A), and 200 μm (panel B). Full size image

The skin biological pathways were studied at molecular level

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The expression of proteic (collagens; Figs. 2B, 3A) as well as glucidic skin markers (GAGs; Fig. 3B, Supplementary Fig. S1B, E) has been measured by immuno-histochemistry in human tissue explants after the different topical treatments. Figure 3 Effects of ex-vivo topical treatment of human skin explants (donor 1) for 10 days with three formulations containing 3 wt% Vit C, upon collagen type I expression in whole skin and GAGs staining at dermal–epidermal junction (DEJ). (A) Collagen type I was labeled by a rabbit anti-human collagen I antibody with biotin/streptavidin amplification and FITC fluorescence revelation (green) and cell nuclei stained with propidium iodide (red). Pictures were realized with a tri CCD DXC 390P camera (Sony) and stored with Leica IM1000 software, version 1.10 (www.leica-microsystems.com). (B) Staining of fixed skin sections with Schiff reagent revealed the neutral GAGs as purple pink near DEJ (arrows), at the bottom of upper epidermis layer. (C) One-way ANOVA with Tukey’s post-test was used to compare collagen I signal (n = 8 images; mean of 3 measures per image), relative to untreated control (not significant). (D) GAGs coloration measured as percent of DEJ surface (n = 8 to 9 images; mean of 3 measures per image) from each explant was compared in formulations and untreated control by one-way ANOVA with Tukey’s post-test (*p < 0.05). Graphs were done with GraphPad Prism version 5.0.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com). Scale bar = 50 µm panel A, and 100 μm panel C. Full size image To gain insights on the molecular pathways behind the observed effects of Vit C–SQ compared to Vit C–Palmitate, to free Vit C or to SQ, a set of 15 genes of interest were selected according to their known relationships with skin physiopathology, which have been documented using the Ingenuity Pathway Analysis (IPA) software40 (Supplementary Fig. S2). The expression of these genes was measured by RT-qPCR in the same skin explants previously treated with the various formulations of Vit C or SQ alone (Supplementary Fig. S3). The expression of some of these genes was also evaluated in epidermal and dermal compartments previously separated by laser capture microdissection. For some genes, the transcriptional effects were higher upon epidermis compared to dermis, which might be related to the epidermis thickening described before (Supplementary Fig. S4). It can be concluded from this study that the Vit C–SQ complex has modified in the strongest extent the transcriptional expression of most of the target genes studied, in a globally reproducible manner for the Vit C–SQ 3 wt% tested in two independent donors. The effects observed in whole skin were also consistent with the sum of the effects measured in separately microdissected epidermis and dermis in the two donors. Moreover, the transcriptional expression data showed consistency with histological data. Hereafter the effects of this bioconjugate upon the various biological functions explored will be reviewed.

Vit C–SQ favors biosynthesis and maintenance of structural fibers of the skin

Collagen is a major constituent of skin where it is structured into fibers representing the major part of dermis mass17. Type III collagen is a homotrimer forming a right-handed triple helix4 which acts as a major structural constituent of the extracellular matrix. The immuno-histochemistry studies performed in donor 1 showed that the collagen III protein content was twice higher in papillary dermis after 10-days treatment with 3 wt% Vit C–SQ formulation comparatively to untreated control, i.e. collagen III signal represented 60 ± 23% versus 34 ± 10% of dermis surface, respectively (p < 0.05). The more limited effects of Vit C–Palmitate (+ 4%) and of free Vit C (+11%) were not significant (Fig. 2B, E). Consistently, this stimulating effect of collagen III biosynthesis by Vit C–SQ upon was also observed in both donors at gene expression level. The COL3A1 mRNA transcript was increased in donor 1 (fourfold) and in donor 2 (11-fold; p < 0.05) after Vit C–SQ 3 wt% treatment versus control, and similarly with Vit C–SQ 5 wt% in donor 2 (tenfold; p < 0.05) (Fig. 4A). Comparatively, the free Vit C produced a smaller and not significant increase in COL3A1 expression in both donors, while Vit C–Palmitate was almost devoid of any effect in donor 1, as well as SQ alone in donor 2. Figure 4 Vitamin C formulations influenced the expression in human skin explants, of the mRNA transcripts of (A) genes encoding collagens type I and type III, (B) genes controlling skin fibrils assembly and (C) genes regulating the extracellular matrix (ECM). Fold changes of each transcript were calculated as 2−ΔCq from Cq normalized by NORMA-gene algorithm56, in whole skin explants treated with 1–3 wt% free or formulated Vit C (in donor 1) or with 3–5 wt% free or squalenized Vit C or with squalene (SQ) alone (in donor 2), relative to the untreated control (set to 1, red dotted line). The data from one of two or three analyzed explants are presented (n = 3 per group). Kruskal–Wallis non-parametric test followed by Dunn’s post-test was used to determine significance between the groups (**p < 0.01, *p < 0.05). Graphs were done with GraphPad Prism version 5.0.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com). Full size image The collagen I protein content was not modified in dermis from skin explants treated for 10 days with any of the 3 wt% Vit C formulations tested, according to immuno-histochemistry results in donor 1 (Fig. 3A, C). Among the genes encoding type I procollagen41, the expression of COL1A1 gene, encoding collagen type I alpha 1 chain, was not modified by Vit C–SQ in donor 1 and was poorly raised in donor 2 (1.6-fold, p < 0.05). The COL1A2 gene encoding collagen type I alpha 2 chain was slightly overexpressed after Vit C–SQ 3 wt% treatment in both subjects studied, i.e. 2.1 to 2.6-fold (p < 0.05). In contrast, free Vit C, Vit C–Palmitate or SQ alone had no significant effect in collagens I gene expression (Fig. 4A). Our transcriptional data obtained from skin layers after microdissection suggest that the overexpression of collagens I and III induced by Vit C–SQ could take place in dermis (Supplementary Fig. S4), consistently with described stimulation of collagen production by Vit C in dermis fibroblasts17. Overall, among the collagen-encoding genes studied here, COL3A1 was the most strongly modified and Vit C–SQ was the most active formulation since it stimulated 2–4 times more its expression than that of COL1A2. These results are in line with studies on liposomal Vit C showing increased production of collagen in pig ear skin35. Our study demonstrates that collagen production is stimulated by Vit C derivatives also in human skin. Finally, the observed effects of Vit C–SQ upon collagens I and III contribute to a limited I / III ratio, which is typical of young skins11,12. This could characterize an anti-aging action for this bioconjugate. Decorin (DCN)42,43 and fibrillin 1 (FBN1)44 are involved in the assembly and maintenance of collagen fibrils and elastic microfibrils, thus contributing to skin strength and suppleness. The corresponding genes were significantly overexpressed in whole skin explants from both subjects, treated with Vit C formulations, with a higher effect of the Vit C–SQ conjugate, i.e. DCN twofold to threefold and FBN1 fourfold to tenfold (p < 0.05), while other formulations had lower or no effect (Fig. 4B). In microdissected skin layers, the transcriptional effect of Vit C–SQ was observed in epidermis for FBN1, thus this might be related to the epidermis thickening described above; however, it occurred at a similar extent in dermis and epidermis for DCN (Supplementary Fig. S4). Moreover, the gene encoding peptidylprolyl isomerase B (PPIB) which contributes to collagen binding, accelerates proteic folding and favors tensile strength of skin45, was twice overexpressed with Vit C–SQ 3 wt% (p < 0.05), while it was not affected by free Vit C or Vit C–Palmitate 3 wt% or by SQ (Fig. 4B).

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Vit C–SQ positively regulates the components of ECM

The matrix metalloproteinases (MMPs) are known to contribute to basement membrane and ECM degradation through the fragmentation of collagens, during skin aging and ultraviolet irradiation46. In our study, the Vit C–SQ complex stimulated 6-times more in donor 1 the transcriptional expression of TIMP metallopeptidase inhibitor of MMPs (TIMP1; 12-fold, p < 0.05; Fig. 4C) and twice more the expression of COL3A1 transcript (fourfold; Fig. 4A), than the expression of MMP1 transcript (1.7-fold, p < 0.05; Fig. 4C) which encodes the collagenase involved in the degradation of type I and III collagens41. Such effects can contribute to preserve the existing collagen. This is consistent with the observed favorable action of Vit C–SQ upon the production of collagens I (Fig. 4A) and III (Figs. 2B, E, 4A) and on the expression of decorin (Fig. 4B), which locates at the surface of collagen fibers thus protecting them against cleavage by MMPs43. Moreover, members of two families of ECM glycoproteins, i.e. laminins, which are the major non collagenous constituent of basement membranes, and tenascins, involved in wound healing47, were also positively modulated at gene level in donor 1 by Vit C–SQ 3 wt% only: laminin subunit alpha 5 (LAMA5; fourfold, p < 0.05) and tenascin XB (TNXB; twofold, p < 0.05) (Fig. 5C). Figure 5 Vitamin C formulations influenced the expression in human skin explants, of the mRNA transcripts of (A) genes encoding enzymes of GAGs biosynthesis, (B) genes controlling cell growth and motility, and (C) genes regulating ECM. Fold changes of each transcript were calculated as 2−ΔCq from Cq normalized by NORMA-Gene algorithm56, in whole skin explants treated with 1–3 wt% free or formulated Vit C (in donor 1) or with 3–5 wt% free or squalenized Vit C or with squalene (SQ) alone (in donor 2), relative to the untreated control (set to 1, red dotted line). The data from one of two or three analyzed explants are presented (n = 3 per group). Kruskal–Wallis non-parametric test followed by Dunn’s post-test was used to determine significance between the groups (**p < 0.01, *p < 0.05). Graphs were done with GraphPad Prism version 5.0.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com). Full size image

Vit C–SQ is favorable to GAGs content in skin

Hyaluronic acid (hyaluronan, HA) is an acidic GAG predominantly present in skin where it retains water and promotes cell motility, adhesion, proliferation and tissue organization. Moreover, HA accumulates in ECM in early stages of wound healing48. Hyaluronan synthase 2 (HAS2) mediates the biosynthesis of HA49 and is described as its main synthase in skin50. The biosynthesis of HA could be stimulated by Vit C–SQ 3 wt% in skin, as suggested by the overexpression in whole skin explants, of the HAS2 gene, i.e. twofold (p < 0.05) in donor 1 and fourfold in donor 2 (Fig. 5A). This is also consistent with the observed overexpression to a similar extent after treatment with Vit C–SQ 3 wt% (threefold, p < 0.01 in donor 1), of the gene encoding adiponectin (ADIPOQ), which promotes biosynthesis of HA along with HAS2 transcript51 (Fig. 5B). The effect of Vit C–SQ upon this gene supports the observed thickening of the epidermis (Fig. 2A, C), since adiponectin favors cell growth and tissue remodeling through the binding and sequestration of growth factors. The hyaluronan mediated motility receptor (HMMR), which is a major HA receptor and regulates cell growth, motility and contact inhibition48 was also strongly overexpressed (sixfold in donor 1; 40-fold, p < 0.05 in donor 2) by Vit C–SQ 3 or 5 wt% in whole skin (Fig. 5B). Moreover, histochemistry staining of neutral GAGs, which act as growth factors reservoirs at the dermal–epidermal junction (DEJ)48, showed a stronger signal at DEJ in skin explants treated with Vit C–SQ 3 wt% compared to Vit C–Palmitate 3 wt% in donor 1 (+ 30%, p < 0.05; Fig. 3B, D). Consistently, skin explants treated with Vit C–SQ 3 wt% exhibited a 50%-higher GAGs signal at DEJ, compared to free Vit C 5 wt% in donor 2 (p < 0.05; Supplementary Fig. S1B, E). The genes encoding two enzymes involved in GAGs biosynthesis were consistently overexpressed in both donors after Vit C–SQ 3 wt% treatment, i.e. beta-1,3-galactosyltransferase 652 (B3GALT6; twofold, p < 0.05) and dermatan sulfate epimerase53 (DSE; fourfold, p < 0.05) (Fig. 5A).

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