Label-Free Quantification of Nanoencapsulated Piperonyl Esters in Cosmetic Hydrogels Using Raman Spectroscopy.

Raman spectroscopy is a well-established technique for the molecular characterisation of samples and does not require extensive pre-analytical processing for complex cosmetic products. As an illustration of its potential, this study investigates the quantitative performance of Raman spectroscopy coupled with partial least squares regression (PLSR) for the analysis of Alginate nanoencapsulated Piperonyl Esters (ANC-PE) incorporated into a hydrogel. A total of 96 ANC-PE samples covering a 0.4% w/w-8.3% w/w PE concentration range have been prepared and analysed. Despite the complex formulation of the sample, the spectral features of the PE can be detected and used to quantify the concentrations. Using a leave-K-out cross-validation approach, samples were divided into a training set (n = 64) and a test set, samples that were previously unknown to the PLSR model (n = 32). The root mean square error of cross-validation (RMSECV) and prediction (RMSEP) was evaluated to be 0.142% (w/w PE) and 0.148% (w/w PE), respectively. The accuracy of the prediction model was further evaluated by the percent relative error calculated from the predicted concentration compared to the true value, yielding values of 3.58% for the training set and 3.67% for the test set. The outcome of the analysis demonstrated the analytical power of Raman to obtain label-free, non-destructive quantification of the active cosmetic ingredient, presently PE, in complex formulations, holding promise for future analytical quality control (AQC) applications in the cosmetics industry with rapid and consumable-free analysis.

Confocal Raman Spectroscopic Imaging for Evaluation of Distribution of Nano-Formulated Hydrophobic Active Cosmetic Ingredients in Hydrophilic Films.

Film-forming systems are highly relevant to the topical administration of active ingredients (AI) to the body. Enhanced contact with the skin can increase the efficacy of delivery and penetration during prolonged exposure. However, after the evaporation of volatile solvents to form a thin film, the distribution of the ingredient should remain homogenous in order to ensure the effectiveness of the formula. This is especially critical for the use of hydrophobic molecules that have poor solubility in hydrophilic films. In order to address this concern, hydroxyphenethyl esters (PHE) of Punica granatum seed oil were prepared as a nanosuspension stabilised by poloxamers (NanoPHE). NanoPHE was then added to a formulation containing polyvinyl alcohol (PVA) as a film forming agent, Glycerol as a plasticiser and an antimicrobial agent, SepicideTM HB. Despite their reliability, reference methods such as high-performance liquid chromatography are increasingly challenged due to the need for consumables and solvents, which is contrary to current concerns about green industry in the cosmetics field. Moreover, such methods fail to provide spatially resolved chemical information. In order to investigate the distribution of ingredients in the dried film, Confocal Raman imaging (CRI) coupled to Non-negatively Constrained Least Squares (NCLS) analysis was used. The reconstructed heat maps from a range of films containing systematically varying PHE concentrations highlighted the changes in spectral contribution from each of the ingredients. First, using NCLS scores it was demonstrated that the distributions of PVA, Glycerol, SepicideTM HB and PHE were homogenous, with respective relative standard deviations (RSD) of 3.33%, 2.48%, 2.72% and 6.27%. Second, the respective relationships between ingredient concentrations in the films and their Raman responses, and the spectral abundance were established. Finally, a model for absolute quantification for PHE was be constructed using the percentage of spectral abundance. The prepared %w/w concentrations regressed against predicted %w/w concentrations, displaying high correlation (R2 = 0.995), while the Root Mean Squared Error (0.0869% w/w PHE) confirmed the precision of the analysis. The mean percent relative error of 3.75% indicates the accuracy to which the concentration in dried films could be determined, further supporting the suitability of CRI for analysis of composite solid film matrix. Ultimately, it was demonstrated that nanoformulation of hydrophobic PHE provides homogenous distribution in PVA based film-forming systems independent of the concentration of NanoPHE used in the formula.

Raman mapping coupled to self-modelling MCR-ALS analysis to estimate active cosmetic ingredient penetration profile in skin.

Confocal Raman mapping (CRM) is a powerful, label free, non-destructive tool, enabling molecular characterization of human skin with applications in the dermo-cosmetic field. Coupling CRM to multivariate analysis can be used to monitor the penetration and permeation of active cosmetic ingredients (ACI) after topical application. It is presently illustrated how multivariate curve resolution alternating least squares (MCR-ALS) can be applied to detect and semi-quantitatively describe the diffusion profile of Delipidol, a commercially available slimming ACI, from Raman spectral maps. Although the analysis outcome can be critically dependent on the a priori selection of the number of regression components, it is demonstrated that profiling of the kinetics of diffusion into the skin can be established with or without additionnal spectral equality constraints in the multivariate analysis, with similar results. Ultimately, MCR-ALS, applied without spectral equality contraints, specifically identifies the ACI as one of main spectral components enabling to investigate its distribution and penetration into the stratum corneum and underlying epidermis layers.

Homogeneous distribution of fatty ester-based active cosmetic ingredients in hydrophilic thin films by means of nanodispersion.

OBJECTIVE: Cosmetic films and patches are interesting forms to promote skin penetration of active ingredients as they ensure their long stay on the treated zone of the skin. Nevertheless, currently developed films and patches are most of all hydrophilic and are not adapted to the hydrophobic molecules. The aim of this study was to establish whether nanodispersion of fatty acid-based active cosmetic ingredients (ACI) could be a manner to introduce high concentrations of those ACI in hydrophilic films. METHODS: Punica granatum seed oil hydroxyphenethyl esters (PHE) constitute a commercialized lipolytic cosmetic ingredient obtained by enzymatic conjugation of tyrosol to long-chain fatty acids and to enhance its skin diffusion. Nanodispersions of PHE were prepared by a green emulsion-solvent evaporation process and dispersed in polyvinyl alcohol films. Raman imaging coupled to multivariate analysis was used to study the distribution of PHE in the films. RESULTS: Nanodispersions of PHE combined with antioxidant vitamin E and stabilized by Pluronic® F127 were successfully prepared. The nanodispersions show a spherical shape and a hydrodynamic diameter close to 100 nm. Raman images analysis with multivariate approaches showed a very homogeneous distribution of PHE nanodispersions in the films compared to free PHE introduced as an ethanol solution. CONCLUSION: Nanodispersions of hydrophobic fatty acid-based ingredients seem to be relevant method to introduce this type of ingredient in hydrophilic film matrix. The co-suspension with vitamin E limits their degradation in time.

OBJECTIF: Les films et patchs cosmétiques sont des formes intéressantes pour augmenter la pénétration cutanée des actifs cosmétiques car ils assurent une exposition prolongée de la zone de peau traitée ce qui favorise la diffusion. Néanmoins, les films et patchs actuellement développés sont majoritairement de nature hydrophile et ne sont pas adaptés aux molécules hydrophobes. Le but de cette étude est d'établir si la nanodispersion d'actifs cosmétiques à base d'acides gras peut être un moyen d'introduire des concentrations élevées de ces actifs dans des films hydrophiles. MÉTHODES: Les esters hydroxyphénéthyliques de l'huile de graines de grenade Punica granatum (PHE) sont commercialisés comme un agent lipolytique. Cet actif obtenu par conjugaison enzymatique du tyrosol à des acides gras à longue chaîne ce qui favorise sa pénétration cutanée. Des nanodispersions de PHE ont été préparées par un procédé d'émulsion- évaporation développé avec un solvant vert. Ces nanosystèmes sont ensuite dispersées dans des films d'alcool polyvinylique. L'imagerie Raman couplée à une méthode d'analyse statistique multivariée a été utilisée pour étudier la distribution des PHE dans les films. RÉSULTATS: Des nanodispersions de PHE associées à de la vitamine E antioxydante et stabilisées par Pluronic® F127 ont été préparées avec succès. Les nanodispersions présentent une forme sphérique et un diamètre hydrodynamique proche de 100 nm. L'analyse d'images Raman au moyen d'une approche multivariée a montré une distribution très homogène des nanodispersions dans les films par rapport aux PHE libres introduits sous forme de solution éthanolique. CONCLUSION: Les nanodispersions d'ingrédients hydrophobes à base d'acides gras semblent être une méthode pertinente pour introduire ce type d'ingrédient dans la matrice de film hydrophile. L'introduction de vitamine E dans les nanodispersion ralentit leur dégradation.

Quantification of low-content encapsulated active cosmetic ingredients in complex semi-solid formulations by means of attenuated total reflectance-infrared spectroscopy.

Attenuated total reflectance-infrared (ATR-IR) spectroscopy is a robust tool for molecular characterisation of matter. Applied to semi-solid formulations, it enables rapid and reliable data collection without pre-analytical requirements. Based on nano-encapsulated Omegalight®, a skin-lightening active cosmetic ingredient (ACI), incorporated in a hydrogel, it is first demonstrated that, despite the high water content and the chemical complexity of the samples (i.e. number of ingredients), the spectral features of the ACI can be detected and monitored. Secondly, with a total of 105 samples divided into a training set (n = 60) and an unknown set (n = 45) covering a 0.5% w/w-5% w/w concentration range, the study further investigates the quantitative performance of ATR-IR coupled with partial least squares regression (PLSR). Through a step-by-step approach in testing different cross-validation protocols, accuracy (root mean square error of cross-validation (RMSECV)) and linearity between the experimental and predicted concentrations (R2) of ATR-IR are consistently evaluated to be respectively 0.097% (w/w) and 0.995 with a lower LOD = 0.067% (w/w). Subsequently, further evaluation of the accuracy (relative error of the predicted concentration compared with the true value, expressed as %) of the analysis was undertaken with the 45 unknown samples that were defined as unknown and analysed by PLSR. The outcome of the analysis demonstrates the ruggedness and the consistency of the determination performed using the ATR-IR data. With an average relative error of 2.5% w/w and only 5 samples out of 45 blind samples exhibiting a relative error above the 5% threshold, high accuracy quantification of the nano-encapsulated ACI can be unambiguously achieved by means of the label-free and non-destructive technique of ATR-IR spectroscopy. Ultimately, the study demonstrates that the analytical capabilities of ATR-IR hold significant potential for applications in the cosmetics industry, and although the path remains long, the present study is one step further to support validation of the technique, albeit for the specific case of Omegalight®.

Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus.

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age-related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi-mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro-inflammatory cytokines and extracellular matrix-degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi-related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non-senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.

ATR-IR coupled to partial least squares regression (PLSR) for monitoring an encapsulated active molecule in complex semi-solid formulations.

Attenuated Total Reflectance-Infrared (ATR-IR) spectroscopy holds great promise for industrial applications as a quality control tool for complex galenic formulations. Although the technique is often promoted for the molecular information it delivers in a label free and cost effective fashion, other advantages can emerge compared to the gold standard analytical tools such as liquid chromatography coupled to mass spectrometry. The present study demonstrates how ATR-IR measurements enable accurate quantitative analysis of an active cosmetic ingredient such as Omegalight® encapsulated in a complex alginate based nano-capsule. The study demonstrates how precise concentrations can be obtained without the requirement for fastidious extraction and separation protocols prior to ATR-IR analysis. However, data mining remains a crucial aspect with particular emphasis on the preprocessing of the data that will be subjected to Partial Least Squares Regression (PLSR) analysis. Therefore, different pre-processing methods have been evaluated to investigate the relationship between corrections applied and PLSR outcomes (i.e. precision, ratio of performance to deviation (RPD) and accuracy of the analysis). Ultimately, it has been found that, against all expectations, some of the preprocessing methods do not necessarily lead to improvements in the end result, while Extended Multiplicative Scattering Correction (EMSC) is the only one which delivers satisfying results, as defined by a Root Mean Square Error (RMSEV) of 0.07% (w/w) and a RPD greater than 30 when performing analysis over the range 0.4-8.2% (w/w). Despite the presence of large amounts of additives such as glycerol and preservatives in the formulation, implementing Leave One Out Cross Validation (LOOCV) further validates the method with a RPD of 18 and relative errors for the predicted concentrations below the 5% (w/w) threshold, hence demonstrating that ATR-IR has analytical capabilities for applications in the cosmetic industry.

Confocal Raman spectroscopic imaging for in vitro monitoring of active ingredient penetration and distribution in reconstructed human epidermis model.

Topically applied active cosmetic ingredients (ACI) or active pharmaceutical ingredients (API) efficacy is directly related to their efficiency of penetration in the skin. In vitro reconstructed human epidermis surrogate models offer in vivo like skin samples for transdermal studies. Using Delipidol®, an ACI currently used in the cosmetics industry, the capabilities to deliver accurate distribution maps and penetration profiles of this molecule by means of confocal Raman spectroscopic imaging have been demonstrated. Using a non-negative constrained least squares (NCLS) approach, contribution of specific molecules can be estimated at each point of spectral maps in order to deliver semi-quantitative heat maps representing the ACI levels in the different skin layers. The concentration profiles obtained are approximately single exponential for all 3 time points evaluated, with a consistent decay constant, which is independent of the sublayer structure. Notably, however, there is no significant penetration into the lower basal layers until a critical concentration is built up, after 3 hours. Combination of Raman confocal imaging with spectral unmixing methods such as NCLS is demonstrated to be a relevant approach for in vitro biological evaluation of cosmetic and pharmaceutical active ingredients and could easily be implemented as a screening tool for industrial use.

Qualitative and Quantitative Study of the Potential of Lipid Nanocapsules of One Hundred Twenty Nanometers for the Topical Administration of Hydrophobic Molecules.

In this study, we evaluated the potential of lipid nanocapsules (LNC) of 120 nm as drug nanocarriers to treat skin diseases. As a model molecule, we encapsulated the fluorescent dye curcumin, which also is an antioxidant. Curcumin-loaded LNC showed interesting antioxidant properties and a low toxicity on human skin cells. The penetration of curcumin in the skin was determined by 2 complementary methods: high performance liquid chromatography was used to measure total curcumin accumulation in the skin, whereas fluorescence confocal spectral imaging of skin sections showed that curcumin preferentially accumulates in the stratum corneum and the viable epidermis. These results confirm that LNC of a size above 100 nm can vectorize hydrophobic compounds to the keratinocytes without transdermal delivery. They also demonstrate the interest of combining 2 analytical methods when studying the skin penetration of nanovectorized molecules.

Effects of a skin neuropeptide (substance p) on cutaneous microflora.

BACKGROUND: Skin is the largest human neuroendocrine organ and hosts the second most numerous microbial population but the interaction of skin neuropeptides with the microflora has never been investigated. We studied the effect of Substance P (SP), a peptide released by nerve endings in the skin on bacterial virulence. METHODOLOGY/PRINCIPAL FINDINGS: Bacillus cereus, a member of the skin transient microflora, was used as a model. Exposure to SP strongly stimulated the cytotoxicity of B. cereus (+553±3% with SP 10(-6) M) and this effect was rapid (<5 min). Infection of keratinocytes with SP treated B. cereus led to a rise in caspase1 and morphological alterations of the actin cytoskeleton. Secretome analysis revealed that SP stimulated the release of collagenase and superoxide dismutase. Moreover, we also noted a shift in the surface polarity of the bacteria linked to a peel-off of the S-layer and the release of S-layer proteins. Meanwhile, the biofilm formation activity of B. cereus was increased. The Thermo unstable ribosomal Elongation factor (Ef-Tu) was identified as the SP binding site in B. cereus. Other Gram positive skin bacteria, namely Staphylococcus aureus and Staphylococcus epidermidis also reacted to SP by an increase of virulence. Thermal water from Uriage-les-Bains and an artificial polysaccharide (Teflose®) were capable to antagonize the effect of SP on bacterial virulence. CONCLUSIONS/SIGNIFICANCE: SP is released in sweat during stress and is known to be involved in the pathogenesis of numerous skin diseases through neurogenic inflammation. Our study suggests that a direct effect of SP on the skin microbiote should be another mechanism.