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The cosmetic industry endeavours to strengthen the greener and safer claims of processes to respond to the high demand from customers for natural and environmentally friendly products. High-frequency ultrasonication technology (HFUT) is a physical process enabling the stabilization of emulsions without requiring additional ingredients, such as emulsifying surfactants (ES) to be introduced into the formulations. In this study, key formulation characteristics of an emulsion synthesized by HFUT and a reference emulsion (RE) were compared, as well as the permeation kinetics of caffeine, used as a model active cosmetic ingredient, from both types of emulsions.The pH, droplet size and viscosity of emulsions prepared by the HFUT and the RE were determined and compared. The permeation of caffeine from the HFUT emulsion and the RE applied to the surface of reconstructed human epidermis (RHE) models was compared.The ES-free formulations prepared by HFUT displayed a nearly 2-fold lower average droplet size and over 3-fold greater viscosity, compared to the RE. Despite these differences, the absence of ES in the HFUT emulsion did not significantly alter the permeation kinetics of caffeine through RHE. The caffeine steady-state flux, lag time and permeability coefficients differed by 20%-30% only.This study demonstrates the potential of the HFUT to yield topical cosmetic products with lower requirements ingredients-wise, without losing efficacy, supporting the possible implementation of the technology in the cosmetic industry.l’industrie cosmétique œuvre à renforcer les revendications plus écologiques et plus sûres des processus pour répondre à la forte demande des clients de produits naturels et plus respectueux de l’environnement. La technologie d’ultrasons à haute fréquence (High-Frequency Ultrasonication Technology, HFUT) est un processus physique permettant de stabiliser les émulsions sans qu’il soit nécessaire d’ajouter des ingrédients supplémentaires, tels que des surfactants émulsifiants, aux formulations. Dans cette étude, les principales caractéristiques de formulation d’une émulsion synthétisée par HFUT et d’une émulsion de référence ont été comparées, ainsi que la cinétique de perméation de la caféine, utilisée comme ingrédient cosmétique actif modèle, dans les deux types d’émulsion. MÉTHODES: le pH, la taille des gouttelettes, et la viscosité de l’émulsion préparée par HFUT et de l’émulsion de référence ont été déterminés et comparés. La perméation de la caféine de l’émulsion HFUT et de l’émulsion de référence appliquées à la surface de modèles d’épiderme humain reconstruit a été comparée. RÉSULTATS: la formulation sans surfactants émulsifiants préparée par HFUT présentait une taille moyenne de gouttelettes presque 2 fois plus faible et une viscosité plus de 3 fois supérieure comparée à l’émulsion de référence. Malgré ces différences, l’absence de surfactants émulsifiants dans l’émulsion HFUT n’a pas significativement modifié la cinétique de perméation de la caféine dans l’épiderme humain reconstruit. Le flux à l’état d’équilibre de la caféine, le temps de latence et les coefficients de perméabilité différaient de 20 à 30 % uniquement.cette étude démontre le potentiel de la technologie HFUT à générer des produits cosmétiques topiques possédant des exigences plus faibles en termes d’ingrédients, sans perte d’efficacité, soutenant la mise en œuvre éventuelle de la technologie dans l’industrie cosmétique.

Attenuated total reflectance-infrared spectroscopy (ATR-IR) coupled with partial least squares regression (PLSR) was evaluated as a rapid, label free and cost-effective tool to quantify water content in extracts obtained from spirulina wet biomass using a glucose glycerol natural deep eutectic solvent (NADES). NADESs are green, renewable and biodegradable solvents with unique properties outcompeting existing organic solvents, for instance, for plant or biomass extraction. The properties of NADESs depend critically on their water concentration, and therefore, it is essential to develop methods to monitor it, to ensure optimal extraction efficiency and experimental repeatability to achieve a better standardization of extraction protocols. First, Karl Fischer titration was performed on a set of 20 NADES extracts in order to obtain reference water concentrations. Secondly, ATR-IR spectra were collected and subjected to datamining to construct PLSR predictive models. An

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR—partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400–1500 cm−1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.

International audience; Natural deep eutectic solvents (NADES) are ionic solutions, of great interest for extraction from biomass, biocatalysis, and nanoparticle synthesis. They are easily synthesised and eco-friendly, have low volatility and high dissolution power, and are biodegradable. However, water content in NADES is a critical parameter, affecting their optimal use and extraction efficiency. Vibrational spectroscopic techniques are rapid, label-free, non-destructive, non-invasive, and cost-effective analytical tools that can probe the molecular composition of samples. A direct comparison between a previous study using attenuated total reflectance infrared (ATR-IR) spectroscopy for water quantification in NADES and the same investigation performed with Raman spectroscopy is presently reported. Three NADES systems, namely betaine-glycerol (BG), choline chloride-glycerol (CCG), and glucose-glycerol (GG), containing a range of water concentrations between 0% (w/w) and 40% (w/w), have been analysed with Raman spectroscopy coupled to partial least squares regression multivariate analysis. The values of root mean square error of cross-validation (RMSECV) obtained from analysis performed on the pre-processed spectra over the full spectral range (150–3750 cm−1) are respectively 0.2966% (w/w), 0.4703% (w/w), and 0.2351% (w/w) for BG, GG, and CCG. While the direct comparison to previous ATR-IR results shows essentially similar outcomes for BG, the RMSECV is 33.14% lower and 65.84% lower for CG and CCG. Furthermore, mean relative errors obtained with Raman spectroscopy, and calculated from a set of samples used as independent samples, were 1.452% (w/w), 1.175% (w/w), and 1.188% (w/w). Ultimately, Raman spectroscopy delivered performances for quantification of water in NADES with similar accuracy to ATR-IR. The present demonstration clearly highlights the potential of Raman spectroscopy to support the development of new analytical protocols in the field of green chemistry.

International audience; This work relies on the use of dip-coating at various withdrawal speeds to form nanocomposite films, with a detailed analysis of the influence of the mode of deposition on the nanoparticle (NP) concentration in the dried film. While the deposition of polymer solutions on the one hand and colloidal suspensions of NPs on the other hand have been separately studied by dip coating, their combination is far being a simple superposition of the two separate behaviors. The formation of nanocomposite thin films composed of model water-soluble biocompatible polymers (polyvinyl alcohol - PVA and polyvinylpyrrolidone - PVP) loaded with poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was studied through a dip coating process. Flat silicon substrates were removed at a controlled withdrawal speed from an aqueous colloidal suspension. Thin films of PLGA NPs with concentrations ranging from 1 to 10 % wt./wt. in PVA or PVP matrices were prepared. The presence of nanoparticles on the well-established process of thin film deposition was examined, as well as the influence of the deposition regime on the nanoparticle concentration in the deposited coating. We demonstrate that the presence of colloidal dispersion of PLGA nanoparticles in water solution of PVA and PVP does not modify the process of film deposition by dip coating. A typical “V” shaped curve was observed, with two well-known deposition regimes: capillary and draining modes respectively obtained at low and high withdrawal speeds. Due to crystallization at low withdrawal speed (favored by slow evaporation of the solvent) it was not possible to identify individual PLGA nanoparticles by AFM in the case of the PVA matrice. Amorphous PVP nanocomposite films were successfully prepared by dip coating, and allowed us to identify individual PLGA nanoparticles with AFM. Because of the prevalence of an evaporation-driven phenomenon at low withdrawal speed, incorporation of NPs was observed over the whole range of withdrawal speeds, showing original behavior compared to recent studies relying on a pure Landau-Levich regime (i.e. non-evaporative systems). Our results indicate that the nanoparticles were not equally retrieved from the solution in the capillary and the draining regimes. This suggests that the balance between the viscous drag and the interfacial effects depends on the deposition mode and calls for a more detailed analysis of the physical processes involved in both regimes.

International audience; 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.

The development and characterization of reconstructed human epidermis (RHE) is an active area of R&amp, D. RHE can replace animal tissues in pharmaceutical, toxicological and cosmetic sciences, yielding scientific and ethical advantages. RHEs remain costly, however, due to consumables and time required for their culture and a short shelf-life. Storing, i.e., freezing RHE could help reduce costs but to date, little is known on the effects of freezing on the barrier function of RHE. We studied such effects using commercial EpiSkin&trade, RHE stored at &minus, 20, &minus, 80 and &minus, 150 &deg, C for 1 and 10 weeks. We acquired intrinsic Raman spectra in the stratum corneum (SC) of the RHEs as well as spectra obtained following topical application of resorcinol in an aqueous solution. In parallel, we quantified the effects of freezing on the permeation kinetics of resorcinol from time-dependent permeation experiments. Principal component analyses discriminated the intrinsic SC spectra and the spectra of resorcinol-containing RHEs, in each case on the basis of the freezing conditions. Permeation of resorcinol through the frozen RHE increased 3- to 6-fold compared to fresh RHE, with the strongest effect obtained from freezing at &minus, 20 &deg, C for 10 weeks. Due to the extensive optimization and standardization of EpiSkin&trade, RHE, the effects observed in our work may be expected to be more pronounced with other RHEs.

Natural Deep Eutectic solvents (NADES) are green, ionic solutions prepared by mixing vegetal cellular constituents. They are renewable and biodegradable solvents composed of hydrogen bonded donor and acceptor compounds. NADES have a wide range of applications, mainly as solvents for extraction of plants, biocatalysis, and nanoparticle synthesis. Water content is very critical, impacting significantly on NADES properties. Therefore, controlling H2O concentration appears essential for optimal use of NADES. In the present study, Attenuated Total Reflectance – infrared spectroscopy (ATR-IR) coupled to multivariate analysis, namely Partial Least Square s regression (PLSR), was investigated as a rapid, label free and cost-effective tool for measuring the water concentration of NADES. Betaine_Glycerol (BG), Choline Chloride_Glycerol (CCG) and Glucose_Glycerol (GG) were the selected model systems, each over a range of concentrations between 0% (w/w) and 40% (w/w). PLSR results demonstrate the robustness of the analysis, yielding R2 values of 0.99 and Root Mean Square Error of Cross Validation (RMSECV) of respectively 0.2602% w/w, 0.6883% w/w and 0.7034% w/w. Moreover, the % relative error achieved is below a 5% threshold, further highlighting the suitability of ATR-IR for water content monitoring. This preliminary work demonstrates the appropriateness of developing easily accessible analytical tools to support the development of green chemistry solvents, which currently represent a major focus of interest in both research and industrial environments.

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.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.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.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.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.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.

Biofilm-related infections are a matter of concern especially because of the poor susceptibility of microorganisms to conventional antimicrobial agents. Innovative approaches are needed. The antibiofilm activity of extracts of cyanobacteria Arthrospira platensis, rich in free fatty acids, as well as of extract-loaded copper alginate-based nanocarriers, were studied on single- and dual-species biofilms of Candida albicans and Cutibacterium acnes. Their ability to inhibit the biofilm formation and to eradicate 24 h old biofilms was investigated. Concentrations of each species were evaluated using flow cytometry. Extracts prevented the growth of C. acnes single-species biofilms (inhibition &gt, 75% at 0.2 mg/mL) but failed to inhibit preformed biofilms. Nanovectorised extracts reduced the growth of single-species C. albicans biofilms (inhibition &gt, 43% at 0.2 mg/mL) while free extracts were weakly or not active. Nanovectorised extracts also inhibited preformed C. albicans biofilms by 55% to 77%, whereas the corresponding free extracts were not active. In conclusion, even if the studied nanocarrier systems displayed promising activity, especially against C. albicans, their efficacy against dual-species biofilms was limited. This study highlighted that working in such polymicrobial conditions can give a more objective view of the relevance of antibiofilm strategies by taking into account interspecies interactions that can offer additional protection to microbes.

International audience; 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®.

Core-shell nanocarriers are increasingly being adapted in cosmetic and dermatological fields, aiming to provide an increased penetration of the active pharmaceutical or cosmetic ingredients (API and ACI) through the skin. In the final form, the nanocarriers (NC) are usually prepared in hydrogels, conferring desired viscous properties for topical application. Combined with the high chemical complexity of the encapsulating system itself, involving numerous ingredients to form a stable core and quantifying the NC and/or the encapsulated active without labor-intensive and destructive methods remains challenging. In this respect, the specific molecular fingerprint obtained from vibrational spectroscopy analysis could unambiguously overcome current obstacles in the development of fast and cost-effective quality control tools for NC-based products. The present study demonstrates the feasibility to deliver accurate quantification of the concentrations of curcumin (ACI)-loaded alginate nanocarriers in hydrogel matrices, coupling partial least square regression (PLSR) to infrared (IR) absorption and Raman spectroscopic analyses. With respective root mean square errors of 0.1469 ± 0.0175% w/w and 0.4462 ± 0.0631% w/w, both approaches offer acceptable precision. Further investigation of the PLSR results allowed to highlight the different selectivity of each approach, indicating only IR analysis delivers direct monitoring of the NC through the quantification of the Labrafac®, the main NC ingredient. Raman analyses are rather dominated by the contribution of the ACI which opens numerous perspectives to quantify the active molecules without interferences from the complex core-shell encapsulating systems thus positioning the technique as a powerful analytical tool for industrial screening of cosmetic and pharmaceutical products. Graphical abstract Quantitative analysis of encapuslated active molecules in hydrogel-based samples by means of infrared and Raman spectroscopy.

International audience; The application of small interfering RNA (siRNA) cancer therapeutics is limited by several extra- and intracellular barriers including the presence of ribonucleases that degrade siRNA, the premature clearance, the impermeability of the cell membrane, or the difficulty to escape endo-lysosomal degradation. Therefore, several delivery systems have emerged to overcome these limitations and to successfully deliver siRNA to the tumor site. This review is focused on polymer-based siRNA nanovectors which exploit the negative charge of siRNA, representing a major challenge for siRNA delivery, to their advantage by loading siRNA via electrostatic assembly. These nanovectors are easy to prepare and to adapt for an optimal gene silencing efficiency. The ability of electrostatically assembled polymeric siRNA nanovectors (EPSN) to improve the half-life of siRNA, to favor the specificity of the delivery and the accumulation in tumor and to enhance the cellular uptake and endosomal escape for an efficient siRNA delivery will be discussed. Finally, the influence of the versatility of the structure of these nanovectors on the protein down-regulation will be evaluated.

International audience; The use of small interfering RNA (siRNA) to regulate oncogenes appears as a promising strategy in the context of cancer therapy, especially if they are vectorized by a smart delivery system. In this study, we investigated the cellular trafficking of a siRNA nanovector (called CS-MSN) functionalized with the cell-penetrating peptide gH625 in a triple-negative breast cancer model. With complementary techniques, we showed that siRNA nanovectors were internalized by both clathrin- and caveolae-mediated endocytosis. The presence of gH625 at the surface of the siRNA nanovector did not modify the entry pathway of CS-MSN, but it increased the amount of siRNA found inside the cells. Results suggested an escape of siRNA from endosomes, which is enhanced by the presence of the peptide gH625, whereas nanoparticles continued their trafficking into lysosomes. The efficiency of CS-MSN to inhibit the GFP in MDA-MB-231 cells was 1.7-fold higher than that of the nanovectors without gH625.

The hyperspectral imaging is commonly used in the cosmetic area. Indeed, spectroscopic imaging techniques are usually employed to study the molecular composition of a cosmetic product, notably the Raman imaging. For this matter, Nonnegative Constrained Least Square (NCLS), has been studied previously and has provided accurate distribution maps of Active Cosmetic Ingredient (ACI) with its associated penetration profile. However, it remains a supervised method since it requires an a priori knowledge of the Raman fingerprint of the ACI to track it and the availability of a large number of spectra from control data affects its performance. This work presents the comparison of a Minimum Volume Constrained Non-negative Matrix Factorization (MVC-NMF) with the NCLS and a popular method in the chemometry community, Multivariate Curve Resolution Alternating Least Square (MCR-ALS) for hyperspectral image analysis. MVC-NMF proposes an unsupervised geometric approach to better fit a linear model to the data that provides lower modelling residuals. We also evaluate the parameter selection of the right number of constituent of Raman imaging from skin samples. It is shown that the MVC-NMF was able to accurately estimate the Raman spectrum of the ACI without supervision.

International audience; Cyanobacteria and especially Spirulina platensis are recognized as abundant resources of lipids and polyunsaturated fatty acids (PUFA). The main valorization of these lipids has been the biofuel production but another approach was developed here: the investigation of S. platensis fatty acids as anti-biofilm agent for skin diseases. In fact, chronic wounds represent an important burden for occidental health care systems, and are usually related to the presence of a biofilm, especially of Candida species.In order to develop a biomimetic approach against Candida biofilm formation, free fatty acids (FFA) isolated from microalgae S. platensis were explored. Four solvents, including three bio-sourced ones, and three extraction conditions were screened. Lipid and pigment amounts, combined with FFA profiles were analyzed to select the optimal conditions: 30 min of ultrasonic extraction using dimethylcarbonate or ethylacetate as extraction solvent. A vectorization using a macroalgal-alginate nanocarrier was successfully performed. Vectorized extracts were safe towards keratinocytes and thus compatible with a topical use. Spirulina lipid-enriched extracts showed a high anti-biofilm growth activity at low concentrations (about 80% inhibition after 24 h at 0.2 mg/mL). The combination of extracts in copper-alginate nanocarriers potentiated the anti-biofilm growth activity and exhibited a good anti-biofilm activity (about 50% inhibition after 24 h at 0.1 mg/mL). The combined approach of encapsulated Spirulina lipid extracts represented thus a relevant concept to develop all in one anti-biofilm weapons against Candida and represent a new perspective for algae biomass valorization.

International audience; 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.

International audience; 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.

Methods of femtosecond laser ablation were used to fabricate bare (ligand-free) silicon (Si) nanoparticles in deionized water. The nanoparticles were round in shape, crystalline, free of any impurities, and water-dissolvable, while the dissolution rate depended on the concentration of oxygen defects in their composition. The nanoparticles were then eletrospun with chitosan to form nanoparticle decorated nanofibrous matrices. We found that the functionalization of nanofibers by the nanoparticles can affect the morphology and physico-chemical characteristics of resulting nanostructures. In particular, the presence of Si nanoparticles led to the reduction of fibers thickness, suggesting a potential improvement of fiber’s surface reactivity. We also observed the improvement of thermal stability of hybrid nanofibers. We believe that the incorporated Si nanoparticles can serve as functional elements to improve characteristics of chitosan-based matrices for cellular growth, as well as to enable novel imaging or therapeutic functionalities for tissue engineering applications.

The development of an efficient small interfering RNA (siRNA) delivery system has held scientists interest since the discovery of the RNA interference mechanism (RNAi). This strategy gives hope for the treatment of many severe diseases. Herein, we developed hybrid nanovectors able to deliver siRNA to triple negative breast cancer cells. The nanovectors are based on PEGylated superparamagnetic iron oxide nanoparticles (SPION) functionalized with gH625 peptide, chitosan and poly-l-arginine. Every component has a key role and specific function: SPION is the core scaffolding the nanovector; PEG participates in the colloidal stability and the immune stealthiness; gH625 peptide promotes the nanovector internalization into cancer cells; cationic polymers provide the siRNA protection and favor siRNA endosomal escape and delivery to cytosol. The formulation was optimized by varying the amount of each compound. The efficacy of the siRNA retention and protection were investigated in the presence of high concentration of serum. Optimized nanovectors show a high uptake by MDA-MB-231 cells. The resulting down regulation of GFP expression was 73 ± 3% with our nanovector compared to 59 ± 8% obtained with the siRNA-Oligofectamine™ complex in the same conditions.

International audience; OBJECTIVE: The present study aims at increasing the concentration of a hydrophobic lightening agent, Omegalight® , in a hydrophilic cosmetic product by means of encapsulation in lipid-based submicron capsules. The core of these capsules is entirely made of the commercial lightening agent.METHODS: Lipid-based encapsulation systems (LNC) were prepared by the PIT method. Their physicochemical properties were followed over 6 months by dynamic light scattering and zeta potential measurements, and in parallel the potential degradation of the active ingredient was monitored by HPLC. The stability of the capsules in a cosmetic gel was studied by spectrofluorimetry and rheology measurements. Sensory analysis was used to determine the influence of the presence of capsules in the gel on the consumer's experience.RESULTS: LNC encapsulating Omegalight® were prepared on a laboratory scale and then on a semi-pilot scale. Their hydrodynamic diameters are around 230 nm. The concentration of Omegalight® in the capsules reaches about 84% w/w, which corresponds to 42% of active ingredient. LNC can be dispersed without degradation at concentrations of up to 20% w/w in a hydrogel without modification of the physicochemical or sensory properties of the gel.CONCLUSION: Lipid-based capsules (LNC), an encapsulation system useful for the epidermal delivery of hydrophobic compounds, were adapted to the encapsulation of a commercial lightening agent. The encapsulation permits the dispersion in a stable manner of a very high concentration of a hydrophobic active molecule in a hydrogel while maintaining the physicochemical and sensory properties of the gel. This article is protected by copyright. All rights reserved.

International audience

International audience; Hybrid (organic/inorganic) nanoparticles emerged as a simple solution to build "theranostic" systems. Due to their physical properties, superparamagnetic iron oxide nanoparticles (SPIONs) and plasmonic gold nanoparticles (Au-NPs) are extensively studied as a part of diagnostic and therapeutic strategies in cancer treatments. They can be used as agents for in vitro or in vivo imaging, for magnetic drug targeting and/or thermal therapy. Their functionalization with organic shells enhances their potential performance in tumor targeting and drug delivery. The advances in such hybrid nanocarriers are well illustrated with the example of the anticancer drug doxorubicin (DOX). The aim of this review is to give a multidisciplinary overview of such smart nanosystems loaded with DOX, based on examples taken from recent publications. From a physico-chemical point of view, we discuss the choices for the strategies for loading DOX and the consequences on drug release. From a biological point of view, we analyze the in vitro and in vivo assays concerning tumor imaging, targeted drug delivery and anticancer efficiency. Future opportunities and challenges are also addressed.

International audience; Calcium alginate nanocarriers (CaANCs) were developed as a potential tool for delivery of hydrophobic active molecules such as pharmaceutical and cosmetic active ingredients. In this study, we focused on interactions between CaANCs and keratinocytes in culture and examined toxicity, internalization and drug release. Prior to cellular interactions, cryogenic transmission electron microscopy images showed that CaANCs appear as regular, spherical and dense particles, giving evidence of the surface gelation of CaANCs. Their size, around 200 nm, was stable under tested conditions (temperature, culture media, presence of serum and presence of encapsulated dye), and their toxicity on keratinocytes was very low. Flow cytometry assays showed that CaANCs are internalized into keratinocytes by endocytosis with a predominant implication of the caveolae-mediated route. Förster resonance energy transfer (FRET) demonstrated that after a 2 h contact, the release of CaANC contents in the cytoplasm of keratinocytes was almost complete. The endocytosis of CaANCs by a lysosome-free pathway, and the rapid release of their contents inside keratinocytes, will allow vectorized molecules to fully exhibit their pharmacological or cosmetic activity.

International audience; The aim of this work was to elucidate the impact of polyethylene glycol (PEG) polymeric coating on the in vitro and in vivo stealthiness of magnetic nanocarriers loaded or not with the anticancer drug doxorubicin. The comparison was made between aqueous suspensions of superparamagnetic iron oxide nanoparticles (SPIONs) stabilized by either citrate ions (C-SPIONs) or PEG(5000) (P-SPIONs), the latter being loaded or not with doxorubicin via the formation of a DOX-Fe(2+) complex (DLP-SPIONs). After determination of their relevant physico-chemical properties (size and surface charge), nanoparticle (NP) stealthiness was studied in vitro (ability to activate the complement system and uptake by monocytes and macrophage-like cells) and in vivo in mice (blood half-life; t(1/2), and biodistribution in main clearance organs). These aspects were quantitatively assessed by atomic absorption spectrometry (AAS). Complement activation dramatically decreased for sterically stabilized P-SPIONs and DLP-SPIONs in comparison with C-SPIONs stabilized by charge repulsion. Monocyte and macrophage uptake was also largely reduced for pegylated formulations loaded or not with doxorubicin. The t(1/2) in blood for P-SPIONs was estimated to be 76 ± 6 min, with an elimination mainly directed to liver and spleen. Thanks to their small size (

International audience; One of the new strategies to improve cancer chemotherapy is based on new drug delivery systems, like the polyethylene glycol-coated superparamagnetic iron oxide nanoparticles (PEG-SPION, thereafter called PS). In this study, PS are loaded with doxorubicin (DOX) anticancer drug, using a pre-formed DOX-Fe(2+) complex reversible at lower pH of tumour tissues and cancer cells. The DOX loaded PS (DLPS, 3% w/w DOX/iron oxide) present a hydrodynamic size around 60nm and a zeta potential near zero at physiological pH, both parameters being favourable for increased colloidal stability in biological media and decreased elimination by the immune system. At physiological pH of 7.4, 60% of the loaded drug is gradually released from the DLPS in ∼2h. The intracellular release and distribution of DOX is followed by means of confocal spectral imaging (CSI) of the drug fluorescence. The in vitro cytotoxicity of the DLPS on MCF-7 breast cancer cells is equivalent to that of a DOX solution. The reversible association of DOX to the SPION surface and the role of polymer coating on the drug loading/release are discussed, both being critical for the design of novel stealth magnetic nanovectors for chemotherapy.

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 (RHE) 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 NCLS (Non negative constrained least squares) 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 three 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 screening tool for industrial use.

This work describes a method for preparation of sub-micron poly( d , l -lactide-co-glycolide) (PLGA) particles loaded with magnetite/maghemite nanoparticles to be used as magnetically-controlled drug delivery systems. The methodology of simple emulsion/evaporation technique has been optimized to provide greater iron oxide loading rates. The surface of iron oxide nanoparticles was coated with oleic acid (OA) for better compatibility with organic phase containing the polymer. To increase their loading into polymeric sub-micron particles, we added dried iron oxide nanoparticles in variable ferrite/polymer ratio of 1:1; 1:1.5 and 1:2 w/w. Composition and surface properties of obtained composite sub-micron particles have been studied in comparison with those of ferrite-free PLGA sub-micron particles. Presence of magnetite/maghemite was qualitatively confirmed by characteristic bands in the FT-IR spectra of composite sub-micron particles. Quantification of the incorporated iron was achieved by AAS. The highest incorporation rates of ferrite (up to 13.5% w/w) were observed with initial ferrite/polymer ratio of 1:1 w/w. TEM images indicate that the composite sub-micron particles are nearly spherical. According to laser granulometry data, average hydrodynamic diameter of the composite sub-micron particles is close to 280 nm, independently of ferrite presence. Electrophoretic properties (zeta potential) were very similar for both composite and ferrite-free PLGA sub-micron particles, thus indicating that the polymeric coating should mask the surface of ferrite nanoparticles buried inside. Finally, composite sub-micron particles exhibit superparamagnetic property.

International audience; The cutaneous penetration of hydrophobic active molecules is of foremost concern in the dermatology and cosmetic formulation fields. The poor solubility in water of those molecules limits their use in hydrophilic forms such as gels, which are favored by patients with chronic skin disease. The aim of this work is to design a novel nanocarrier of hydrophobic active molecules and to determine its potential as an ingredient of a topical form. The nanocarrier consists of an oily core surrounded by a protective shell of alginate, a natural polysaccharide isolated from brown algae. These calcium alginate-based nanocarriers (CaANCs) were prepared at room temperature and without the use of organic solvent by an accelerated nanoemulsification-polymer crosslinking method. The size (hydrodynamic diameter ~200 nm) and surface charge (zeta potential ~ − 30 mV) of the CaANCs are both compatible with their application on skin. CaANCs loaded with a fluorescent label were stable in model hydrophilic galenic forms under different storage conditions. Curcumin was encapsulated in CaANCs with an efficiency of ~95%, fully retaining its antioxidant activity. The application of the curcumin-loaded CaANCs on excised human skin led to a significant accumulation of the active molecules in the upper layers of the skin, asserting the potential of these nanocarriers in active pharmaceutical and cosmetic ingredients topical delivery.

International audience; The intracellular distribution of the antiancer drug doxorubicin (DOX) was followed qualitatively by fluorescence confocal spectral imaging (FCSI) and quantitatively by capillary electrophoresis (CE). FCSI permits the localization of the major fluorescent species in cell compartments, with spectral shifts indicating the polarity of the respective environment. However, distinction between drug and metabolites by FCSI is difficult due to their similar fluorochromes, and direct quantification of their fluorescence is complicated by quantum yield variation between different subcellular environments. On the other hand, capillary electrophoresis with fluorescence detection (CE-LIF) is a quantitative method capable of separating doxorubicin and its metabolites. In this paper, we propose a method for determining drug and metabolite concentration in enriched nuclear and cytosolic fractions of cancer cells by CE-LIF, and we compare these data with those of FCSI. Significant differences in the subcellular distribution of DOX are observed between the drug administered as a molecular solution or as a suspension of drug-loaded iron oxide nanoparticles coated with polyethylene glycol. Comparative analysis of the CE-LIF vs FCSI data may lead to a tentative calibration of this latter method in terms of DOX fluorescence quantum yields in the nucleus and more or less polar regions of the cytosol.

International audience; We report here the development of stable aqueous suspensions of biocompatible superparamagnetic iron oxide nanoparticles (SPIONs). These so-called ferrofluids are useful in a large spectrum of modern biomedical applications, including novel diagnostic tools and targeted therapeutics. In order to provide prolonged circulation times for the nanoparticles in vivo, the initial iron oxide nanoparticles were coated with a biocompatible polymer poly(ethylene glycol) (PEG). To permit covalent bonding of PEG to the SPION surface, the latter was functionalized with a coupling agent, 3-aminopropyltrimethoxysilane (APS). This novel method of SPION PEGylation has been reproduced in numerous independent preparations. At each preparation step, particular attention was paid to determine the physico-chemical characteristics of the samples using a number of analytical techniques such as atomic absorption, Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy, transmission electron microscopy (TEM), photon correlation spectroscopy (PCS, used for hydrodynamic diameter and zeta potential measurements) and magnetization measurements. The results confirm that aqueous suspensions of PEGylated SPIONs are stabilized by steric hindrance over a wide pH range between pH 4 and 10. Furthermore, the fact that the nanoparticle surface is nearly neutral is in agreement with immunological stealthiness expected for the future biomedical applications in vivo.

International audience; The clinical use of the anticancer drug doxorubicin (DOX) is limited by strong side effects and phenomena of cell resistance. Drug targeting by binding DOX to nanoparticles could overcome these limitations. We recently described a method to associate DOX to superparamagnetic iron oxide nanoparticles (SPION) in view of magnetic drug targeting (Munnier et al. in Int J Pharm 363:170–176, 2008). DOX is bound to the nanoparticle surface through a pre-formed DOX–Fe2+ complex. The DOX-loaded SPION present interesting properties in terms of drug loading and biological activity in vitro. The purpose of this study is to explore the possible mechanisms of the in vitro cytotoxicity of DOX-loaded SPION. The uptake of SPION was followed qualitatively by conventional optical microscopy after Prussian blue staining and quantitatively by iron determination by atomic absorption spectroscopy. The subcellular distribution of intrinsically fluorescent DOX was followed by confocal spectral imaging (CSI) and the subsequent cytotoxicity by the MTT method. We reveal modifications of DOX intracellular interactions for SPION-delivered drug and increased cytotoxicity. These results are discussed in terms of internalization route of the drug and of a potential role of iron oxide nanoparticles in the observed cytotoxicity.

International audience; We report a one-pot synthesis protocol for highly efficient and stable covalent binding of both the fluorescent drug doxorubicin (DOX) and the biocompatible polymer poly(ethylene glycol) (PEG) to the surface of superparamagnetic iron oxide nanoparticles (SPIONs). The final aim is to obtain a biocompatible, injectable nanosystem combining anticancer activity (magnetically targeted drug delivery) and nondestructive imaging of the treated cancer cells and tissues by means of fluorescence and magnetic resonance imaging (MRI). Our protocol employs silane and epoxide chemistry, which could also be useful to bind other molecules possessing a primary or secondary amine group, such as drugs, proteins, and fluorescent labels. The suspensions of SPIONs-DOX-PEG (iron concentration of 17 mg/L) obtained in this study are stable at physiological pH values. This stability coupled with the PEG surface neutrality makes these nanoparticles compatible with their application in vivo, via systemic administration. Efficient binding of DOX to the SPIONs surface via the amine group of the sugar moiety of the drug, i.e., outside of the aromatic pharmacophore-fluorophore, preserves the fluorescence activity of DOX. Confocal fluorescence spectral imaging of treated MCF7 cancer cells indicates that, in spite of the accumulation of SPIONs-DOX-PEG in the cytosol, only a minor fraction of the drug reaches the nucleus in 24 h. As a result, no in vitro cytotoxicity against MCF7 cancer cells was detected (the highest iron and drug concentrations were 2.7 mg/L and 8.1 µM, respectively). Interestingly, SPIONs-DOX particles noncoated with PEG were cytotoxic. We conclude that cellular enzymes can cleave the amine drug-particle linkage, but the PEG shell hinders the cleavage, possibly by sterical repulsion. Therefore, the developed chemistry is useful for stable coating of SPIONs with polymers and fluorescent labels, while an alternative strategy will be needed for more efficient drug release.

A new method of reversible association of doxorubicin (DOX) to superparamagnetic iron oxide nanoparticles (SPION) is developed for magnetically targeted chemotherapy. The efficacy of this approach is evaluated in terms of drug loading, delivery kinetics and cytotoxicity in vitro. Aqueous suspensions of SPION (ferrofluids) were prepared by coprecipitation of ferric and ferrous chlorides in alkaline medium followed by surface oxidation by ferric nitrate and surface treatment with citrate ions. The ferrofluids were loaded with DOX using a pre-formed DOX-Fe(2+) complex. The resulting drug loading was as high as 14% (w/w). This value exceeds the maximal loading known from literature up today. The release of DOX from the nanoparticles is strongly pH-dependent: at pH 7.4 the amount of drug released attains a plateau of approximately 85% after 1h, whereas at pH 4.0 the release is almost immediate. At both pH, the released drug is iron-free. The in vitro cytotoxicity of the DOX-loaded SPION on the MCF-7 breast cancer cell line is similar to that of DOX in solution or even higher, at low-drug concentrations. The present study demonstrates the potential of the novel method of pH-sensitive DOX-SPION association to design novel magnetic nanovectors for chemotherapy.

During the last decade, the application of nanotechnologies for anticancer drug delivery has been extensively explored, hoping to improve the efficacy and to reduce side effects of chemotherapy. The present review is dedicated to a certain kind of anticancer drug nanovectors developed to target tumors with the help of an external magnetic field. More particularly, this work treats anticancer drug nanoformulations based on superparamagnetic iron oxide nanoparticles coated with biocompatible polymers. The major purpose is to focus on the specific requirements and technological difficulties related to controlled delivery of antitumoral agents. We attempt to state the problem and its possible perspectives by considering the three major constituents of the magnetic therapeutic vectors: iron oxide nanoparticles, polymeric coating and anticancer drug.

International audience; Increase of lipophilicity of cationic doxorubicin (DOX) by its association with a fatty acid ion is of interest for pharmaceutical formulations and could have an impact on the drug delivery into cancer cells. On the basis of spectroscopic analysis of intrinsic DOX fluorescence, this study provides an experimental evidence of DOX-oleate interactions as function of ion/drug molar ratio (R) and pH. An electrostatic attraction to oleates is dominant for the cationic form of DOX (pH 6.5) and a hydrophobic interaction is characteristic of the molecular form of DOX (pH 8.6). A high content of sodium oleate vesicles ([oleate]>/=0.2 mM, R>/=20) limits the electrostatic and hydrophobic interactions at pH 6.5 while favoring the hydrophobic interactions at pH 8.6. The influence of these interactions on the lipophilicity of the cationic form of DOX is analyzed by measuring the apparent partition coefficient (aqueous buffer pH 6.5/methylene chloride). The results show a lipophilicity gain for the cationic form of DOX in presence of 10 : 1 ion/drug molar ratio, while no lipophilicity increase is observed at 50 : 1 molar ratio.

International audience; This study describes how the control of doxorubicin (DOX) polarity allows to encapsulate it inside poly(lactide-co-glycolide) (PLGA) nanoparticles formulated either by a single oil-in-water (O/W) or a double water-in-oil-in-water (W/O/W) emulsification method (SE and DE, respectively). DOX is commercially available as a water soluble hydrochloride salt, which is useful for DE. The main difficulty related to DE approach is that the low affinity of hydrophilic drugs to the polymer limits entrapment efficiency. Compared to DE method, SE protocol is easier and should provide an additional gain in entrapment efficiency. To be encapsulated by SE technique, DOX should be used in a more lipophilic molecular form. We evaluated the lipophilicity of DOX in terms of apparent partition coefficient (P) and modulated it by adjusting the pH of the aqueous phase. The highest P values were obtained at pH ranging from 8.6 to 9, i. e. between two DOX pK(a) values (8.2 and 9.6). The conditions favorable for the drug lipophilicity were then used to formulate DOX-loaded PLGA nanoparticles by SE method. DOX encapsulation efficiency as well as release profiles were evaluated for these nanoparticles and compared to those with nanoparticles formulated by DE. Our results indicate that the encapsulation of DOX in nanoparticles formulated by SE provides an increased drug entrapment efficiency and decreases the burst effect.