Design, optimization, characterization, and in vitro evaluation of metformin-loaded liposomes for triple negative breast cancer treatment.

Recently, metformin (Met) has shown to have antineoplastic properties in cancer treatment by improving hypoxic tumor conditions, and causing reduction in the synthesis of biomolecules, which are vital for cancer growth. However, as an orally administered drug, Met has low bioavailability and rapid renal clearance. Thus, the goal of this study was to vectorize Met inside liposomes in the context of triple negative breast cancer (TNBC), which currently lacks treatment options when compared to other types of breast cancer. Vectorization of Met inside liposomes was done using Bangham method by implementing double design of experiment methodology to increase Met drug loading (minimum-run resolution V characterization design and Box-Behnken design), as it is generally extremely low for hydrophilic molecules. Optimization of Met-loaded liposome synthesis was successfully achieved with drug loading of 190 mg/g (19% w/w). The optimal Met-liposomes were 170 nm in diameter with low PdI (< 0.1) and negative surface charge (-20 mV), exhibiting sustained Met release at pH 7.4. The liposomal Met delivery system was stable over several months, and successfully reduced TNBC cell proliferation due to the encapsulated drug. This study is one the first reports addressing liposome formulation through thin-film hydration using two design of experiment methods aiming to increase drug loading of Met.

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

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

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

Formation of miRNA Nanoprobes-Conjugation Approaches Leading to the Functionalization.

Recently, microRNAs (miRNA) captured the interest as novel diagnostic and prognostic biomarkers, with their potential for early indication of numerous pathologies. Since miRNA is a short, non-coding RNA sequence, the sensitivity and selectivity of their detection remain a cornerstone of scientific research. As such, methods based on nanomaterials have emerged in hopes of developing fast and facile approaches. At the core of the detection method based on nanotechnology lie nanoprobes and other functionalized nanomaterials. Since miRNA sensing and detection are generally rooted in the capture of target miRNA with the complementary sequence of oligonucleotides, the sequence needs to be attached to the nanomaterial with a specific conjugation strategy. As each nanomaterial has its unique properties, and each conjugation approach presents its drawbacks and advantages, this review offers a condensed overview of the conjugation approaches in nanomaterial-based miRNA sensing. Starting with a brief recapitulation of specific properties and characteristics of nanomaterials that can be used as a substrate, the focus is then centered on covalent and non-covalent bonding chemistry, leading to the functionalization of the nanomaterials, which are the most commonly used in miRNA sensing methods.

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

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.

Comparison of Vibrational Spectroscopic Techniques for Quantification of Water in Natural Deep Eutectic Solvents.

Vibrational spectroscopic techniques, i.e., attenuated total reflectance infrared (ATR-IR), near infrared spectroscopy (NIRS) and Raman spectroscopy (RS), coupled with Partial Least Squares Regression (PLSR), were evaluated as cost-effective label-free and reagent-free tools to monitor water content in Levulinic Acid/L-Proline (LALP) (2:1, mol/mol) Natural Deep Eutectic Solvent (NADES). ATR-IR delivered the best outcome of Root Mean Squared Error (RMSE) of Cross-Validation (CV) = 0.27% added water concentration, RMSE of Prediction (P) = 0.27% added water concentration and mean % relative error = 2.59%. Two NIRS instruments (benchtop and handheld) were also compared during the study, respectively yielding RMSECV = 0.35% added water concentration, RMSEP = 0.56% added water concentration and mean % relative error = 5.13% added water concentration, and RMECV = 0.36% added water concentration, RMSEP = 0.68% added water concentration and mean % relative error = 6.23%. RS analysis performed in quartz cuvettes enabled accurate water quantification with RMECV = 0.43% added water concentration, RMSEP = 0.67% added water concentration and mean % relative error = 6.75%. While the vibrational spectroscopic techniques studied have shown high performance in relation to reliable determination of water concentration, their accuracy is most likely related to their sensitivity to detect the LALP compounds in the NADES. For instance, whereas ATR-IR spectra display strong features from water, Levulinic Acid and L-Proline that contribute to the PLSR predictive models constructed, NIRS and RS spectra are respectively dominated by either water or LALP compounds, representing partial molecular information and moderate accuracy compared to ATR-IR. However, while ATR-IR instruments are common in chemistry and physics laboratories, making the technique readily transferable to water quantification in NADES, Raman spectroscopy offers promising potential for future development for in situ, sample withdrawal-free analysis for high throughput and online monitoring.

Highlighting the efficiency of ultrasound-based emulsifier-free emulsions to penetrate reconstructed human skin.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

OBJECTIF: 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. CONCLUSION: 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.

Comparison of Raman and attenuated total reflectance (ATR) infrared spectroscopy for water quantification in natural deep eutectic solvent.

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.

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.

In Situ Water Quantification in Natural Deep Eutectic Solvents Using Portable Raman Spectroscopy.

Raman spectroscopy is a label-free, non-destructive, non-invasive analytical tool that provides insight into the molecular composition of samples with minimum or no sample preparation. The increased availability of commercial portable Raman devices presents a potentially easy and convenient analytical solution for day-to-day analysis in laboratories and production lines. However, their performance for highly specific and sensitive analysis applications has not been extensively evaluated. This study performs a direct comparison of such a commercially available, portable Raman system, with a research grade Raman microscope system for the analysis of water content of Natural Deep Eutectic Solvents (NADES). NADES are renewable, biodegradable and easily tunable "green" solvents, outcompeting existing organic solvents for applications in extraction from biomass, biocatalysis, and nanoparticle synthesis. Water content in NADES is, however, a critical parameter, affecting their properties, optimal use and extraction efficiency. In the present study, portable Raman spectroscopy coupled with Partial Least Squares Regression (PLSR) is investigated for rapid determination of water content in NADES samples in situ, i.e., directly in glassware. 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 28.5% (w/w), were studied. The results are directly compared with previously published studies of the same systems, using a research grade Raman microscope. PLSR results demonstrate the reliability of the analysis, surrendering R2 values above 0.99. Root Mean Square Errors Prediction (RMSEP) of 0.6805%, 0.9859% and 1.2907% w/w were found for respectively unknown CCG, BG and GG samples using the portable device compared to 0.4715%, 0.3437% and 0.7409% w/w previously obtained by analysis in quartz cuvettes with a Raman confocal microscope. Despite the relatively higher values of RMSEP observed, the comparison of the percentage of relative errors in the predicted concentration highlights that, overall, the portable device delivers accuracy below 5%. Ultimately, it has been demonstrated that portable Raman spectroscopy enables accurate quantification of water in NADES directly through glass vials without the requirement for sample withdrawal. Such compact instruments provide solvent and consumable free analysis for rapid analysis directly in laboratories and for non-expert users. Portable Raman is a promising approach for high throughput monitoring of water content in NADES that can support the development of new analytical protocols in the field of green chemistry in research and development laboratories but also in the industry as a routine quality control tool.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

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®.

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.

Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly.

Healthcare-associated infections resulting from bacterial attachment and biofilm formation on medical implants are posing significant challenges in particular with the emergence of bacterial resistance to antibiotics. Here, we report the design, synthesis and characterization of self-assembled nanostructures, which integrate on their surface antibacterial peptides. The antibacterial WMR peptide, which is a modification of the native sequence of the myxinidin, a marine peptide isolated from the epidermal mucus of hagfish, was used considering its enhanced activity against Gram-negative bacteria. WMR was linked to a peptide segment of aliphatic residues (AAAAAAA) containing a lipidic tail (C19H38O2) attached to the ε-amino of a terminal lysine to generate a peptide amphiphile (WMR PA). The self-assembly of the WMR PA alone, or combined with coassembling shorter PAs, was studied using spectroscopy and microscopy techniques. The designed PAs were shown to self-assemble into stable nanofiber structures and these nanoassemblies significantly inhibit biofilm formation and eradicate the already formed biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Candida albicans (pathogenic fungus) when compared to the native WMR peptide. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity, and establish an innovative strategy to develop self-assembled antimicrobial materials for biomedical applications.

gH625 Cell-Penetrating Peptide Promotes the Endosomal Escape of Nanovectorized siRNA in a Triple-Negative Breast Cancer Cell Line.

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.

Raman spectroscopy as a potential tool for label free therapeutic drug monitoring in human serum: the case of busulfan and methotrexate.

A methodology is proposed, based on Raman spectroscopy coupled with multivariate analysis, to determine the Limit of Detection (LOD) and Limit of Quantification (LOQ) for therapeutic drug monitoring in human serum, using the examples of Busulfan, a cell cycle non-specific alkylating antineoplastic agent, and, Methotrexate, a chemotherapeutic agent and immune system suppressant. In this study, ultrafiltration is employed to fractionate spiked human pooled serum to efficiently recover the drug in the filtrate prior to performing Raman analysis. The drug concentration ranges were chosen to encompass the recommended therapeutic ranges and toxic levels in patients. Raman spectra were collected from the filtrates in the liquid form, using an inverted backscattering microscopic geometry, using 532 nm as source. Finally, prediction models were built by using Partial Least Squares Regression (PLSR) and LOD and LOQ were calculated directly from the linear prediction models. The LOD calculated for Busulfan is 0.0002 ± 0.0001 mg mL-1, 30-40 times lower than the level of toxicity, enabling the application of this method in target dose adjustment of Busulfan for patients undergoing, for example, bone marrow transplantation. The LOD and LOQ calculated for Methotrexate are 7.8 ± 5 µM and 26 ± 5 µM, respectively, potentially enabling high dose monitoring. The promising results obtained from this study suggest the potential of Raman spectroscopy for therapeutic drug monitoring of drugs in bodily fluids.

Raman spectroscopic screening of high and low molecular weight fractions of human serum.

This study explores the suitability of Raman spectroscopy as a bioanalytical tool, when coupled with ultra-filtration and multivariate analysis, to detect imbalances in both high molecular weight (total protein content, γ globulins and albumin) and low molecular weight (urea and glucose) fractions of the same samples of human patient serum, in the native liquid form. Ultracentrifugation was employed to separate and concentrate the high and low molecular weight fractions of the serum. Initially, aqueous solutions of the respective molecular species, covering physiologically relevant concentration ranges, were analysed to optimise the measurement protocols. An adapted Extended Multiplicative Signal Correction (EMSC) algorithm was applied to raw spectra to remove water background signal and spectral interferents (ß-carotene). Using a validated partial least squares regression modelling method, R2 values, Root Mean Square Error of Cross Validation (RMSECV) and standard deviations were established for the quantification of γ globulin, total protein, albumin, urea and glucose content of the patient serum samples. The study demonstrates that Raman spectroscopy in the liquid form is a viable alternative and/or adjunct to current clinical practice for the parallel analysis of high and low molecular weight fractions, and simultaneous analysis of multiple analytes in the low molecular weight fraction, of human serum for diagnostic applications.

Analysis of bodily fluids using vibrational spectroscopy: a direct comparison of Raman scattering and infrared absorption techniques for the case of glucose in blood serum.

Analysis of biomarkers present in the blood stream can potentially deliver crucial information on patient health and indicate the presence of numerous pathologies. The potential of vibrational spectroscopic analysis of human serum for diagnostic purposes has been widely investigated and, in recent times, infrared absorption spectroscopy, coupled with ultra-filtration and multivariate analysis techniques, has attracted increasing attention, both clinical and commercial. However, such methods commonly employ a drying step, which may hinder the clinical work flow and thus hamper their clinical deployment. As an alternative, this study explores the use of Raman spectroscopy, similarly coupled with ultra-filtration and multivariate analysis techniques, to quantitatively monitor diagnostically relevant changes of glucose in liquid serum samples, and compares the results with similar analysis protocols using infrared spectroscopy of dried samples. The analysis protocols to detect the imbalances in glucose using Raman spectroscopy are first demonstrated for aqueous solutions and spiked serum samples. As in the case of infrared absorption studies, centrifugal filtration is utilised to deplete abundant analytes and to reveal the spectral features of Low Molecular Weight Fraction analytes in order to improve spectral sensitivity and detection limits. Improved Root Mean Square Error of Cross Validation (RMSECV) was observed for Raman prediction models, whereas slightly higher R2 values were reported for infrared absorption prediction models. Summarising, it is demonstrated that the Raman analysis protocol can yield accuracies which are comparable with those reported using infrared absorption based measurements of dried serum, without the need for additional drying steps.

Site-Specific Conjugation of Auristatins onto Engineered scFv Using Second Generation Maleimide to Target HER2-positive Breast Cancer in Vitro.

Antibody-drug conjugates (ADC) are spearheading vectorized chemotherapy against cancer, with 4 FDA-approved ADCs and 79 in clinical trials. However, most ADCs are produced using a stochastic bioconjugation method, target hematological cancers, and are derived from a full immunoglobulin-G (IgG). These factors limit their efficacy, especially against solid tumors which remain difficult to treat. Here we report the site-specific conjugation of a single auristatin derivative onto an engineered anti-HER2 single chain fragment variable (scFv) of the trastuzumab antibody, generating new scFv-drug conjugates (SDC). Two cysteines were judiciously incorporated at the beginning of the scFv hexahistidine tag, in order to allow controlled bioconjugation of a heterobifunctional linker including a second generation maleimide (SGM), either cleavable (for monomethyl auristatin E) or noncleavable (for monomethyl auristatin F). Our data indicated that both SDCs conserved their affinity to HER2 in comparison to the native scFv, and were efficiently able to kill in vitro HER2-positive SK-BR-3 cells at subnanomolar concentrations (EC50 of 0.68 nM and 0.32 nM). No effect was observed on HER2-negative MCF-7 cells. Ours results showed efficient targeting of site-specific SDCs against HER2-positive breast cancer cells. This work represents a first important step in the design of more effective small conjugates, paving the way for future in vivo translation to evaluate their full potential.

Impact of Site-Specific Conjugation of ScFv to Multifunctional Nanomedicines Using Second Generation Maleimide.

Biocompatible multifunctional nanomedicines (NMs) are known to be an attractive platform for targeted anticancer theranosis. However, these nanomedicines are of interest only if they efficiently target diseased cells and accumulate in tumors. Here we report the synthesis of a new generation of immunotargeted nanomedicines composed of a superparamagnetic iron oxide nanoparticle (SPION) core, polyethylene glycol coating and the anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. We developed two novel bioengineered scFv carrying two cysteines located (i) at the end (4D5.1-cys2) or (ii) at the beginning (4D5.2-cys2) of its hexahistidine tag. The scFv bioconjugation was controlled via heterobifunctional linkers including a second generation maleimide (SGM). Our data indicated that the insertion of cysteines at the beginning of the hexahistidine tag was allowed to obtain nearly 2-fold conjugation efficiency (13 scFv/NP) compared to NMs using classical maleimide. As a result, the NMs-4D5.2 built using the optimal 4D5-cys2 and linkers equipped with SGM showed the enhanced recognition of HER2 in an ELISA format and on the surface of SK-BR-3 breast cancer cells in vitro. Their stability in serum was also significantly improved compared to the NMs-4D5. Our results showed the fundamental importance of the controlled ligand conjugation in the perspective of rational design of NMs with tailored physicochemical and biological properties.

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.