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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.

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; Analytical Quality Control (AQC) in centralised preparation units of oncology centers is a common procedure relying on the identification and quantification of the prepared chemotherapeutic solutions for safe intravenous administration to patients. Although the use of Raman spectroscopy for AQC has gained much interest, in most applications it remains coupled to a flow injection analyser (FIA) requiring withdrawal of the solution for analysis. In addition to current needs for more rapid and cost-effective analysis, the risk of exposure of clinical staff to the toxic molecules during daily handling is a serious concern to address. Raman spectroscopic analysis, for instance by Confocal Raman Microscopy (CRM), could enable direct analysis (non-invasive) for AQC directly in infusion bags. In this study, 3 anticancer drugs, methotrexate (MTX), 5-fluorouracil (5-FU) and gemcitabine (GEM) have been selected to highlight the potential of CRM for withdrawal free analysis. Solutions corresponding to the clinical range of each drug were prepared in 5% glucose and data was collected from infusion bags placed under the Raman microscope. Firstly, 100% discrimination has been obtained by Partial Least Squares Discriminant Analysis (PLS-DA) confirming that the identification of drugs can be performed. Secondly, using Partial Least Squares Regression (PLSR), quantitative analysis was performed with mean % error of predicted concentrations of respectively 3.31%, 5.54% and 8.60% for MTX, 5-FU and GEM. These results are in accordance with the 15% acceptance criteria used for the current clinical standard technique, FIA, and the Limits of Detection for all drugs were determined to be substantially lower than the administered range, thus highlighting the potential of confocal Raman spectroscopy for direct analysis of chemotherapeutic solutions.

International audience; Recently, active targeting using nanocarriers with biological ligands has emerged as a novel strategy for improving the delivery of therapeutic and/or imaging agents to tumor cells. The presence of active targeting moieties on the surface of nanomedicines has been shown to play an important role in enhancing their accumulation in tumoral cells and tissues versus healthy ones. This property not only helps to increase the therapeutic index but also to minimize possible side effects of the designed nanocarriers. Since the overexpression of epidermal growth factor receptors (EGFR) is a common occurrence linked to the progression of a broad variety of cancers, the potential application of anti-EGFR immunotherapy and EGFR-targeting ligands in active targeting nanomedicines is getting increasing attention. Henceforth, the EGFR-targeted nanomedicines were extensively studied in vitro and in vivo but exhibited both satisfactory and disappointing results, depending on used protocols. This review is designed to give an overview of a variety of EGFR-targeting ligands available for nanomedicines, how to conjugate them onto the surface of nanoparticles, and the main analytical methods to confirm this successful conjugation.

In a clinical setting, analytical quality control of the administration of chemotherapeutic preparations is required to ensure patient safety with respect to the dose and more importantly the correct anticancer drug. As such, analytical tools enabling both qualitative and quantitative verification of the composition of prepared solutions can greatly benefit the hospital workflow, reducing cost and ultimately ensuring optimum patient care and outcome. Raman and infrared spectroscopy are rapid and cost-effective techniques, delivering label free molecular characterisation of samples. A comparative study has been conducted using four commercial intravenous formulations, DOXORUBICINE TEVA®, CERUBIDINE®, HOLOXAN® and METHOTREXATE MYLAN®, respectively containing doxorubicin, daunorubicin, ifosfamide and methotrexate as active drugs. Using clinically relevant concentration ranges prepared in 0.9 % NaCl or 5% glucose solutions, it is demonstrated that 100 % discrimination can be achieved for all formulations using either Raman or IR spectroscopic techniques, combined with multivariate discriminant analysis. Employing a partial least square regression analysis, Raman spectroscopy performed on liquid samples delivers a better accuracy compared to infrared for quantification, based on the mean squared error of cross validation. However, it is demonstrated that, despite the strong contribution of glucose and mannitol, an excipient found in CERUBIDINE®, infrared spectroscopy remains an equally viable option for translation into clinics.

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.

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.

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®.

International audience; Changes in protein concentrations within human blood are used as an indicator for nutritional state, hydration and underlying illnesses. They are often measured at regular clinical appointments and the current analytical process can result in long waiting times for results and the need for return patient visits. Attenuated total reflectance – Fourier transform infrared (ATR-FTIR) spectroscopy has the ability to detect minor molecular differences, qualitatively and quantitatively, in biofluid samples, without extensive sample preparation. ATR-FTIR can return an analytical measurement almost instantaneously and therefore could be deemed as an ideal technique for monitoring molecular alterations in blood within the clinic.To determine the suitability of using ATR-FTIR spectroscopy to enable protein quantification in a clinical setting, pooled human serum samples spiked with varying concentrations of human serum albumin (HSA) and immunoglobulin G (IgG) were analysed, before analysing patient clinical samples. Using a validated partial least squares method, the spiked samples (IgG) produced a linearity as high as 0.998 and a RMSEV of 0.49 ± 0.05 mg mL−1, with the patient samples producing R2 values of 0.992 and a corresponding RMSEV of 0.66 ± 0.05 mg mL−1. This claim was validated using two blind testing models, leave one patient out cross validation and k-fold cross validation, achieving optimum linearity and RMSEV values of 0.934 and 1.99 ± 0.79 mg mL−1, respectively.This demonstrates that ATR-FTIR is able to quantify protein within clinically relevant complex matrices and concentrations, such as serum samples, rapidly and with simple sample preparation. The ability to provide a quantification step, along with rapid disease classification, from a spectroscopic signature will aid clinical translation of vibrational spectroscopy to assist with problems currently faced with patient diagnostic pathways.

Gene therapy and particularly small interfering RNA (siRNA) is a promising therapeutic method for treatment of various human diseases, especially cancer. However the lack of an ideal delivery system limits its clinical applications. Effective anticancer drug development represents the key for translation of research advances into medicines. Previously we reported, the optimization of magnetic siRNA nanovectors (MSN) formulation based on superparamagnetic iron oxide nanoparticles (SPION) and chitosan for systemic administration. This work aimed at using rational design to further optimize and develop MSN. Therefore, formulated MSN were first purified, then their physical and chemical properties were studied mainly through capillary electrophoresis. 95% of siRNA was found enclosed within the purified MSN (pMSN). pMSN showed colloidal stability at pH 7.4, effective protection of siRNA against ribonuclease degradation up to 24 hours and few siRNA release (less than 10%) at pH 7.4. These findings push toward further evaluation studies in vitro and/or in vivo, indicating the appropriateness of pMSN for cancer theranostics.

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.

Section spéciale du Meeting SFNano 2018 : Nanomedicine- from particle design to applications (2018/12/03_2018/12/05, Montpellier, FRA), edited by Nathalie Mignet, Chantal Pichon et Marie-Pierre Rols; International audience; The association between superparamagnetic iron oxide nanoparticles (SPION), carrying small interfering RNA (siRNA) as therapeutic agents and humanized anti- human epidermal growth factor receptor-2 (HER2) single-chain antibody fragments (scFv) for the active delivery into HER2-overexpressing cells appears as an interesting approach for patients with HER2-overexpressing advanced breast cancer. The obtained Targeted Stealth Magnetic siRNA Nanovectors (TS-MSN) are formulated by combining: (i) the synthesis protocol of Targeted Stealth Fluorescent Particles (T-SFP) which form the core of TS-MSN and (ii) the formulation protocol allowing the loading of T-SFP with polyplexes (siRNA and cationic polymers). TS-MSN have suitable physico-chemical characteristics for intravenous administration and protect siRNA against enzymatic degradation up to 24 h. The presence of HER2-targeting scFv on TS-MSN allowed an improved internalization (3-4 times more compared to untargeted S-MSN) in HER2-overexpressing breast cancer cells (BT-474). Furthermore, anti-survivin siRNA delivered by TS-MSN in HER2-negative breast-cancer control cells (MDA-MB-231) allowed significant down-regulation of the targeted anti-apoptotic protein of about 70%. This protein down-regulation increased in HER2 + cells to about 90% (compared to 70% with S-MSN in both cell lines) indicating the contribution of the HER2-active targeting. In conclusion, TS-MSN are promising nanocarriers for the specific and efficient delivery of siRNA to HER2-overexpressing breast cancer cells.

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; 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.

International audience; 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 (β-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.

International audience; 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.

International audience; 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.

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.

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; Anticancer drugs are prescribed and administrated to an increasing number of patients on a daily basis. As a consequence, a number of concerns have been raised about the patient health and safety in the case that the drugs administered are not at the required concentration or even worse not the correct ones. Quality control of therapeutic solutions has therefore been extensively implemented in hospital environments, in order to avoid any failure in the intense workflow faced by administering pharmacists. In the present study, infrared (IR) and Raman spectroscopy have been employed for the analysis of 3 commercially available therapeutic solutions TEVA®, MYLAN®, CERUBIDINE®, respectively containing doxorubicin, epirubicin and daunorubicin. They perfectly illustrate the analytical difficulties encountered, as these 3 chemotherapeutic drugs are isomers, hardly distinguishable with conventional approaches such as UV/VIS spectrometry. Any analytical failure to identify these molecules can lead to delays in patient treatment. While Partial Least Squares Regression analysis demonstrates that both Raman and IR can deliver satisfactory quantitative analysis in the clinical range, with respective Root Mean Square Error of Cross Validation (RMSECV) between 0.0127 – 0.0220 g·L−1 and 0.0573 – 0.0759 g·L−1, the identification rate between the 2 techniques differs substantially. Indeed, Principal Component Analysis – Factorial Discriminant Analysis (PCA-FDA) highlights that, depending on the data preprocessing applied to Raman spectra, the discrimination between the 3 drugs is decreased, with in some cases specificity and sensitivity below 50%. However, IR analysis displays encouraging results with an overall specificity and sensitivity between 99 and 100%, suggesting that reliable validation of the therapeutic solution for administration to patients can be achieved. IR and Raman spectroscopy could assist and support quality control of chemotherapeutic solutions prepared in personalised concentrations for each patient. The effective and reliable characterisation of therapeutic solutions could have a lot to offer to improve current practices in a near future.

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; We describe a novel protocol for a one-step, seed-less, organic solvent- and surfactant-free synthesis of optically dense aqueous colloids of gold nanoflowers (AuNF), with tunable absorption wavelength between 620 and 800 nm. We demonstrate that simple variation of the ratio of two reagents allows the plasmonic band position to be tuned to any desired wavelength ± 5 nm, namely to those of the laser sources commonly used for SERS spectroscopy. The AuNF size distribution was sufficiently narrow, comparable to that known with seed-mediated synthesis. The AuNF have been validated as efficient aggregation-free substrates for surface-enhanced Raman scattering (SERS) spectroscopy using two common fluorescent dyes, Nile Blue and Crystal Violet, both thiol-free. Their fluorescence was quenched and SERS signal intensity was a linear function of the dye concentration, from nanomolar to micromolar range. Easy to prepare and to use, these AuNF appear as a particularly user-friendly and efficient way to obtain plasmonic substrates for SERS in the red and deep red spectral range.

The use of hybrid materials, where a matrix sustains nanoparticles controlling the release of the chemotherapeutic drug, could be beneficial for the treatment of primary tumors prior or after surgery. This localized chemotherapy would guarantee high drug concentrations at the tumor site while precluding systemic drug exposure minimizing undesirable side effects. We combined bacterial cellulose hydrogel (BC) and nanostructured lipid carriers (NLCs) including doxorubicin (Dox) as a drug model. NLCs loaded with cationic Dox (NLCs-H) or neutral Dox (NLCs-N) were fully characterized and their cell internalization and cytotoxic efficacy were evaluated in vitro against MDA-MB-231 cells. Thereafter, a fixed combination of NLCs-H and NLCs-N loaded into BC (BC-NLCs-NH) was assayed in vivo into an orthotopic breast cancer mouse model. NLCs-H showed low encapsulation efficiency (48%) and fast release of the drug while NLCs-N showed higher encapsulation (97%) and sustained drug release. Both NLCs internalized via endocytic pathway, while allowing a sustained release of the Dox, which in turn rendered IC50 values below of those of free Dox. Taking advantage of the differential drug release, a mixture of NLCs-N and NLCs-H was encapsulated into BC matrix (BC-NLCs-NH) and assayed in vivo, showing a significant reduction of tumor growth, metastasis incidence and local drug toxicities., Centro de Investigación y Desarrollo en Fermentaciones Industriales, Centro de Química Inorgánica

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.

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.

Infrared spectroscopy is a reliable, rapid and cost effective characterisation technique, delivering a molecular finger print of the sample. It is expected that its sensitivity would enable detection of small chemical variations in biological samples associated with disease. ATR-IR is particularly suitable for liquid sample analysis and, although air drying is commonly performed before data collection, just a drop of human serum is enough for screening and early diagnosis. However, the dynamic range of constituent biochemical concentrations in the serum composition remains a limiting factor to the reliability of the technique. Using glucose as a model spike in human serum, it has been demonstrated in the present study that fractionating the serum prior to spectroscopic analysis can considerably improve the precision and accuracy of quantitative models based on the partial least squares regression algorithm. By depleting the abundant high molecular weight proteins, which otherwise dominate the spectral signatures collected, the ability to monitor changes in the concentrations of the low molecular weight constituents is enhanced. The Root Mean Square Error for the Validation set (RMSEV) has been improved by a factor of 5 following human serum processing with an average relative error in the predictive values below 1% being achieved. Moreover, the approach is easily transferable to different bodily fluids, which would support the development of more efficient and suitable clinical protocols for exploration of vibrational spectroscopy based ex vivo diagnostic tools.

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; The endogenous mechanism of RNA interference is more and more used in research to obtain specific down-regulation of gene expression in diseases such as breast cancer. Currently, despite the new fields of study open up by RNA interference, the rapid degradation of siRNA by nucleases and their negative charges prevent them from crossing cell membranes. To overcome these limitations, superparamagnetic iron oxide nanoparticles (SPIONs) represent a promising alternative for nucleic acid delivery. Previously, we reported the magnetic siRNA nanovectors (MSN) formulation using electrostatic assembly of (1) SPIONs, also able to act as contrast agents for magnetic resonance imaging (MRI), (2) siRNA and (3) chitosan aiming at their protection and enhancing their transfection efficacy. However, these nanoparticles displayed low stability in biological suspensions and inefficient transfection of active siRNA.This work aimed at upgrading MSN to Stealth MSN (S-MSN) by adding a polyethylene glycol coating to ensure colloidal stability and stealth properties. Furthermore, another polymer (poly-L-arginine) was added for efficient siRNA transfection and the quantitative composition of the formulation was adapted for biological purposes. Results showed that S-MSN provide high siRNA complexation and protection against enzymatic degradation. Green fluorescent protein (GFP) specific down-regulation on MDA-MB231/GFP cells was comparable to that of commercially available reagents, without observable cytotoxicity. According to our works, S-MSN appears as an effective formulation for in vitro siRNA specific delivery.

International audience; We synthesized rationally designed multifunctional nanoparticles (NPs) composed of a superparamagnetic iron oxide nanoparticle (SPION) core, cyanine fluorescent dye emitting in far red, polyethylene glycol (PEG5000) coating, and the membranotropic peptide gH625, from the cell-penetrating peptides (CPP) family. The peptide sequence was enriched with an additional cysteine so it can be involved as a reactive moiety in a certain orientation- and sequence-specific coupling of the CPP to the PEG shell of the NPs. Our data indicate that the presence of approximately 23 peptide molecules per SPION coated with approximately 137 PEG chains minimally changes the overall NP characteristics. The final CPP-capped NP hydrodynamic diameter was 98nm, the polydispersity index was 0.192, and the zeta potential was 4.08mV. The in vitro evaluation, performed using an original technique fluorescence confocal spectral imaging, showed that after a short incubation duration (maximum 30min), SPIONs-PEG-CPP uptake was 3-fold higher than that for SPIONs-PEG. The CPP also drives the subcellular distribution of a higher NP fraction towards low polarity cytosolic locations. Therefore, the major cellular uptake mechanism for the peptide-conjugated NPs should be endocytosis. Enhancement/acceleration of this mechanism by gH625 appears promising because of potential applications of SPIONs-PEG-gH625 as a multifunctional nanoplatform for cancer theranosis involving magnetic resonance imaging, optical imaging in far red, drug delivery, and hyperthermia.

International audience

International audience; Short interfering RNAs (siRNAs) can downregulate the synthesis of proteins and thus be used to treat certain diseases where the protein synthesis is upregulated, such as cancer. The challenge is to deliver siRNAs in the target cell as they are rapidly degraded by nucleases and have difficulties to cross the cellular membranes. Superparamagnetic iron oxide nanoparticles (SPIONs) are widely studied as platforms for smart biocompatible nanosystems which can be used for magnetic drug targeting and magnetic resonance imaging. The aim of this work was to combine siRNAs, SPIONs, and chitosan, to develop new magnetic siRNA nanovectors suitable for systemic administration. In a first time, the one factor at a time (OFAT) methodology was used to adjust different formulation parameters and to test the feasibility of such a formulation. In a second time, design of experiment (DOE) methodology was used to analyze the influence of these formulation parameters on the physicochemical characteristics hydrodynamic diameter (DH) and ζ-potential. Finally, four MSNs suitable for systemic administration could be identified using the OFAT method. The DOE method showed a significant effect of CR and [NaNO3] on the DH and a significant effect of MR and [siRNA] on the ζ-potential of the nanocarriers.

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.

Background This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. Methods SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. Results The SFP-FA have zeta potential below − 6 mW and stable hydrodynamic diameter close to 100 nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22–27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. Conclusions The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. General significance Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.

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; In the present study, we describe the synthesis and characterization of new generation of cancer-targeting magnetic nanoprobes: superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylene glycol (PEG) shell functionalized with recombinant anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. An anti-HER2 scFv with terminal cysteine (scFv 4D5-Cys) has been rationally engineered in order to favor its orientation- and site-directed covalent conjugation to the polymeric surface of PEGylated SPIONs. Optimization of scFv and nanoparticles production allowed to obtain well-characterized SPIONs-PEG–scFv nanoparticles carrying ∼7 fragments per nanoparticle, having a hydrodynamic diameter of ca. 86 nm and nearly neutral surface. The nanoprobes-scFv capability to recognize the HER2 protein has been confirmed by enzyme-linked immunosorbent assay (ELISA). Compared to non-targeted PEGylated SPIONs, the SPIONs–PEG–scFv nanoprobes showed an enhanced binding to HER2-overexpressing cells (SK-BR3) in vitro as it was shown by immunofluorescence. Finally, ICP-AES measurements shown that in 1 hour the uptake of SPIONs–PEG–scFv in HER2-overexpressing cells is 2.1 times greater than non-targeted PEGylated SPIONs. Therefore, both due to their physico-chemical characteristics and the immunotargeting of HER2-positive breast cancer cells, the SPIONs–PEG–scFv appear as promising nanoplatforms for future applications in theranostic treatment of cancers.

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.

International audience; We report the efficient one-step synthesis and detailed physicochemical evaluation of novel biocompatible nanosystems useful for cancer therapeutics and diagnostics (theranostics). These systems are the superparamagnetic iron oxide nanoparticles (SPIONs) carrying the anticancer drug doxorubicin and coated with the covalently bonded biocompatible polymer poly(ethylene glycol) (PEG), native and modified with the biological cancer targeting ligand folic acid (PEG-FA). These multifunctional nanoparticles (SPION-DOX-PEG-FA) are designed to rationally combine multilevel mechanisms of cancer cell targeting (magnetic and biological), bimodal cancer cell imaging (by means of MRI and fluorescence), and bimodal cancer treatment (by targeted drug delivery and by hyperthermia effect). Nevertheless, for these concepts to work together, the choice of ingredients and particle structure are critically important. Therefore, in the present work, a detailed physicochemical characterization of the organic coating of the hybrid nanoparticles is performed by several surface-specific instrumental methods, including surface-enhanced Raman scattering (SERS) spectroscopy, X-ray photoelectron spectrometry (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). We demonstrate that the anticancer drug doxorubicin is attached to the iron oxide surface and buried under the polymer layers, while folic acid is located on the extreme surface of the organic coating. Interestingly, the moderate presence of folic acid on the particle surface does not increase the particle surface potential, while it is sufficient to increase the particle uptake by MCF-7 cancer cells. All of these original results contribute to the better understanding of the structure–activity relationship for hybrid biocompatible nanosystems and are encouraging for the applications in cancer theranostics.

International audience; Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a two-dimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.

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.

International audience; In the field of oncology, research is now focused on the development of theranostic nanosystems that combine the functions of drug delivery and imaging for diagnosis/monitoring. In this context, we designed polyethylene glycol (PEG)ylated superparamagnetic iron oxide nanoparticles (SPIONs) for the delivery of doxorubicin (DOX), an antineoplastic agent. These DOX-loaded PEGylated SPIONs, or DLPS, should be useful for the delivery of DOX in vivo, as well as for magnetic drug targeting (MDT) and magnetic resonance imaging (MRI). The aim of this study was to evaluate the potential applications of DLPS in vivo as drug carrier systems for the reduction of xenograft breast tumors induced in nude mice. Prior to the animal model experiments, the main internalization pathways for the nanovectors in MDA-MB435 breast cancer cells were determined to be based on caveolae- and clathrin-mediated endocytosis. The time- and quantity-dependence of the nanoparticle uptake by the cells altered the in vitro cytotoxicity of the DLPS. The in vitro antiproliferative effect of the DLPS was dependent not only on DOX concentration, but also on the efficacy of nanoparticle internalization. Evaluation of the effect of DLPS treatment on xenograft tumors in nude mice showed that DLPS limited tumor growth in a manner comparable to that of free DOX under normal conditions of tumor growth. The application of an external magnetic field on tumors, i.e., MDT, did not improve the efficacy of the DLPS treatment. Nevertheless, the vectorization of DOX with DLPS appears to limit the hematologic side effects usually associated with DOX treatment.

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; Metallic nanoparticles (MNPs) such as iron oxide and gold nanoparticles are interesting platforms to build theragnostic nanocarriers which combine both therapeutic and diagnostic functions within a single nanostructure. Nevertheless, their surface must be functionalized to be suitable for in vivo applications. Surface functionalization also provides binding sites for targeting ligands, and for drug loading. This review focuses on the materials and surface chemistry used to build hybrid nanocarriers that are inorganic cores functionalized with organic materials. The surface state of the MNPs largely depends on their synthesis routes, and dictates the strategies used for functionalization. Two main strategies can be found in the literature: the design of core-shell nanosystems, or embedding nanoparticles in organic materials. Emerging tendencies such as the use of clusters or alternative coating materials are also described. To present both hydrophilic and lipophilic nanosystems, we chose the doxorubicin anticancer agent as an example, as the molecule presents an affinity for both types of materials.

International audience; A study of the complex drying dynamics of polymeric mixtures with optical microscopy and gravimetric measurement is presented. Droplet formation is observed, followed by a collapse that leads to the residual craters in the dried film. The process is followed in situ under well-defined temperature and hygrometric conditions to determine the origin and nature of these droplets and craters. The drying process is usually completed within 1 h. The observations are explained using a simple diffusion model based on experimental results collected from mass and optical measurements as well as Raman confocal microspectrometry. Although the specific polymeric mixtures used here are of interest to the cosmetic industry, the general conclusions reached can apply to other polymeric aqueous solutions with applications to commercial and artistic painting.

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.

The aim of this tutorial review is to give an overview of the state of the art of intracellular applications of analytical SERS spectroscopy. We pay particular attention to nanoparticle-based SERS spectroscopy since this currently dominates the published literature on non-disturbing analysis of live cells. We describe recent advances in this domain due to the development of multispectral imaging and to the combined use of SERRS (surface-enhanced resonance Raman scattering) and fluorescence spectroscopy. Finally, a perspective view is given on the tip-based approaches like tip-enhanced Raman spectroscopy (TERS) which allow micrometric and nanometric resolution.

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.