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BackgroundCerium (Ce) is a rare earth element, rapidly oxidizing to form CeO2, and currently used in numerous commercial applications, especially as nanoparticles (NP). The potential health effects of Ce remain uncertain, but literature indicates the development of rare earth pneumoconiosis accompanied with granuloma formation, interstitial fibrosis and inflammation. The exact underlying mechanisms are yet not completely understood, and we propose that autophagy could be an interesting target to study, particularly in macrophages. Therefore, the objective of our study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO2 NP in mice. Mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO2 NP by single oropharyngeal administration and sacrificed up to one month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFβ1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype). Results: Such pulmonary exposure to CeO2 NP induces a progressive and dose-dependent lung fibrosis in the bronchiolar and alveolar walls, together with the activation of autophagy. Blockade of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, via the modulation of macrophage polarization towards M2 phenotype. Conclusion: In conclusion, our findings bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as potential new therapeutic targets for future anti-fibrotic therapies.

International audience; RNA interference discovery has opened up new fields of study, including the understanding of dysregulation occurring in pathogenesis, especially in cancerogenesis. Through this mechanism, the controlled specific down-regulation improves the identification of gene function involved in a biological mechanism and their contribution in an altered phenotype. In this way, high-throughput screening (HTS) of RNAi has emerged as a potent tool in order to study a large number of gene functions for the identification of new biomarkers and/or therapeutic targets. However, HTS requires for that purpose generic carriers with highly efficient transfection. Recently, progresses have been made regarding the design of non-viral vectors (lipids, peptides, polymers) as delivery agents. The efficiency and specificity of gene-based expression of siRNA have been improved, while at the same time reducing toxicity. Among these carriers, lipid-based agents, already used as commercial reagents for in vitro transfection, are considered as a promising approach for siRNA delivery.Here, we have developed novel nanostructured lipid carriers through a design of experiment approach which promote high transfection efficiency in several human cell lines. These lipid nanoparticles present an outstanding colloidal stability, whereas their cationic shell promotes ready- to-use features for fast and simple complexation with nucleic acids by electrostatic bonds establishment, without inducing cytotoxicity. As evidenced by gel retardation assay, complexation is stable over time, even in cell culture media, enabling thus stable transfection efficiency for all samples in a protocol of large-scale screening. Compared to the commercially available lipoplexes, these lipid nanoemulsions offer the possibility to combine several functions into a unique carrier, such as fluorescent particle tracking through the encapsulation of lipophilic dyes and/or the co-delivery of other drugs. Thereby, the distribution monitoring into cells with nanocarriers entrapping lipophilic cyanine derivatives demonstrates an improved internalization of nucleic acids. Furthermore, preliminary experiments performed on primary cells in culture demonstrated also an improved transfection compared to commercially available lipoplexes, opening thus the way for RNAi screening in that cells recognised as hard to transfectBesides, ligand moieties can be grafted onto the particle surface in order to selectively target a specific subset of cells. Such cationic lipid nanodroplets open new avenues in the development of high throughput RNAi screening as well as their translation to clinics.

International audience; In this study, an empirical modelling-based method is proposed and implemented to identify the critical quality attributes and speed up the formulation optimization of a nanostructured lipid carrier. Design of experiments. A mixture design with five factors, each associated with the nanoparticle formulation, was used: cationic lipid concentration (X1), fusogenic lipid concentration (X2), PEG surfactant concentration (X3), lecithin concentration (X4) and the hydrodynamic diameter (X5). The first four factors are dependant of each other and obey to a constraint equation about the nanoparticle composition. The nanoparticle properties considered were polydispersity (Y1), stability (Y2) and transfection efficacies on the Hela (Y3) and PC3 cell lines (Y4). Rational methodology of nanoparticle design. The empirical modelling methodology was split up into three consecutive steps. In the first part, we show that only the hydrodynamic diameter of the nanoparticle has a significant influence on the polydispersity response and we deduce its design space. In the second step, an empirical model of the nanoparticle stability is obtained, which allows us to identify two main contributors: the nanoparticle size and the concentration of surfactant PEG. A stability region in the (X3, X5) space is derived from this model. In the final part of this study, two response surface models are computed from the experimental data and are used to determine the optimal values of three formulation factors of the nanostructured lipid carrier.Results. Two different formulations have been synthetized and their in vitro properties have corroborated the predicted values provided by the previous models. This study confirms that a rational and rigorous engineering of nanoparticles is possible, owing to statistical design of experiments and empirical modelling techniques. Such approaches can drastically reduce the preclinical development duration of nanotechnologies in medical applications.

Current efforts in nanofluidics aimed at detecting scarce molecules or particles are focused mainly on the development of electrokinetic-based devices. However, these techniques require either integrated or external electrodes, and a potential drop applied across a carrier fluid. One challenge is to develop a new generation of electroless passive devices involving a simple technological process and packaging without embedded electrodes for micro- and nanoparticles enrichment with a view to applications in biology such as the detection of viral agents or cancers biomarkers. This paper presents an innovative technique for particles handling and enrichment based exclusively on a pressure-driven silicon bypass nanofluidic device. The device is fabricated by standard silicon micro–nanofabrication technology. The concentration operation was demonstrated and quantified according to two different actuation modes, which can also be combined to enhance the concentration factor further. The first, “symmetrical” mode involves a symmetric cross-flow effect that concentrates nanoparticles in a very small volume in a very local point of the device. The second mode, “asymmetrical” mode advantageously generates a streaming potential, giving rise to an Electroless Electropreconcentration (EL-EP). The concentration process can be maintained for several hours and concentration factors as high as ~200 have been obtained when both symmetrical and asymmetrical modes are coupled. Proof of concept for concentrating E. coli bacteria by the manual actuation of the EL-EP device is also demonstrated in this paper. Experiments demonstrate more than a 50-fold increase in the concentration of E. coli bacteria in only ~40 s.

Unlike Quality by Testing approach, where products were tested only after drug manufacturing, Quality by Design (QbD) is a proactive control quality paradigm, which handles risks from the early development steps. In QbD, regression models built from experimental data are used to predict a risk mapping called Design Space in which the developers can identify values of critical input factors leading to acceptable probabilities to meet the efficacy and safety specifications for the expected product. These empirical models are often limited to quantitative responses. Moreover, in practice the smallness and incompleteness of datasets degrade the quality of predictions. In this study, a Bayesian approach including variable selection, parameter estimation and model quality assessment is proposed and assessed using a real case study devoted to the development of a Cationic Nano-Lipid Structures for siRNA Transfection. Two original model structures are also included to describe both binary and percentage response variables. The results confirm the practical relevance and applicability of the Bayesian implementation of the QbD analysis.