95 research outputs found

    Electron microscopy contribution in the comprehension of interaction mechanisms between nanoparticles and biological cells

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    Parmi les nanoparticules aptes à accompagner la radiothérapie en clinique, les nanoparticules à base d oxyde de gadolinium paraissent pertinentes, de part leur multimodalité en imagerie et leur effet radiosensibilisant prouvé in vitro et in vivo. Cet effet de radiosensibilisation est exceptionnel notamment sur des cellules cancéreuses radiorésistantes de la lignée SQ20B (carcinome squameux tête et cou) et uniquement pour des doses modérées de nanoparticules (aux alentours de 0.6 mM en Gd). Les clichés de microscopie électronique ont montré que ce maximum de radiosensibilisation est dû à une internalisation maximale des particules dans le cytoplasme, notamment par macropinocytose. Ce mécanisme d internalisation est caractérisé par la formation de vésicules de grandes tailles, ou macropinosomes. Il se produit suivant deux étapes : la formation d agglomérats de nanoparticules à proximité de la membrane cellulaire puis la récupération de ceux-ci par les lamellipodes de la cellule. La première étape est fortement dépendante des caractéristiques physicochimiques des particules, plus particulièrement leur potentiel zêta qui détermine la taille de l agglomérat, et de la distance les séparant de la cellule. Dans des gammes de taille et de distance à la membrane optimales aux concentrations modérées, l agglomérat peut être récupéré par les lamellipodes de la cellule. Il s en suit une protubérance sur la membrane plasmique formant un macropinosome contenant les agglomérats de nanoparticules. Cet endosome précoce suivra ensuite le schéma d endocytose classique dans le cytoplasme en fusionnant avec des corps multivésiculaires, uniquement visible en microscopie électronique à transmission, pouvant contenir des enzymes de dégradation détruisant leur contenu. Ces enzymes rendent le pH acide à l intérieur de la vésicule. Plus les nanoparticules sont proches du noyau cellulaire plus leur effet radiosensibilisant sera efficace. Les espèces oxygénées réactives (ROS) et les électrons Auger et secondaires peuvent atteindre l ADN du noyau plus facilement. A faibles doses ( 0.7 mM) les nanoparticules forment une couronne autour de la membrane cellulaire agissant comme écran, empêchant ainsi les ROS et les électrons générés de pouvoir atteindre l ADN et induire des cassures, le noyau étant situé à quelques micromètres de la membrane cellulaire. Les résultats obtenus ouvrent la voie sur la nécessité de contrôler l'internalisation cellulaire des nanoparticules en contrôlant leur chimie, laissant envisager ainsi des opportunités prometteuses dans le domaine de la radiothérapie assistée par nanoparticules délivrant de faibles doses de radiation aux patients.Over the last few decades, nanoparticles have been studied in theranostic field with the objective of exhibiting a long circulation time through the body coupled to major accumulation in tumor tissues, rapid elimination, therapeutic potential and contrast properties. In this context, we developed sub-5 nm gadolinium-based nanoparticles that possess in vitro efficient radiosensitizing effects at moderate concentration when incubated with head and neck squamous cell carcinoma cells (SQ20B). Two main cellular internalization mechanisms were evidenced and quantified: passive diffusion and macro- pinocytosis. Whereas the amount of particles internalized by passive diffusion is not sufficient to induce in vitro a significant radiosensitizing effect, the cellular uptake by macropinocytosis leads to a successful radiotherapy in a limited range of particles incubation concentration. Macropinocytosis processes in two steps: formation of agglomerates at vicinity of the cell followed by their collect via the lamellipodia (i.e. the arms ) of the cell. The first step is strongly dependent on the physicochemical characteristics of the particles, especially their zeta potential that determines the size of the agglomerates and their distance from the cell. These results should permit to control the quantity of particles internalized in the cell cytoplasm, promising ambitious opportunities towards a particle-assisted radiotherapy using lower radiation doses.VILLEURBANNE-DOC'INSA-Bib. elec. (692669901) / SudocSudocFranceF

    Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro

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    International audienceBackground: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. Methods: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Results and conclusion: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed

    Internalization pathways into cancer cells of gadolinium-based radiosensitizing nanoparticles

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    International audienceOver the last few decades, nanoparticles have been studied in theranostic field with the objective of exhibiting a long circulation time through the body coupled to major accumulation in tumor tissues, rapid elimination, therapeutic potential and contrast properties. In this context, we developed sub-5 nm gadolinium-based nanoparticles that possess in vitro efficient radiosensitizing effects at moderate concentration when incubated with head and neck squamous cell carcinoma cells (SQ20B). Two main cellular internalization mechanisms were evidenced and quantified: passive diffusion and macropinocytosis. Whereas the amount of particles internalized by passive diffusion is not sufficient to inducein vitro a significant radiosensitizing effect, the cellular uptake by macropinocytosis leads to a successful radiotherapy in a limited range of particles incubation concentration. Macropinocytosis processes in two steps: formation of agglomerates at vicinity of the cell followed by their collect via the lamellipodia (i.e. the "arms") of the cell. The first step is strongly dependent on the physicochemical characteristics of the particles, especially their zeta potential that determines the size of the agglomerates and their distance from the cell. These results should permit to control the quantity of particles internalized in the cell cytoplasm, promising ambitious opportunities towards a particle-assisted radiotherapy using lower radiation doses

    Nanostructured gold surfaces as biosensors (surface-enhanced chemiluminescence and double detection by surface plasmon resonance and luminescence)

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    This thesis is devoted to develop two biological detection systems based on nanostructured gold surfaces to detect the binding of streptavidin/biotin. In the first part, we have studied a multimodal detection system using local surface plasmon resonance of the gold substrate and the luminescence of labelling core-shell Gd2O3/SiOx nanoparticles, which profits from the plasmonic property of nanostructure gold. In a second part, we have focused in a surface-enhanced chemiluminescence system based on chemiluminescence of luminal/hydrogen peroxide (H2O2) enhanced by gold nanostructures in the vicinity, which benefits from the catalytic property of nanostructure gold. Several parameters influencing the chemiluminescence of luminal were investigated. Enhancing mechanism of luminal chemiluminescence was proved to be not related to Plasmon-assisted process but originates from catalytic properties of the metal induced by corrugationCette thèse est consacré à développer deux systèmes pour la détection biologique à la base de la surface d or pour détecter la liaison entre les molécules de biotine et de streptavidine. Premièrement, nous avons étudié un système de détection multimodal utilisant la résonance des plasmons de surface localisé sur substrats d or et la luminescence de nanoparticules labellisées de coeur-écorce Gd2O3/SiOx, qui bénéficie de la propriété plasmonic d or en nanostructure. Deuxièmement, nous avons focalisé sur un système qui se fonde sur le phénomène d exaltation de chimiluminescence par la surface. La chimiluminescence de luminophore/ peroxyde d hydrogène (H2O2) est exaltée par nanostructure d or à proximité, qui bénéficie de la propriété catalytique de nanostructure d or. Plusieurs paramètres ont été étudiés de manière systématique. Finalement, il est prouvée que le mécanisme de l exaltation de chimiluminescence est originaire de la propriété catalytique du métal induit par la rugositéVILLEURBANNE-DOC'INSA LYON (692662301) / SudocSudocFranceF

    Recent progress on elaboration of undoped and doped Y2O3, Gd2O3 rare-earth nano-oxide

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    Evidence for the verwey transition in highly nonstoichiometric nanometric fe-based ferrites

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    Evidence for the verwey transition in highly nonstoichiometric nanometric fe-based ferrites

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    Apparent magic numbers in embedded Ti-O clusters

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    International audienceStable clusters constituted of four TiO2 entities have been evidenced in nanometer-sized spinel oxides by a combination of diffraction and x-ray-absorption expts. Contrary to free clusters, the size of the embedded clusters is strongly detd. by the constraints imposed by the surrounding matrix so that the magic no. of four is in fact specific to the matrix and only then apparent

    Apparent magic numbers in embedded Ti-O clusters

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    International audienceStable clusters constituted of four TiO2 entities have been evidenced in nanometer-sized spinel oxides by a combination of diffraction and x-ray-absorption expts. Contrary to free clusters, the size of the embedded clusters is strongly detd. by the constraints imposed by the surrounding matrix so that the magic no. of four is in fact specific to the matrix and only then apparent

    Reactivity of nanosized iron-vanadium spinels towards oxygen and cation distribution

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    International audienceBecause of the division state of nanosized iron-vanadium spinels, iron and vanadium ions are oxidized within the spinel structure. Calorimetric and thermogravimetric analyses, such as infrared spectroscopy, show that Fe 2 + B , V 3 + B and Fe 3 + are successively oxidized into Fe 3 + and V 5 + ions below 450 °C. The mixed valency cation-deficient spinels resulting from these oxidations have a vacancy content that increases with x, which may be higher than those found as yet for this type of oxide.L'etat de division des spinelles de fer-vanadium nanometriques V x Fe 3 - x O 4 (0 x 2) permet d'oxyder au sein-meme de la structure spinelle les ions du fer et du vanadium. Les analyses calorimetriques et thermogravimetriques, ainsi que la spectroscopie infrarouge, montrent que les ions Fe 2 + B , V 3 + B et Fe 2 + A sont oxydes successivement en ions Fe 3 + et V 5 + a des temperatures inferieures a 450 °C. Les spinelles lacunaires a valence mixte qui en resultent, ont un taux en lacunes croissant avec x, qui peut etre superieur a ceux determines jusqu'alors pour ce type d'oxydes
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