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

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

Improvements for imaging ceramics sintering in situ in ESEM

International audienceSintering of green samples of alumina produced by ice-templating was followed in situ in an environmental scanning electron microscope (ESEM) up to temperatures as high as 1375 degrees C. These alumina samples with well-defined architectures are of great interest in the field of materials science due to their high specific strength (especially in compression), low density and adaptable porosity. For the present study, they also have the advantage to exhibit an important topography, inducing interesting contrast when imaged in an ESEM. Improvements of the imaging conditions in the ESEM were essential to really follow the sintering process involving formation of necks between grains or shift of the centre of grains. This paper describes the improvements made and the results observed on the sintering process of alumina green samples processed by ice-templating

Deformation mechanism of cerium oxide nanocubes - an in situ transmission electron microscopy study

Cerium oxide nanoparticles are used in many industrial products, among which solid oxide fuel cell electrodes or catalysts. However, their mechanical properties are rarely taken into account and few studies dealt with the determination of their deformation mechanism [1, 2]. Please click Download on the upper right corner to see the full abstract

In situ nanocompression tests in an environmental TEM to study plasticity of cerium oxides

Cerium oxide plays an important role in several fields, among which catalysis, gas detection or fuel cells [1]. Cerium oxide nanoparticles are also used as superior abrasive particles in chemical mechanical planarization (CMP), which is a key process in semiconductor device fabrication [2]. Most of the current research focus on the synthesis of cerium oxide to optimize CMP, but analysing its deformation mechanisms is also a promising research direction [3]. Please click Additional Files below to see the full abstract

TEM observation and in situ compression tests of transition alumina prepared by high pressure compaction at room temperature

The behavior of ceramics at the nanometer scale strongly differs from the one of the corresponding bulk material. For instance, strong plastic deformation has recently been reported in isolated nanometer-sized alumina nanoparticles or MgO nanocubes, when tested in situ in a transmission electron microscope (TEM). This plastic behavior may also occur in a powder during the compaction process, even at room temperature. Controlling plastic deformation of nanoparticles during the ceramics processing might be a way to enhance their properties or to improve the processing route (compaction and sintering steps, for instance). We present here a comprehensive study of the mechanical behavior of transition alumina in the compacted powder. Please click Additional Files below to see the full abstract

Evidence for the formation of distorted nanodomains involved in the phase transformation of stabilized zirconia by coupling convergent beam electron diffraction and in situ TEM nanoindentation

International audienceThe transformation of zirconia from its tetragonal to its monoclinic phase is an important feature of the zirconia system. First found to be an advantage due to its important toughening effect, it can also be very detrimental when it occurs in the framework of low-temperature degradation, particularly in the case of biomaterial applications. One way to avoid or to control this phase transformation is to understand how it initiates and more particularly the stress states that can trigger it. A new technique available inside a transmission electron microscope seems to be particularly well suited for that type of study: convergent beam electron diffraction, a well-known technique to reveal stresses, was coupled to in situ transmission electron microscopy mechanical nanoindentation. The experiments reveal the presence of sheared nanoregions at grain boundaries. These could act as embryos for tetragonal-to-monoclinic phase transformations. This is an important first step in the understanding of the earliest stage of zirconia phase transformation

Accurate characterization of pure silicon-substituted hydroxyapatite powders synthesized by a new precipitation route

International audienceThis paper presents a new aqueous precipitation method to prepare silicon-substituted hydroxyapatites Ca10(PO4)6-y(SiO4)y(OH)2-y(VOH)2-y (SiHAs) and details the characterization of powders with varying Si content up to y = 1.25 mol molSiHA−1. X-ray diffraction, transmission electron microscopy, solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopy were used to accurately characterize samples calcined at 400°C for 2 h and 1000°C for 15 h. This method allows the synthesis of monophasic SiHAs with controlled stoichiometry. The theoretical maximum limit of incorporation of Si into the hexagonal apatitic structure is y < 1.5. This limit depends on the OH content in the channel, which is a function of the Si content, temperature and atmosphere of calcination. These results, particularly those from infrared spectroscopy, raise serious reservations about the phase purity of previously prepared and biologically evaluated SiHA powders, pellets and scaffolds in the literature

Internalization pathways into cancer cells of gadolinium-based radiosensitizing nanoparticles

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

Nanoparticules lubrifiantes à structure fermée

Les additifs à action tribologique (MoDTC et ZnDTP) actuellement utilisés sont des molécules organiques à base de soufre et de phosphore. Pour des raisons environnementales, il est important de diminuer nettement voire d'éliminer la présence de ces deux éléments dans les huiles. Nous nous sommes intéressés à des nanoparticules à structure fermée (sphérique, cylindrique) comme remplaçants potentiels de ces additifs. Leur taille, nanométrique, leur permet d'entrer facilement dans le contact et leur structure, fermée, leur confère une grande inertie chimique. Les fullerènes de MS2 ont montré des propriétés réductrices de frottement et anti-usure très intéressantes. Leur mécanisme d'action est basé sur la libération de monofeuillets de MS2 hexagonal dans le contact tribologique. Les propriétés tribologiques des nanotubes de carbone sont reliées à certains paramètres (nombe de feuillets, présence de catalyseur). Une modification structurale des nanotubes dans le contact est nécessaire.Additives with tribological action (MoDTC and ZnDTP) used at present are organic molecules containing sulphur and phosphorous. For environmental reasons, it is important to reduce or eliminate the presence of these two elements in oil. In this study, we are being interested in nanoparticles with closed structure (spherical, cylindrical) as potential substitutes of these additives. Their size, nanometric, enables them to enter easily the contact and their structure, closed, confers a great chemical inertia to them. Fullerenes of MS2 show very good friction reducing and anti wear properties. Their lubrication mechanism is based on the release of hexagonal MS2 sheets in the contact area. Tribological properties of carbon nanotubes depend on several parameters (number of wall, presence of catalyst). A structural modification of nanotubes inside the contact area is necessary.LYON-Ecole Centrale (690812301) / SudocSudocFranceF

Contribution de la nanoindentation in situ en Microscopie Electronique en Transmission à l'étude des céramiques

La connaissance du comportement et des propriétés des matériaux est d une grande importance pour optimiser leur mise en forme et adapter leur utilisation. Pour étudier ces propriétés de nombreuses techniques sont couramment utilisées : les essais de traction, la microindentation, la nanoindentation instrumentée Aujourd hui, un intérêt particulier est porté sur les nanomatériaux et matériaux nanostructurés car ils présentent souvent des propriétés différentes et plus intéressantes. La nanoindentation instrumentée, notamment, permet de déterminer des paramètres matériaux de manière locale. Cependant, le comportement en temps réel ne peut être observé et l échantillon ne doit pas être de dimension trop faible (typiquement, l étude de nanoparticules n est pas envisageable). Le principal atout de la nanoindentation in situ en Microscopie Electronique en Transmission vis-à-vis des autres techniques existantes est la possibilité d étudier le comportement de nano-objets ou des comportements très locaux et en temps réel, tout en observant les transformations subies par le matériau. Dans cette étude, nous avons évalué les potentialités de cette nouvelle technique via l analyse de céramiques très étudiées au laboratoire notamment en tant que biomatériaux : la zircone stabilisée et l alumine. Dans le cas de la zircone (stabilisée à l yttrium ou au cérium), le but était de localiser à l échelle nanométrique les contraintes responsables ou inhérentes à la transformation de phase quadratique-monoclinique, phénomène ayant une très grande influence sur les propriétés du matériau massif. Pour ce faire, après avoir déterminé une technique de préparation adaptée, nous proposons une voie d étude pour la localisation des contraintes liées à la transformation de phase : le CBED (Convergent Beam Electron Diffraction) couplé à la nanoindentation in situ. Dans le cas de l alumine, l objectif était d étudier le matériau (commercial et non un matériau modèle) dans sa forme originelle à savoir sous forme de nanoparticules d alumine de transition. L idée était d étudier le comportement de ces nanoparticules sous compression. Nous avons notamment constaté que ces particules pouvaient subir une grande déformation plastique à température ambiante. Nous avons pu également, sur quelques particules, obtenir une série d images en cours de compression ainsi que la courbe de charge-déplacement correspondante. Ces résultats ont ensuite été soumis à une analyse des images couplée à une simulation de type Eléments Finis (réalisées par le LAMCOS).Knowledge of the behavior and properties of materials is of great importance to optimize their processing and adapt their use. To study these properties, many techniques are commonly used: tensile tests, microindentation, instrumented nanoindentation ... Today, particular interest is focused on nanomaterials and nanostructured materials because they often have different and more interesting properties. Instrumented nanoindentation allow to determine material parameters. However, the real-time behavior can not be observed and the study of nano-objects is difficult (nanoparticles for example). The main advantage of in situ TEM (Transmission Electron Microscopy) nanoindentation is the ability to study the behavior of nano-objects in real time. In this study, we evaluated the potential of this new technique by analyzing ceramics extensively studied in the laboratory such as biomaterials: stabilized zirconia and alumina. In the case of zirconia (stabilized with yttrium or cerium), the goal was to locate at the nanoscale, the constraints responsible for the tetragonal to monoclinic phase transformation. This phenomenon having a great influence on the bulk material properties. To do this, after having determined a suitable preparation method, we suggest a way to study the localization of constraints: the CBED (Convergent Beam Electron Diffraction) coupled with in situ TEM nanoindentation. In the case of alumina, the goal was to study the material in its original form (nano powder of transition alumina). The idea was to study the behavior of these nanoparticles under compression. We particularly observed that these particles could undergo large plastic deformation at room temperature. We have also obtained during compression on few particles, series of images and the corresponding load-displacement curve. These results were then analyzed by image analysis coupled with Finite Element simulations (performed in LAMCOS lab).VILLEURBANNE-DOC'INSA-Bib. elec. (692669901) / SudocSudocFranceF