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

Quantification of the Distribution of Carbon Black in Natural Rubber/Polybutadiene Blends by Differential Scanning Calorimetry

The distribution of carbon black (CB) in uncured blends of natural rubber (NR) and polybutadiene (PB) can be quantified using DSC. The crystallization temperature of PB containing CB, measured during cooling from the melt, increases with the CB loading. This phenomenon was used to calculate the CB content in PB and conversely in NR in NR/PB/CB mixtures. Two different blends containing N550 CB were studied. A deeper analysis was performed over the entire composition range of NR/PB blends filled with N234 CB. Two different trends were highlighted depending on composition. Below 50 wt% of NR in the elastomer phase, the crystallization temperature of PB is higher than that of pure PB indicating that a part of the CB is dispersed in PB. Above 50 wt% of NR, the crystallization temperature of PB is lower than that of pure PB, indicating that the whole CB is in the NR phase and that fractionated crystallization of PB simultaneously occurs. Both phenomena are consistent as the dispersed phase morphology is favored by a large increase of the matrix viscosity by the filler. For both CBs, the results indicate that CB has larger affinity for NR than for PB. This tendency was confirmed by transmission electron microscopy

Utilisation de la rhéologie pour la description de la morphologie de mélanges caoutchouc naturel/polybutadiène

International audienceL'objectif de cette étude est de présenter comment la rhéologie a pu être utilisée pour caractériser la morphologie de mélanges de deux élastomères incompatibles, le caoutchouc naturel (NR) et le polybutadiène (PB). Les mesures rhéologiques à l'état fondu en cisaillement oscillatoire dans le domaine linéaire ont été réalisées pour obtenir des informations sur la structure de ces coupages. A basse fréquence et pour de faibles taux de la phase minoritaire, le module de conservation présente un excès d'élasticité qui augmente avec la teneur en phase minoritaire. Pour de plus fortes concentrations en phase minoritaire, cette extra-élasticité diminue en relation avec la morphologie. Des mesures rhéologiques à l'état solide en torsion rectangulaire ont été conduites sur les coupages NR/PB. L'évolution du module de conservation à la température de cristallisation du PB est reliée à la morphologie de cet élastomère. Le phénomène de cristallisation fractionnée est évoqué pour expliquer la structure du PB dans les coupages NR/PB. Enfin, les mélanges NR/PB ont été ultra-cryo-microtomés et analysés par microscopie électronique en transmission, afin de confirmer les résultats des deux tests précédents. La superposition de ces différentes procédures permet une description de la morphologie des coupages NR/PB non chargés et non vulcanisés sur l'ensemble du domaine de composition

Etude de la morphologie des coupages d'élastomères

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Coupling of Various Methods for the Investigation of the Morphology of Blends ofNatural Rubber and Polybutadiene

International audienceThe aim of this work is to develop a set of experimental methods to investigate the morphology of blends of two immiscible elastomers, namely natural rubber(NR) and polybutadiene(PB). Selective extraction, dynamic mechanical spectroscopy in the melt, and transmission electron microscopy (TEM) were used to get informa- tion on the structure of un-fillled and un-cured blends

3D investigation of a PS/ABS polymer using different microscopy techniques

International audienc

Effects of Siglec-7 ligand, ganglioside, on platelet function.

<p>(<b>A</b>) Platelet activation upon stimulation with GD2 (n = 10). *: Significant difference, <i>t</i>-test, <i>p</i><0.05, changes in membrane expression of CD62P in platelets stimulated by TRAP <i>vs</i> resting platelets. There was no significant change in the expression of CD62P between platelets stimulated with GD2 or vehicle control (<i>t</i>-test, <i>p</i>>0.05). Similar results were observed for GD3 and GT1b stimuli. Thus, gangliosides show no effect on platelet activation. (<b>B</b>) Platelet aggregation (n = 3) analyses. Incubation of platelets with either GD2 or vehicle did not induce platelet aggregation or alter their response to ADP stimulation (10 µM). (<b>C</b>) Platelet secretion analysis: Platelet secretion of serotonin, sCD40L, and RANTES induced by GD2 stimulation (n = 10). The secretion of resting platelets was slightly lower than both the vehicle and GD2-stimulated platelets; however, there was no significant difference between the latter two groups (<i>t</i>-test, <i>p</i>>0.05).</p

Mechanism of GD2-induced platelet apoptosis.

<p>Both platelets pretreated with blocking anti-Siglec-7 PAb and untreated platelets were incubated with vehicle or GD2. A23187 was used as an apoptosis positive control. Different parameters of apoptosis pathways in platelets were analyzed. (<b>A</b>) Expression of TRAIL R1 in platelets stimulated with GD2 (n = 12). *: significant difference (<i>t</i>-test, <i>p</i><0.05) <i>vs</i> resting platelets. The extrinsic pathway may not be involved in platelet apoptosis induced by GD2. (<b>B</b>) Analyses of phosphatidylserine (PS) exposure (n = 13). The extent of PS exposure by GD2 was reduced by blocking anti-Siglec-7 pAb in a concentration-dependent manner. *, #: Significant difference (<i>t</i>-test, <i>p</i><0.05) <i>vs</i> vehicle or GD2 stimulated conditions respectively. (<b>C</b>) Platelet microparticle assay (n = 10). PMP formation was calculated with respect to the concentration in vehicle conditions (arbitrarily designated 100%). *, #: significant difference (Wilcoxon paired test, <i>p</i><0.05) <i>vs</i> vehicle or GD2 stimulated conditions, respectively. (<b>D</b>) ΔΨm depolarization. ΔΨm depolarization resulted in decreased DIOC6(3) accumulation. *, #: Significant difference (<i>t</i>-test, <i>p</i><0.05) <i>vs</i> vehicle or GD2 stimulated conditions, respectively. Results are representative of five independent experiments. GD2-induced mitochondrial depolarization in platelets treated with anti-FcγRII mAb or PBS control. *, ** significant difference (<i>t</i>-test, <i>p</i><0.05) of ΔΨm between GD2 stimulated platelets vs unstimulated platelets in the presence or absence of anti-FcγRII mAb. #, ¥: Anti-Siglec-7 PAb + GD2 vs only-GD2 stimulated platelets in the presence or absence of anti-FcγRII mAb (n = 5). NS: Not significant. (<b>E</b>) Western blot demonstrates strong expression of Bax and Bak in GD2-treated human platelets and this expression was prevented by blocking anti Siglec-7 pAb. Results are representative of three independent experiments.</p

Characterization of Siglec-7 as a novel marker of platelet activation.

<p>(<b>A</b>),(<b>C</b>). Agonist-induced platelet activation (n = 10). Platelets were stimulated with different agonists: TRAP (PAR-1), PAR-4 activator, collagen (β₂α₁ integrin and GPVI-FcR gamma). Membrane MFI expression of CD62P (<b>A</b>) and Siglec-7 (<b>B</b>) was correlated under all experimental conditions (PCC = 0.995, <i>p</i> = 0.002) (<b>C</b>). (<b>D</b>),(<b>E</b>). Kinetics of CD62P (<b>D</b>) and Siglec-7 (<b>E</b>) expression on platelet membranes (n = 5). Membrane expression of Siglec-7 and CD62P in platelets under un-stimulated and TRAP stimulated conditions was analyzed by flow cytometry. MFI changes between CD62P and Siglec-7 were correlated over time (unstimulated: PCC = 0.842; <i>p</i> = 0.016; stimulated by TRAP: PCC = 0.958; <i>p</i> = 0.01). *, #, ¥, ¤: significant differences (analysis of variance, <i>p</i><0.05) between MFI of Siglec-7 or CD62P marker over time <i>vs</i> 0, 30, 60, and 180 min respectively; ‡: <i>t</i>-test, <i>p</i><0.05) MFI of Siglec-7 or CD62P marker in TRAP-induced platelets activation <i>vs</i> resting platelets (expression of CD62P and Siglec-7 in MFI was reported for unstimulated conditions at 0 min, which was considered as 100%).</p

PI3K inhibitors reduce GD2-induced platelet apoptosis (n = 15).

<p>(<b>A</b>) Platelets were pre-treated with varying concentrations of intracellular pathway inhibitors (DPI for NAPDH oxidase, LY294,002 for PI3K, BIM I for PKC and BAY-11 for NFκB) or DMSO (negative control) followed by stimulation with GD2. Cell death rate (%) was calculated by the percentage of diminution in platelet number in comparison to unstimulated platelets. DPI, BIM I, and LY294,002 prevented cell death in a concentration-dependent manner. *: Significant difference (<i>t</i>-test, <i>p</i><0.05) between inhibitor <i>vs</i> DMSO treated. The lowest concentration of inhibitors with a significant effect (DPI 1 µM, LY294,002 50 µM, BIM I 10 µM) (no difference at a higher concentration) was selected to treat platelets. (<b>B</b>) Loss of ΔΨm resulted in reduced accumulation of DIOC6(3), and was calculated as the percentage of diminution in DIOC6(3) MFI compared with unstimulated platelets. (<b>C</b>) Phosphatidylserine exposure (left panel: percentage of CD41a⁺, Annexin V⁺; right panel: MFI). *: Significant difference (<i>t</i>-test, <i>p</i><0.05) between inhibitor <i>vs</i> DMSO pretreated platelets. Thus, PI3K inhibitor prevented both ΔΨm depolarization and PS exposure in platelets, while NAPDH oxidase inhibitor prevented only ΔΨm depolarization.</p