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S. Annexe-a-:-partie-expérimentale and . Synthèse-de-l'amorceur,

, Sous atmosphère d'argon, 3,40 g de 2,2-dithioéthanol ainsi que 4,2 g de pyridine anhydre sont dissous

, RMN 1 H (CDCl3) ? (ppm) : 4,44 (t, OCH2, 4H), vol.2, p.98

, Les solutions de monomères, de diméthylformamide (DMF), de pentaméthyldiéthylène triamine (PMDETA) et de l'amorceur précédemment synthétisé (DS-Br) sont dégazées à l'argon pendant 30 min

, Un tricol surmonté d'un réfrigérant est mis sous vide puis conditionné sous argon. Le bromure de cuivre (Cu I Br) et le DMF sont introduits et mis sous agitation jusqu

. Le, suivi des monomères. L'amorceur est introduit en dernier et le milieu réactionnel est chauffé à 60°C, sous agitation et sous atmosphère inerte

, Le nombre de cellules final est estimé à 1 million par échantillon (1 échantillon = 1 puits), d'après un comptage effectué sur plusieurs échantillons, en utilisant une cellule de Malassez. OBSERVATION DE CELLULES EN MEB Pour préparer les échantillons, les cellules B16F10 sont cultivées sur des morceaux de wafer de silicium recouverts d'une couche d'oxyde de 150 nm d'épaisseur environ. Ces wafers sont préalablement nettoyés dans divers solvants et par traitement à l'UV-ozone

. Le, Après une nuit d'incubation, le milieu de culture est remplacé par 1 mL d'une solution de NPs à 0 ou 22 µg/mL d'or, préparée la veille dans du milieu de culture contenant 0,25 ?g/mL d'amphotéricine B et 10 ?g/mL de gentamicine. Après une incubation de 18h avec les NPs

/. 100%, Pour sécher les échantillons, deux derniers bains sont réalisés dans de l'hexaméthyldisilazane. Le séchage est effectué à l'air libre, sous hotte

, ?g/mL d'amphotéricine B et 10 ?g/mL de gentamicine. Après une incubation de 18h avec les NPs

, Les cellules doivent ensuite être fixées, déshydratées, puis incluses dans une résine époxy

A. Tableau, 2 -Premier protocole testé pour la fixation, déshydratation et inclusion des cellules pour l'observation en TEM Cellules contrôles et exposées aux NPs Rinçages DPBS x3

, 5% dans tampon sodium cacodylate 0,2M à pH 7, vol.2, p.30

, L'objectif est notamment d'améliorer le contraste des structures lipidiques avec l'utilisation d'osmium, mais aussi d'augmenter la taille des AuNPs par une amplification à l'argent (silver) dans certains cas (Silver enhancer kit, D'autres protocoles sont testés dans un deuxième temps

, L'amplification à l'argent a parfois été précédée d'un traitement avec de la saponine

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