caa a22 4500 605480273 CHVBK 20210128100414.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s10544-015-9947-8 doi (NATIONALLICENCE)springer-10.1007/s10544-015-9947-8 Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles [Elektronische Daten] [Andréa Kurtz-Chalot, Jean-Philippe Klein, Jérémie Pourchez, Delphine Boudard, Valérie Bin, Odile Sabido, Laurence Marmuse, Michèle Cottier, Valérie Forest] Amorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodoTM were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodoTM emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes ). Using this model, the endocytosis of 60nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification. Springer Science+Business Media New York, 2015 Silica particles nationallicence Macrophages nationallicence Size nationallicence Cellular uptake nationallicence Cytotoxicity nationallicence Kurtz-Chalot Andréa LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Klein Jean-Philippe LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Pourchez Jérémie LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Boudard Delphine LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Bin Valérie LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Sabido Odile LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Marmuse Laurence Nano-H, 2 Place de l'Europe, 38070, Saint Quentin Fallavier, France aut Cottier Michèle LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Forest Valérie LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut Biomedical Microdevices Springer US; http://www.springer-ny.com 17/2(2015-04-01), 1-12 1387-2176 17:2<1 2015 17 10544 https://doi.org/10.1007/s10544-015-9947-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10544-015-9947-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kurtz-Chalot Andréa LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Klein Jean-Philippe LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Pourchez Jérémie LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Boudard Delphine LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Bin Valérie LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Sabido Odile LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Marmuse Laurence Nano-H, 2 Place de l'Europe, 38070, Saint Quentin Fallavier, France aut NATIONALLICENCE 700 1- Cottier Michèle LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 700 1- Forest Valérie LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France aut NATIONALLICENCE 773 0- Biomedical Microdevices Springer US; http://www.springer-ny.com 17/2(2015-04-01), 1-12 1387-2176 17:2<1 2015 17 10544