title: Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles.
abstract: 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 pHrodo(TM) were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodo(TM) 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 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm 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.
authors.affiliation: LINA Laboratoire Interdisciplinaire d'étude des Nanoparticules Aérosolisées (EA 4624), 15 rue Ambroise Paré, 42023, Saint-Étienne, Cedex 2, France.
authors.forename: Andréa
authors.initials: A
authors.lastname: Kurtz-Chalot
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Klein
authors.forename: Jérémie
authors.initials: J
authors.lastname: Pourchez
authors.forename: Delphine
authors.initials: D
authors.lastname: Boudard
authors.forename: Valérie
authors.initials: V
authors.lastname: Bin
authors.forename: Odile
authors.initials: O
authors.lastname: Sabido
authors.forename: Laurence
authors.initials: L
authors.lastname: Marmuse
authors.forename: Michèle
authors.initials: M
authors.lastname: Cottier
authors.forename: Valérie
authors.initials: V
authors.lastname: Forest
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects; D004705:Endocytosis / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D018384:Oxidative Stress; D000067996:RAW 264.7 Cells / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D012822:Silicon Dioxide; D059008:Time-Lapse Imaging / Q000295:instrumentation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D012822:Silicon Dioxide; D007770:L-Lactate Dehydrogenase; D016650:Fluorescein-5-isothiocyanate