title: Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing.
abstract: The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods.
  
  
  
  This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane.
  
  
  
  The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Grigore
authors: G
authors: Rischitor
authors: Present address : Nanoimmunotech S.L., Av. de la Autonomía, 50003, Zaragoza, Spain.
authors: Mariantonietta
authors: M
authors: Parracino
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Rita
authors: R
authors: La Spina
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Patricia
authors: P
authors: Urbán
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Isaac
authors: I
authors: Ojea-Jiménez
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Elena
authors: E
authors: Bellido
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Andrea
authors: A
authors: Valsesia
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Sabrina
authors: S
authors: Gioria
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Robin
authors: R
authors: Capomaccio
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Agnieszka
authors: A
authors: Kinsner-Ovaskainen
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: Douglas
authors: D
authors: Gilliland
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy.
authors: François
authors: F
authors: Rossi
authors: European Commission Joint Research Centre, Institute for Health and Consumer and Protection, Nanobiosciences Unit, Via E. Fermi 2749, 21027, Ispra, VA, Italy. pascal.colpo@ec.europa.eu.
authors: Pascal
authors: P
authors: Colpo
mesh_terms: D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D053758:Nanoparticles / Q000633:toxicity*; D013056:Spectrophotometry, Ultraviolet
keywords: Cell uptake; Colloidal stability; Dosimetry; In vitro assay; Nanoparticle characterization; Nanoparticle transport; Nanoparticles