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Quantification of nanoparticle dose and vesicular inheritance in proliferating cells.

ABSTRACT: Assessing dose in nanoparticle-cell interactions is inherently difficult due to a complex multiplicity of possible mechanisms and metrics controlling particle uptake. The fundamental unit of nanoparticle dose is the number of particles internalized per cell; we show that this can be obtained for large cell populations that internalize fluorescent nanoparticles by endocytosis, through calibration of cytometry measurements to transmission electron microscopy data. Low-throughput, high-resolution electron imaging of quantum dots in U-2 OS cells is quantified and correlated with high-throughput, low-resolution optical imaging of the nanoparticle-loaded cells. From the correlated data, we obtain probability distribution functions of vesicles per cell and nanoparticles per vesicle. Sampling of these distributions and comparison to fluorescence intensity histograms from flow cytometry provide the calibration factor required to transform the cytometry metric to total particle dose per cell, the mean value of which is 2.4 million. Use of the probability distribution functions to analyze particle partitioning during cell division indicates that, while vesicle inheritance is near symmetric, highly variable vesicle loading leads to a highly asymmetric particle dose within the daughter cells.

SUBMITTER: Summers HD

PROVIDER: S-EPMC3722614 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Quantification of nanoparticle dose and vesicular inheritance in proliferating cells.

Summers Huw D HD Brown Martyn R MR Holton Mark D MD Tonkin James A JA Hondow Nicole N Brown Andrew P AP Brydson Rik R Rees Paul P

ACS nano 20130625 7

Assessing dose in nanoparticle-cell interactions is inherently difficult due to a complex multiplicity of possible mechanisms and metrics controlling particle uptake. The fundamental unit of nanoparticle dose is the number of particles internalized per cell; we show that this can be obtained for large cell populations that internalize fluorescent nanoparticles by endocytosis, through calibration of cytometry measurements to transmission electron microscopy data. Low-throughput, high-resolution e ...[more]

PMID: 23773085

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Project description: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.

Dataset Information

Quantification of nanoparticle dose and vesicular inheritance in proliferating cells.

Publications

Quantification of nanoparticle dose and vesicular inheritance in proliferating cells.

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