The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by bench-top centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro.

In vitro toxicity assays are efficient and inexpensive tools for screening the increasing number of engineered nanomaterials (ENMs) entering the consumer market. However, the data produced by in vitro studies often vary substantially among different studies and from in-vivo data. In part these discrepancies may be attributable to lack of standardization in dispersion protocols and inadequate characterization of particle-media interactions which may affect the particle kinetics and the dose delivered to cells. In this study, a novel approach for preparation of monodisperse, stabilized liquid suspensions was presented and coupled with a numerical model which estimates delivered dose values. Empirically derived material- and media-specific functions were derived for each media- ENM system that can be used to convert administered to delivered doses. The interactions of ENMs with a variety of physiologic media were investigated and the importance of this approach was demonstrated by in vitro cytotoxicity assays using THP-1 macrophages.

Summary Evidence continues to grow of the importance of in vitro and in vivo dosimetry in the hazard assessment and ranking of engineered nanomaterials (ENMs). Accurate dose metrics are particularly important for in vitro cellular screening to assess the potential health risks or bioactivity of ENMs. In order to ensure meaningful and reproducible quantification of in vitro dose, with consistent measurement and reporting between laboratories, it is necessary to adopt standardized and integrated methodologies for 1) generation of stable ENM suspensions in cell culture media, 2) colloidal characterization of suspended ENMs, particularly properties that determine particle kinetics in an in vitro system (size distribution and formed agglomerate effective density), and 3) robust numerical fate and transport modeling for accurate determination of ENM dose delivered to cells over the course of the in vitro exposure. Here we present such an integrated comprehensive protocol based on such a methodology for in vitro dosimetry, including detailed standardized procedures for each of these three critical steps. The entire protocol requires approximately 6-12 hours to complete.

BackgroundThere is a great need for screening tools capable of rapidly assessing nanomaterial toxicity. One impediment to the development of reliable in vitro screening methods is the need for accurate measures of cellular dose. We present here a methodology that enables accurate determination of delivered to cell dose metrics. This methodology includes (1) standardization of engineered nanomaterial (ENM) suspension preparation; (2) measurement of ENM characteristics controlling delivery to cells in culture; and (3) calculation of delivered dose as a function of exposure time using the ISDD model. The approach is validated against experimentally measured doses, and simplified analytical expressions for the delivered dose (Relevant In Vitro Dose (RID)f function) are derived for 20 ENMs. These functions can be used by nanotoxicologists to accurately calculate the total mass (RIDM), surface area (RIDSA), or particle number (RIDN) delivered to cells as a function of exposure time.ResultsThe proposed methodology was used to derive the effective density, agglomerate diameter and RID functions for 17 industrially-relevant metal and metal oxide ENMs, two carbonaceous nanoparticles, and non-agglomerating gold nanospheres, for two well plate configurations (96 and 384 well plates). For agglomerating ENMs, the measured effective density was on average 60% below the material density. We report great variability in delivered dose metrics, with some materials depositing within 24 hours while others require over 100 hours for delivery to cells. A neutron-activated tracer particle system was employed to validate the proposed in vitro dosimetry methodology for a number of ENMs (measured delivered to cell dose within 9% of estimated).ConclusionsOur findings confirm and extend experimental and computational evidence that agglomerate characteristics affect the dose delivered to cells. Therefore measurement of these characteristics is critical for effective use of in vitro systems for nanotoxicology. The mixed experimental/computational approach to cellular dosimetry proposed and validated here can be used by nanotoxicologists to accurately calculate the delivered to cell dose metrics for various ENMs and in vitro conditions as a function of exposure time. The RID functions and characterization data for widely used ENMs presented here can together be used by experimentalists to design and interpret toxicity studies.

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