Estimating effective density of engineered nanomaterials for accurate in vitro dosimetry

The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials (ENMs). An important key to accurate in vitro dosimetry is the characterization of ENM sedimentation and diffusion rates in physiological fluid, which depend upon the agglomeration state of the ENM in suspension (agglomerate diameter and effective density). In this project we demonstrate that the effective density of a suspended ENM, from which the sedimentation rate can be directly calculated, can be accurately estimated from the volume of the pellet obtained by simple bench-top centrifugation of ENM suspensions in packed cell volume (PCV) tubes. Because the pellet contains a known volume of ENM, the balance of its volume can be assumed to consist of media trapped within and between ENM particles or agglomerates, and the effective density of the suspended ENM can therefore be calculated as a volume-weighted average of the raw ENM and media densities. This proposed method will enable nanotoxicologists to accurately model ENM transport, and thereby obtain accurate dosimetry in cell culture systems. This simple and cost-effective method could substantially forward the development of reliable and efficient methods for toxicological testing of nano-bio interactions in-vitro.

a) ENM primary particles suspended in cell culture media exist as agglomerates consisting of multiple primary particles, which may be enveloped by a corona of proteins from the media, and media trapped between primary particles (intra-agglomerate media). b) ENM agglomerates within suspensions applied to cells settle toward the cells over time as a result of mass transport (sedimentation and diffusion). The initial administered dose is the concentration of ENM in the initially homogeneous suspension. As transport progresses agglomerates are concentrated near or deposited onto the cells. The mass of ENM deposited per area is the delivered dose. c) In volumetric centrifugation a sample of ENM suspension is centrifuged in a packed cell volume (PCV) tube to produce a pellet, the volume of which can be used to estimate the effective density of the ENM in suspension. The pellets contain both packed agglomerates and the media remaining between them (inter-agglomerate media). We refer to fraction of the pellet volume occupied by agglomerates as the stacking factor (SF).

a) ENM primary particles suspended in cell culture media exist as agglomerates consisting of multiple primary particles, which may be enveloped by a corona of proteins from the media, and media trapped between primary particles (intra-agglomerate media). b) ENM agglomerates within suspensions applied to cells settle toward the cells over time as a result of mass transport (sedimentation and diffusion). The initial administered dose is the concentration of ENM in the initially homogeneous suspension. As transport progresses agglomerates are concentrated near or deposited onto the cells. The mass of ENM deposited per area is the delivered dose. c) In volumetric centrifugation a sample of ENM suspension is centrifuged in a packed cell volume (PCV) tube to produce a pellet, the volume of which can be used to estimate the effective density of the ENM in suspension. The pellets contain both packed agglomerates and the media remaining between them (inter-agglomerate media). We refer to fraction of the pellet volume occupied by agglomerates as the
stacking factor (SF).