Quantification of
SiO2
nanoparticle sedimentation on
A549
cells
Authors: Kowoll Thomas , Fritsch‐Decker Susanne , Fertig Regina , Mueller Erich , Weiss Carsten , Gerthsen Dagmar 
Published: December 2016

In recent years many studies were published on toxicological effects of nanoparticles (NPs) on human tissue. Many dose‐response studies rely on in vitro assays, in which cultured cells are exposed to a suspension of cell‐culture medium and NPs. Limbach et al. [1] stated, that the sedimentation process of NPs in this setup is more complex than in the case of microparticles (MPs). While sedimentation of NPs is primarily driven by diffusion, it is mainly gravitational force, which influences MPs. To compare and quantify the sedimentation of particles of both scales, sedimentation studies and simulations with SiO
2
particles were performed in this work. Scanning electron microscopy (SEM) was applied to measure the direct cellular dose, i.e., the sedimented areal densities of particles (AD) on the cells, as a more appropriate definition of dose according to Teeguarden et al. [2]. Simulations are based on ISDD, a computational model by Hinderliter et al. [3].

The samples were prepared as follows: A549 lung cancer cells were seeded onto indium‐tin‐oxide coated glass substrates in culture plates containing Dulbecco's Modified Eagle's Medium supplemented with fetal calf serum (FCS). After adhering overnight, the cells were incubated with SiO
2
particles (from 70 nm up to 500 nm diameter) in cell culture medium for 1 and 4 hours. Using ISDD and assuming, that sedimented particles are homogeneously distributed, targeted ADs were calculated according to predefined incubation concentrations. After fixation with paraformaldehyde the cells were dehydrated with graded ethanol series, dried by critical point drying and investigated in a FEI Quanta 650 SEM. Particles were imaged with secondary electrons (SE) in intercellular regions between cells. Backscattered electron (BSE) images were taken to detect particles on cells. To increase the BSE contrast, a retarding bias was applied to the sample stage.

Fig. 1a shows a representative SE image of an intercellular region with 200 nm SiO
2
particles after 1 h incubation. The particles appear homogeneously distributed. Fig. 1b depicts a cell surface of the same specimen with a substantially smaller AD (0.15 NP/µm² compared to 0.84 NP/µm² in Fig. 1a) and an inhomogeneous NP distribution. A smaller cellular AD is also observed for all other particles sizes and incubation times. This is shown in Fig. 2a, where measured cellular and intercellular ADs for each specimen are compared. In Fig. 2b, the simulated AD is plotted against the measured intercellular AD for all samples. Calculated and measured ADs agree well within the error bars, which can be considered as a verification of the ISDD model. However, the cellular ADs are significantly lower indicating that another effect must be taken into account. Cellular uptake can be ruled out as an explanation, because focused‐ion‐beam sectioning of whole cells did not show a high particle density in cells.

Fluorescence microscopy (FM) investigations with rhodamine labelled SiO
2
NPs (Ø = 70 nm) and A549 cells qualitatively confirm the differences between cellular and intercellular ADs observed by SEM (Fig. 3a). The red fluorescence is much stronger in intercellular regions than on cells, however the signals stem from small agglomerates. Dynamic light scattering shows, that this agglomeration is caused by FCS coating of NPs applied before incubation. To study the impact of FCS in more detail, further in vitro experiments with and without FCS precoatings of NPs and/or substrates were performed. Preliminary results indicate, that protein coatings induce an attractive interaction between NPs with their protein corona and surface proteins. Since FM is unable to resolve single NPs, SEM is best suited for confirmation.

SEM is convenient to quantitatively determine ADs of NPs and reveals distinct differences between cellular and intercellular ADs. Our results highlight a major problem of bulk investigation methods relying on lysates, because intercellular and cellular regions cannot be distinguished.