Abstract
For any living cell the exchange with its environment is vital. Therefore, many different kinds of cargo are able to enter cells via energy-dependent or -independent routes. Nanoparticles are no exemption. It is known that small silica nanoparticles with a diameter below 50 nm are taken up by cells and that their uptake exerts pronounced toxic effects beyond a certain concentration threshold. However, neither the exact uptake mechanism of these particles nor the actual reason for their toxicity has yet been elucidated. In this study we examined the uptake of silica nanoparticles with a diameter of 7, 12 and 22 nm by means of transmission electron microscopy, accompanied by toxicological assays. We show that for every particle diameter tested a different membrane morphology during uptake can be observed and that the amount of particles entering in one event is different for the three sizes. Silica particles with a diameter of 22 nm show single-particle internalization with a membrane wrapped around the particles in the cytosol, whereas 12 nm particles display row-like multi-particle uptake into elongated membrane structures and those with a diameter of 7 nm or less end up in tubular endocytic structures containing many particles. These membrane morphologies proved to be highly reproducible as we found them in five different cell lines. Additionally, we performed ATP and LDH assays to determine particle toxicity. Exceeding a certain concentration threshold the nanoparticles showed a high toxic potential both in the biochemical assay measurements and from morphological findings. We could not find any hint at the induction of apoptosis, neither morphologically nor biochemically. In this regard we discuss membrane damage and consumption as one possible mechanism of toxicity, linking morphological observations to toxicological findings to bridge the gap in understanding the mechanism of toxicity of small nanoparticles.
Highlights
Silicon dioxide nanoparticles (SiNPs) are used in a wide range of commercially available products to improve product features
For SiNP-12 and SiNP-22, even after only 2 h of incubation we found a significant number of cells with a leakage in the outer membrane and general signs of necrosis like vacuolization, nuclear membrane disintegration and mitochondria containing agglomerates of dark appearance in transmission electron microscopy (TEM) brightfield micrographs (Figure 8)
In the present study we show that small silica NPs (1) tend to agglomerate in a protein containing environment; (2) reveal a size-dependent mode of uptake into cultured cells, regardless of NP concentration, medium or temperature; (3) show similar uptake morphologies for all investigated cell lines and (4) induce a size-dependent cytotoxicity most likely mediated by a necrotic process
Summary
Silicon dioxide nanoparticles (SiNPs) are used in a wide range of commercially available products to improve product features. As free-flow or anti-caking agent they are contained, e.g., in powdery products of the food industry, serve as fragrance carriers in cosmetics or as the abrasive component in toothpaste This results in an exposure to SiNPs on a more or less regular basis. Lesniak et al report free SiNPs (50 nm diameter) in the cytosol after treatment with higher particle concentrations [8]. It has been repeatedly measured by different groups that the number of particles entering a cell in a given time is dependent on particle size and the serum content in the culture medium [11,12]. Studies using cell models like artificial liposomes or polymersomes report the uptake of SiNPs into these structures in a size-dependent manner [15]
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