Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties

Abstract

Cellular internalisation of industrial engineered nanoparticles is undesired and a reason for concern. Here we investigated and compared the ability of seven different mammalian cell cultures in vitro to incorporate six kinds of engineered nanoparticles, focussing on the role of cell type and particle properties in particle uptake. Uptake was examined using light and electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) for particle element identification. Flow cytometry was applied for semi-quantitative analyses of particle uptake and for exploring the influence on uptake by the phagocytosis inhibitor Cytochalasin D (CytoD). All particles studied were found to enter each kind of cultured cells. Yet, particles were never found within cell nuclei. The presence of the respective particles within the cells was confirmed by EDX. Live-cell imaging revealed the time-dependent process of internalisation of technical nanoparticles, which was exemplified by tungsten carbide particle uptake into the human skin cells, HaCaT. Particles were found to co-localise with lysosomal structures within the cells. The incorporated nanoparticles changed the cellular granularity, as measured by flow cytometry, already after 3 h of exposure in a particle specific manner. By correlating particle properties with flow cytometry data, only the primary particle size was found to be a weakly influential property for particle uptake. CytoD, an inhibitor of actin filaments and therewith of phagocytosis, significantly inhibited the internalisation of particle uptake in only two of the seven investigated cell cultures. Our study, therefore, supports the notion that nanoparticles can enter mammalian cells quickly and easily, irrespective of the phagocytic ability of the cells

Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles

“…This result contrasts with some studies that have demonstrated that side scatter could differentiate among concentrations of titanium-, tungsten-, and diamond-based particles [6,11]. However, these studies did not examine PSPs, and it is possible that the granularity (and side scatter) generated by uptake of PSPs was different from that of NPs they tested.…”

“…Polystyrene NPs could not be detected by the flow technique. The assay, termed phagocytosis-coupled flow cytometry, uses the fact that monocytes have a strong capacity for particle uptake compared with other cell types [11]. J774A.1 cells were chosen for this study because, similar to other monocytic cells, they have an inherent ability to internalize particles differing in size and composition.…”

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

“…This would suggest that, in our case, the internalization pathway depends mostly on NP properties and on the properties of the protein corona formed on the NP surface in the cell culture media, 67 although several other studies showed that the internalization pathway vastly depends on the cell type as well. 34,68,69 Consistent with the involvement of macropinocytosis and CME, the NPs underwent the main intracellular trafficking route; from early endosomes through late endosomes to lysosomes (Figure 3), where the degradation of endocytosed cargo normally occurs. 11 NPs were also observed in amphisomes ( Figure 3G-I), which indicates that the presence of NPs in the endosomes does not interfere with the normal intertwining of the two intracellular digestion pathways.…”

Mechanisms of Nanotoxicity

“…Harush-Frenkel et al (2008) demonstrated, by means of polylactic acid-based model ENM, that negatively charged particles preferentially localize in the lysosomes while positively charged particles escape this route and are more dominantly found in the cytosol. The vast majority of environmentally relevant ENM tested thus far are negatively charged and a variety of them have indeed been found colocalized with lysosomes (e.g., Busch et al, 2011). Accumulation in lysosomes may render these organelles, particularly susceptible due to locally increased exposure.…”