Quantification of Internalized Silica Nanoparticles via STED Microscopy

Abstract

The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 × 1010 particles mL−1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20–27% of the particles sedimented. In comparison, 102-103 NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 1012 particles mL−1) of the smaller particles induced cytotoxicity.

Super-resolution microscopy as a powerful tool to study complex synthetic materials

“…STED was first used to study nanoparticle-cell interactions to demonstrate the nuclear localization and clustering of 30 nm fluorescent silica nanoparticles (SNPs) in Caco-2 cells 115,116 . More recently, Peuschel et al used 3D STED to quantify the internalization of 25 nm and 85 nm SNPs in A549 cells 117 and differentiate between membraneattached and internalized nanoparticles. A similar approach was also used to study the internalization and colocalization of carbon dots 118 and protein-based fluorescent nanoparticles 119 .…”

<p>Distribution of SiO₂&nbsp;nanoparticles&nbsp;in 3D liver microtissues</p>

“…73 Furthermore, the protein corona, consisting of proteins adsorbed at the NP surface, influences NP internalization and cytotoxicity. 71 The SiO 2 NPs used in this study did not affect the viability of HepG2 spheroids ( Figure 4). Also Dubiak-Szepietowska et al reported no cytotoxicity of SiO 2 NPs in HepG2 spheroids after 24 hours exposure.…”

“…70 In contrast, Schübbe et al and Peuschel et al described that SiO 2 NPs, which were prepared by the same synthesis, cause neither damage of membrane integrity, reduction of metabolic activity nor DNA damage in A549 and Caco-2 cells. 71,72 The contradictory results are due to differences in particle properties, cell type, and experimental conditions. SiO 2 NPs that have been prepared by different synthesis methods vary in their shape, size, and porosity which might cause different biological effects.…”

Interaction of zinc peroxide nanoparticle with fibroblast cell

“…The physico-chemical features of the nanoparticles are the determining factors for the nanoparticle-living cell interactions and consequently influence cell behaviour. The size and shape of the nanomaterials and chemical functionalities on the surface play a critical role in binding the nanomaterials to cell membrane and subsequent cellular uptake [4][5][6]. This motivates pursuing research towards the biological interference of nanoparticles and development of multi-model diagnostic probes that could facilitate the combination dignostics, preferably covering both the anatomical and physiological aspect of various disease [7-8] including the deadly cancer.…”