Observation of Positively Charged Magnetic Nanoparticles inside HepG2 Spheroids Using Electron Microscopy

Abstract

Magnetic resonance imaging (MRI) using magnetic nanoparticles has been used to diagnose vascular diseases as well as to monitor transplanted cells and tissues. In this study, we synthesized magnetic iron oxide nanoparticles (TMADM-03), electrically charged by the presence of a cationic end-group substitution of dextran, and observed these nanoparticles inside three-dimensional models of HepG2 spheroids, which mimic tissues. Patterned cell array glass disks were prepared to visualize the presence of TMADM-03 uptaken by HepG2 spheroids using transmission electron microscopy (TEM). The HepG2 cells (2 × 10(5) cells) were inoculated onto Cell-able™ 12-well plates. After 48 h of culture, the cells were incubated with 75 µg Fe/ml TMADM-03 in culture medium for 24 h. To investigate the cellular function of the HepG2 spheroids, the albumin secretion was evaluated by an ELISA. The albumin secretion after incubation for 24 h was reduced compared with the secretion prior to the addition of TMADM-03. TEM image samples were prepared in a planar direction or a vertical direction to the HepG2 spheroids on patterned cell array glass disks. The incorporation of TMADM-03 inside the HepG2 spheroids was confirmed. In addition, TMADM-03 could be observed in the deeper layers of the spheroids, and this was localized in the lysosomes. These data suggest that the novel magnetic iron oxide nanoparticles invade three-dimensional HepG2 spheroids.

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

“…Miyamoto et al reported the localization of positively charged magnetic iron oxide NPs in deeper layers inside HepG2 spheroids using TEM analysis. 38 In comparison with the present study, the spheroid size was considerably smaller with a diameter of about 50-100 µm. Internalized NPs were mainly localized in the lysosomes.…”

“…32,35 In recent years, liver microtissues were used as a model for NP toxicity assessment. 34,[36][37][38][39] Dubiak-Szepietowska et al observed a stronger cytotoxic effect of Ag, ZnO, and SiO 2 NPs on HepG2 cells when grown in 2D compared to cells grown in 3D hydrogels. 34 The lower sensitivity to toxic concentrations of Ag NPs was also detected in HepaRG cell spheroids.…”

Utility of Three-Dimensional Cultures of Primary Human Hepatocytes (Spheroids) as Pharmacokinetic Models

“…Further, cells in spheroid cultures secrete an extracellular matrix in which they reside, and they can interact with cells from their original microenvironment [ 31 ]. Spheroids can also be formed from human cancer-derived hepatocytes, such as HepaRG cells [ 32 , 33 , 34 ], HepG2 cells [ 35 , 36 , 37 ], and Huh-7 cells [ 38 ]. Cultured human cancer-derived hepatocytes have long been used for human-specific toxicity assessments, but they generally have lower expression levels of drug-metabolizing enzymes than PHHs [ 39 , 40 ].…”

Ultrastructural Features of Gold Nanoparticles Interaction with HepG2 and HEK293 Cells in Monolayer and Spheroids

“…Morphological changes in spheroids treated with NPs or chemical compounds are studied mainly in transmitted light and various fluorescence methods [ 9 , 19 , 20 , 21 ]. The use of electron microscopy is rare and mostly is limited to registration of NPs presence in a cell [ 22 , 23 , 24 ] or TEM-illustration of NPs used in a study [ 9 , 15 , 21 , 25 , 26 ]. However, the size of NPs requires studying their interaction with cells at subcellular level, which is realized in a transmission electron microscopy (TEM) of ultrathin sections.…”