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

Abstract

Introduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO 2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results: Spheroids were exposed to 100 µg mL −1 SiO 2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO 2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO 2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO 2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.

Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model

“…Fleddermann et al found that SiO 2 -NPs were distributed through the whole HepG2 spheroid when the NPs were mixed with cells before spheroid formation. However, when exposing the already formed spheroids for 24 h, NPs were seen only to a depth of 20 µm [26]. Cell types, cell densities, physicochemical characteristics of the NPs (including size distribution) and ion release may influence the penetration inside the spheroid [73][74][75][76][77].…”

“…This increases the cell-to-cell contacts and intercellular communication [22] and changes the protein expression and metabolic status of the cells [21][22][23]. HepG2 cells in 3D cultures show upregulation of genes involved in liver-specific xenobiotic and lipid metabolism, whereas genes related to the extracellular matrix, cytoskeleton and cell adhesion have higher expression in 2D cultures [22,24].The use of spheroids as 3D cultures in hepatotoxicity assessment is an increasing field of interest, and HepG2 spheroids, prepared with and without using scaffolds, have been applied for toxicity experiments with both NPs [6,25,26] and chemicals [27,28]. However, the differences in toxic responses between cells cultured in 2D and 3D are not yet clear.…”

“…The use of spheroids as 3D cultures in hepatotoxicity assessment is an increasing field of interest, and HepG2 spheroids, prepared with and without using scaffolds, have been applied for toxicity experiments with both NPs [6,25,26] and chemicals [27,28]. However, the differences in toxic responses between cells cultured in 2D and 3D are not yet clear.…”

Gelatin interpenetration in poly N‐isopropylacrylamide network reduces the compressive modulus of the scaffold: A property employed to mimic hepatic matrix stiffness

“…FDA was used to mark the live cells; FDA is taken up by the cells, cleaved to fluorescein by esterases which is impermeable to the cell membranes and hence stays within the cells. PI, on the other hand, is impermeable to the cell membranes and passes through the disintegrated membrane of dying cells, thereby probing dead cells (Fleddermann et al, ). Similar to resazurin reduction and LDH release, more PI stained (red), that is, dead cells were visible in the N0.5 group as compared to other scaffolds wherein FDA stained (green), that is, viable cells are visible (Figure c).…”

Head-to-head comparison of the penetration efficiency of lipid-based nanoparticles in a 3D tumor spheroid model

“…rapid proliferation at the surface and slow metabolism -or necrosis -in the center of spheroids 25 . The majority of studies of NP distributions in tumor spheroids use tumor spheroid fixation [26][27][28][29][30][31][32][33][34] , often in combination with histological sectioning 7,[35][36][37][38] . Nevertheless, there are also reports of NP penetration in live tumor spheroids using confocal-scanning microscopy [39][40][41][42][43][44][45][46][47][48][49] .…”