Distribution of SiO₂ nanoparticles in 3D liver microtissues

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, SiO₂ 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 SiO₂ 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, SiO₂ NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO₂ NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO₂ 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.

Keywords:

• silica nanoparticles
• human hepatocarcinoma cells
• spheroids
• penetration

Supplementary materials

Figure S1 Packing density of HepG2 spheroids at day 3 and 7.

Notes: Several HepG2 spheroids were analyzed at day 3 (left) or day 7 (right) after seeding of 1,000 cells per well. In representative confocal fluorescence micrographs the cell membrane (green) and cell nucleus (cyan) are presented. Exemplarily overview images of the whole spheroids are shown.

Figure S2 Cell viability of HepG2 spheroids in the presence of SiO₂ NPs.

Notes: After their formation, HepG2 spheroids were incubated with varying SiO₂ NP concentrations (1, 10, 100, 200 µg mL⁻¹) for 24 hours. Cell viability was determined by Resazurin assay. Results are presented as mean ± SD from three independent experiments. Dashed line indicates control value (100%).

Abbreviation: NPs, nanoparticles.

Figure S3 Immunostaining of whole HepG2 spheroid.

Notes: Prior to staining, HepG2 spheroid was permeabilized with triton X-100 for 30 minutes at room temperature. After blocking with 5% BSA, the sample was immunostained for nuclear lamina (Lamin B). Embedding of the spheroid under a cover slide resulted in a flattened form. Confocal image of HepG2 spheroid immunostained for nuclear lamina is depicted in yellow. Orthogonal views (xy, xz, and yz) showing the intersection planes at the position of the green cross-hair.

Figure S4 Nanoparticle localization in spheroids.

Notes: HepG2 spheroids were exposed to 100 µg mL⁻¹ SiO₂ NPs either after spheroid formation (A) or during spheroid formation at day 0 (B) or day 2 (C). In representative confocal fluorescence micrographs, the cell membrane (green) and SiO₂ NPs (magenta) are presented. Overview images of the whole spheroid (left) are shown. White frame indicates the position of the detailed z-stacks. Exemplary, orthogonal views (xy, xz, yz) were derived from z-stacks at a selected layer. Arrows highlight the localization of SiO₂ NPs in the spheroid.

Abbreviation: NPs, nanoparticles.

Figure S5 Localization of ATTO 647N-APTES dye conjugate in spheroids.

Notes: HepG2 spheroids were exposed to 0.83 µM ATTO 647N-APTES dye conjugate after spheroid formation (A) or during spheroid formation at day 0 (B). In representative confocal fluorescence micrographs, the actin cytoskeleton (green, left) or ATTO 647N-APTES dye conjugate (magenta, right) are presented.

Figure S6 Spheroid diameter in dependence of the silica nanoparticle exposure scenario.

Notes: HepG2 spheroids were either untreated or exposed to 100 µg mL⁻¹ SiO₂ NPs either after spheroid formation or during spheroid formation (day 0, day 2). Spheroid diameter was determined for five spheroids (n=5). Results are presented as mean + SD.

Abbreviation: NPs, nanoparticles.

Acknowledgments

The authors would like to thank Anika Krieger for the particle synthesis and characterization as well as Dominique Piché for the review of the final paper. The authors thank Eduard Arzt for continuing support of their work at INM. The publication of this article was funded by the Open Access Fund of the Leibniz Association. The work was funded by the German Federal Ministry for Economic Affairs and Energy, in the framework of the Zentrale Innovationsprogramm Mittelstand (ZIM)-cooperation network NanoPharm in the project ‘MORPHEUS’.

Author contributions

JF, HP, and AK designed the study. JF and HP performed microscopy imaging and analysis. JS carried out cell viability and oxidative stress experiments. MK performed electron microscopy investigations. IT participated in particle synthesis and characterization. All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.