A reliable approach for assessing size-dependent effects of silica nanoparticles on cellular internalization behavior and cytotoxic mechanisms

Abstract

Background

The size of nanoparticles is considered to influence their toxicity, as smaller-sized nanoparticles should more easily penetrate the cell and exert toxic effects. However, conflicting results and unstandardized methodology have resulted in controversy of these size-dependent effects. Here, we introduce a unique approach to study such size-dependent effects of nanoparticles and present evidence that reliably supports this general assumption along with elucidation of the underlying cytotoxic mechanism.

Methods

We prepared and physically characterized size-controlled (20–50 nm) monodispersed silica nanoparticles (SNPs) in aqueous suspensions. Then, a variety of biochemical assessments are used for evaluating the cytotoxic mechanisms.

Results

SNP treatment in three cell lines decreased cell viability and migration ability, while ROS production increased in dose- and size-dependent manners, with SNPs <30 nm showing the greatest effects. 30- and 40-nm SNPs were observed similar to these biological activities of 20- and 50-nm, respectively. Under the conventionally used serum-free conditions, both 20-nm and 50-nm SNPs at the IC₅₀ values (75.2 and 175.2 μg/mL) induced apoptosis and necrosis in HepG2 cells, whereas necrosis was more rapid with the smaller SNPs. Inhibiting endocytosis impeded the internalization of the 50-nm but not the 20-nm SNPs. However, agglomeration following serum exposure increased the size of the 20-nm SNPs to approximately 50 nm, preventing their internalization and cell membrane damage without necrosis. Thus, 20-nm and 50-nm SNPs show different modes of cellular uptake, with smaller SNPs capable of trafficking into the cells in an endocytosis-independent manner. This approach of using non-overlapping size classes of SNPs under the same dose, along with serum-induced agglomeration analysis clarifies this long-standing question about the safety of small SNPs.

Conclusion

Our results highlight the need to revise safety guidelines to account for this demonstrated size-dependent cytotoxicity under serum-free conditions, which may be similar to the microenvironment after tissue penetration.

Keywords:

• silica nanoparticles
• size-dependent cytotoxicity
• cellular internalization
• necroptosis
• serum agglomeration

View correction statement:

A Reliable Approach for Assessing Size-Dependent Effects of Silica Nanoparticles on Cellular Internalization Behavior and Cytotoxic Mechanisms [Corrigendum]

Acknowledgment

The authors thank Drs. Sang Chul Lee, Baek-Soo Han, and Kyoung-Jin Oh for their helpful advice. This study was supported by a grant from the Korea Research Institute of Bioscience and Biotechnology and by grants from the National Research Foundation of Korea, which was funded by the Ministry of Science, Information & Communication Technology and Future Planning (NRF-2015M3A9C6030284, 2015M3A9D7029882, 2015M3A7B6027948).

Disclosure

The authors report no conflicts of interest in this work.