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

Authors Kim W, Kim WK, Lee K, Son MJ, Kwak M, Chang WS, Min JK, Song NW, Lee J, Bae KH

Received 22 July 2019

Accepted for publication 18 August 2019

Published 10 September 2019 Volume 2019:14 Pages 7375—7387


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Prof. Dr. Thomas J. Webster

Wooil Kim, 1,* Won Kon Kim, 1,* Kyungmin Lee, 1 Min Jeong Son, 1 Minjeong Kwak, 2 Won Seok Chang, 3 Jeong-Ki Min, 1 Nam Woong Song, 2 Jangwook Lee, 1 Kwang-Hee Bae 1

1Division of Biomedical Research, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; 2Center for Nano-Bio Measurement, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Republic of Korea; 3Department of Nanoprocess, Korea Institute of Machinery & Materials (KIMM), Daejeon 34103, Republic of Korea

*These authors contributed equally to this work

Correspondence: Jangwook Lee;Kwang-Hee Bae
Division of Biomedical Research, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yusung-gu, Daejeon 34141, Republic of Korea
Tel +82 42 860 4268
Fax +82 42 860 4149

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 IC 50 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

Corrigendum for this paper has been published

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