| Summary: | 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 Bae1 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 workCorrespondence: Jangwook Lee;Kwang-Hee BaeDivision of Biomedical Research, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yusung-gu, Daejeon 34141, Republic of KoreaTel +82 42 860 4268Fax +82 42 860 4149Email jlee@kribb.re.kr; khbae@kribb.re.krBackground: 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
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