A Reliable Approach For Assessing Size-Dependent Effects Of Silica Nanoparticles On Cellular Internalization Behavior And Cytotoxic Mechanisms (Vol 14, Pg 7375, 2019)

Background: The size of nanoparticles is considered to influence their toxicity, as smallersized 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 mu g/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.