Silica nanoparticles (SiNPs) are produced on an industrial scale and used in various fields including health care, because silica is stable, inexpensive, and easy to handle. Despite these benefits, there is concern that exposure to SiNPs may lead to adverse effects in certain types of cells or tissues, such as hemolysis, immune responses, and developmental abnormalities in the brain and developing embryos. Although investigations on the toxicity of SiNPs against neurons are essential for medicinal use, only a few studies have assessed the direct effects of SiNPs on cells derived from the central nervous system. In this study, we investigated the toxic effects of SiNPs on primary cultures of hippocampal cells, using SiNPs with diameters of 10-1500 nm. We showed that treatment with SiNPs caused oxidative stress and cell death. Furthermore, we found that these cytotoxicities were dependent on the particle size, concentration, and surface charge of SiNPs, as well as the treatment temperature. The toxicity was reduced by SiNP surface functionalization or protein coating and by pretreating cells with an antioxidant, suggesting that contact-induced oxidative stress may be partially responsible for SiNP-induced cell death. These data will be valuable for utilizing SiNPs in biomedical applications.

Nanomaterials have a variety of unique physical and chemical properties, and are being studied for biotechnological, pharmacological, and medical applications. Silica nanoparticles (SiNPs) are produced on an industrial scale and used in various fields. Despite these benefits, there is concern that exposure to SiNPs may lead to adverse effects in certain types of cells or tissues, such as developmental abnormalities in the brain and developing embryos. Although investigations on the toxicity of SiNPs against neurons are essential for medicinal use, a few studies have assessed the direct effects of SiNPs on cells derived from the central nervous system. In this study, we investigated the toxic effects of SiNPs on primary cultures of hippocampal cells. We showed that treatment with SiNPs caused oxidative stress and cell death. Furthermore, we found that these cytotoxicities were dependent on the particle size, concentration, and surface charge of SiNPs. The toxicity was reduced by SiNP surface functionalization or protein coating and by pretreating cells with an antioxidant, suggesting that contact-induced oxidative stress may be responsible for SiNPinduced cell death. These data will be valuable for utilizing SiNPs in biomedical applications.

Various nanomaterials have been produced with the development of nanotechnology, some of which have been reported to have adverse effects on several types of cells, organs, and the environment. It has been suggested that some small nanoparticles can cross the blood-brain barrier and accumulate in the brain, which may be a potential cause of brain diseases. Neuronal cells are vulnerable to hypoxia, hypotrophy, and mechanical and oxidative stress. Therefore, it is essential to assess the toxicity of nanoparticles to neurons accurately. In this report, we describe a primary culture protocol to evaluate the toxicity of nanoparticles on neurons, a potential high-throughput method for assessing the cytotoxicity, and a method for evaluating the effect on neuronal maturation. This report assessed the toxicity of silicon dioxide, zinc oxide, and iron nanoparticles using rat hippocampal neurons, which are used frequently in pharmacological and physiological studies. Based on the methods and protocols we reported in this report, it may be possible to evaluate nanotoxicity to various neurons by using primary cultures of other brain regions (cerebral cortex, cerebellum, thalamus, etc.), spinal cord, and peripheral nerves.