Induction of Oxidative Stress and Cell Death in Neural Cells by Silica

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Induction of oxidative stress and cell death in neural cells by silica nanoparticles. Yuji Kamikubo, Tomohito Yamana, Yoshie Hashimoto, and Takashi Sakurai ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00248 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 20, 2018

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ACS Chemical Neuroscience

Induction of oxidative stress and cell death in neural cells by silica nanoparticles.

Yuji Kamikubo1*, Tomohito Yamana1, Yoshie Hashimoto1, and Takashi Sakurai1

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Department of Pharmacology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku,

Tokyo 113-8421, Japan.

All correspondence should be addressed to: Yuji Kamikubo, PhD Department of Pharmacology, Juntendo University School of Medicine Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan E-mail: [email protected] Fax: +81-(0)3-5802-0419 Phone: +81-(0)3-5802-1035

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ABSTRACT: 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 to 1,500 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.

Keywords nanoparticle, silica, reactive oxygen species, cytotoxicity, neuron, stress

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Introduction With the development of nanoscience, nanomaterials with various sizes, chemical elements, shapes, and surface modifications have been produced for industries and consumers. Although bulk materials have constant features regardless of their size, nanomaterials possess novel size-dependent physical and chemical properties, and have been used in a broad range of processes and products including paints, foods, cosmetics, clothes, polishing of fine structures, and pharmaceuticals.1 Because of their interesting optical and electrical properties, inorganic nanomaterials (e.g., nanorods, nanowires, and quantum dots) have been used in optoelectronics.2 Nanomaterials including nanoparticles (NPs) composed of titanium dioxide, alumina, zinc oxide, and silicon dioxide (silica) are well studied and have been applied in various fields. Because silica nanoparticles (SiNPs) are inexpensive, innocuous, and easy to produce and functionalize, they are often applied as adsorbents, catalyst carriers, materials for bio-imaging, and drug-delivery systems.3, 4 However, some investigators have raised concerns regarding the biocompatibility and potential toxicity of SiNPs when they contact tissues and the cell surface. Data from recent studies showed that SiNP treatment induced cytotoxicity and inflammatory responses in lung epithelial and endothelial cell lines.5, 6 Numerous reports have shown the in vivo distribution and bioactivity of several types of nanomaterials, including SiNPs. Some reports have indicated that NPs can cross the blood–brain barrier and injure brain tissues.7 Recent findings showed that NPs exerted toxic effects on neuronal cell lines or primary glial cell cultures.8-10 However, neuron-targeting NPs have been successfully

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used for gene delivery to neurons.11 It is well known that neural cells are vulnerable to stresses, although it remains unclear whether SiNPs negatively impact central neurons. To address this issue, we investigated the biological effects of SiNPs with various sizes at various concentrations on primary cultures of rat hippocampal cells in comparison to those in the kidney cell line, HEK293. Neurons are electrically excitable, have a highly polarized structure composed of dendrites and an axon, and show vulnerability to hypoxia, hyperthermia, and mechanical and oxidative stress.12 Thus, we considered various relevant parameters such as the concentration, administration period, surface modification, and temperature when studying the effects of SiNPs on neurons. Our analysis indicated that SiNPs exert concentration-dependent toxicity on hippocampal cells and that hippocampal cells were more vulnerable than HEK293 cells. Surface carboxylation and adsorption of albumin reduced cytotoxicity, suggesting that interactions between SiNPs and the cell surface are key to their cytotoxicity. Similar to other cell types,13-15 we showed that treatment with SiNPs generated reactive oxygen species (ROS) and that pretreatment with an antioxidant reduced the cytotoxic effects. This study represents the first step in elucidating the biochemical and biophysical consequences of SiNPs to neurons. Our data provide insights into the safe and effective use of SiNPs.

RESULTS AND DISCUSSION Cytotoxic effects of SiNP. Concerns regarding the potential health risks of nanomaterials have been raised with their increased use and the development of nanotechnology. Toxic effects of 4


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NPs were observed in previous experimental studies exploring potential biomedical uses. Basic studies of the biological activities of nanomaterials are essential to ensure the safe use of nanomaterials. While SiNPs have been tested in biomedical applications, the biological effects and physical features of the chemical groups coated on SiNPs have not been elucidated. Here we examined whether treatment with SiNPs caused cytotoxic effects in neural cells. In addition, we investigated a method for reducing the adverse effects of SiNPs in biomedical applications. In previous reports,5, 8 nanomaterials induced cell damage within several hours after administration. Thus, we used the CellTiter-Glo2.0 (CTG) Luminescent Cell Viability Assay (Promega, WI, USA), which enables quick estimation of the number of viable cells by quantifying ATP.16 The ATP-based measurement is more sensitive and rapid than other methods involving 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alamarBlue, and Calcein-AM.17 The MTT assay and other commonly used cell-viability assays require prolonged incubation times (e.g., 4 h for the MTT assay) to convert indicator molecules into detectable signals. In contrast, the CTG assay requires a much shorter incubation time in a one-step format and enables measurement of time-dependent changes in cell viability with higher time resolution. Using hippocampal primary cultures (mixed cultures of neurons and glial cells) and HEK293 cells, we confirmed the linear correlation between the number of viable cells and luminescence in CTG assays (Figure S1A, B). A previous report indicated that SiNPs, tens of nanometers in diameter, are cytotoxic to HEK293 cells.18 Furthermore, recent findings showed that NPs exerted toxic effects on neuronal 5



cell lines or primary cultures of neuron. 19 20 21 Thus, we first examined the toxicity of SiNPs with a diameter of 30 nm (SiNP30) to HEK293 cells and cultured hippocampal cells. HEK293 cells or cultured hippocampal cells seeded in 96-well microplates were treated with SiNP30 for 2 h, and cell viability was assessed with CTG assays. The analysis showed a concentration-dependent cytotoxicity of SiNP30 to HEK293 and hippocampal cells, and indicated that neurons were more vulnerable to SiNP30 than HEK293 cells (Figure 1A). We independently confirmed the cytotoxicity of SiNP30 by performing MTT assays (the most commonly used colorimetric assay) to measure the number of viable cells.17 In both CTG and MTT assays, signals were consistently reduced by SiNP30 administration in a concentration-dependent manner (Figure S1C). To further confirm cell damage induced by SiNPs, we also performed propidium iodide (PI) staining. PI is a plasma membrane-impermeable dye with red fluorescence that is commonly used to detect dead or dying cells. PI fluorescence signals were clearly increased by SiNP30 treatment (Figure S2). To evaluate cell damage and morphology, we observed primary hippocampal neurons using phase-contrast (Figure 1B, C) and fluorescence microscopy (Figure 1D–F). Phase-contrast images of cultured neurons revealed that treatment with 0.1 mg/mL SiNP30 induced neurite collapse within 30 min (Figure 1B, C). To visualize the cytotoxic effects of SiNPs on cells, we treated cultured neurons with fluorescently labeled SiNP30. The results suggested that SiNP contact with the cell surface, including dendrites and axons, was cytotoxic to the neurons (Figure 1D–F). Next, we performed time-lapse analysis of SiNP cytotoxicity to cultured hippocampal neurons. To visualize morphological changes, we transfected hippocampal neurons with a plasmid 6


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encoding the modified yellow fluorescent protein, Venus. Treatment with a high concentration (1 mg/mL) of SiNP30 collapsed axons and dendrites rapidly, and induced soma swelling after several min (Figure 2A). Because dystrophic neurite and soma swelling were exclusively associated with neuronal damages,22 we examined the correlation of the SiNP30-administration period with cell viability. The CTG assay indicated that high SiNP30 concentrations (0.1 to 1 mg/mL) reduced luminescent signals within 60 min (Figure 2B), whereas lower SiNP30 concentrations (

Induction of Oxidative Stress and Cell Death in Neural Cells by Silica

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