Elsevier

Toxicology in Vitro

Volume 24, Issue 3, April 2010, Pages 872-878
Toxicology in Vitro

Silver nanoparticles induce cytotoxicity by a Trojan-horse type mechanism

https://doi.org/10.1016/j.tiv.2009.12.001Get rights and content

Abstract

Silver nanoparticles (AgNPs) are widely applied in many household products and medical uses. However, studies on the effects of AgNPs on human health and environmental implications are in the beginning stage. Furthermore, most data on the toxicity of AgNPs have been generated using nanoparticles modified with detergents to prevent agglomeration, which may alter their toxicities. In this study, we studied toxicity using AgNPs prepared by dispersing them in fetal bovine serum (FBS), biocompatible materials. AgNPs (average size; 68.9 nm, concentrations; 0.2, 0.4, 0.8, and 1.6 ppm, exposure time; 24, 48, 72, and 96 h) showed cytotoxicity to cultured RAW264.7 cells by increasing sub G1 fraction, which indicates cellular apoptosis. AgNPs decreased intracellular glutathione level, increased NO secretion, increased TNF-α in protein and gene levels, and increased gene expression of matrix metalloproteinases (MMP-3, MMP-11, and MMP-19). When cells were treated with AgNPs, they were observed in the cytosol of the activated cells, but were not observed in the dead cells. It seemed that AgNPs were ionized in the cells to cause cytotoxicity by a Trojan-horse type mechanism suggested by previously reported studies.

Introduction

Silver nanoparticles (AgNPs) have been widely used in personal care products, food service, building materials, medical instruments, and textiles because of their antibacterial effect. Furthermore, the application of AgNPs has become more widespread for sensing, catalysis, transport, and other application in biological and medical sciences. Unlike the unintentional exposure to nanoparticles through the uses of consumer products, emerging biomedical applications of nanoparticles such as drug-delivery agents, biosensors, and imaging contrast agents involve deliberate, direct ingestion/injection of nanoparticles into the human body. That is why toxicity is a critical factor to be considered when evaluating their potential for biomedical purposes (Lewinski et al., 2008, Ju-Nam and Lead, 2008).

Several studies have reported that AgNPs significantly induced cell necrosis or apoptosis in several cell types. For example, AgNP less than 3 nm induced cytotoxicity in macrophages (Yen et al., 2009). A decrease of cell viability was also observed in liver and neuron cells treated with AgNPs (Hussain et al., 2005, Hussain et al., 2006). Twenty-eight-day oral toxicity, genotoxicity, and gender-related tissue distribution of AgNPs in rat was investigated (Kim et al., 2008). Subchronic inhalation toxicity of AgNPs was also investigated (Sung et al., 2009). In those reports, histopathological examinations indicated dose-dependent increases in lesions related to silver nanoparticle exposure, including mixed inflammatory cell infiltrate, chronic alveolar inflammation, and small granulomatous lesions. However, direct evidence on toxic effects of unmodified AgNPs has not been fully documented at the cellular and molecular levels. This is partially because unmodified AgNPs are unstable in the culture media of cell lines. To generate the toxicity data for AgNPs, particles modified with chemicals such as hydrocarbons, sucrose, and decanethiol have been used. Detergents such as Tween, dimethylsulfoxide, carboxymethyl cellulose, and dipalmitoyl phosphatidyl choline have also been added to the culture media to prevent agglomeration, which might alter the toxicities of the nanoparticles (Arora et al., 2009, Murdock et al., 2008, Ahamed et al., 2008, Bothun, 2008, Carlson et al., 2008, Foucaud et al., 2007).

The characterizations of the physico-chemical properties of the nanoparticles are essential for assessing the potential toxicity of nanoparticles, and ensuring data reproducibility. However, the key parameters affecting the biological activity of AgNPs have not been fully elucidated. Complete characterization of AgNPs may include measurements of size distribution, shape and other morphological features, chemistry of the material, solubility, surface area, state of dispersion, surface chemistry, and other physico-chemical properties (Royal Society, 2004, Hood, 2004, Powers et al., 2006, Tsuji et al., 2006).

In this study, we studied the toxicity of unmodified AgNP by dispersing them in FBS (fetal bovine serum) which is not exogenous but endogenous biocompatible material, and also examined the mechanisms by which these particles exert toxicity.

Section snippets

Preparation and characterization of AgNPs suspension

Silver nanoparticles (AgNPs) purchased from Sigma–Aldrich (St. Louis, MO, USA, Cat. No. 484059, particle size <150 nm) were suspended at a concentration of 10 mg/ml in fetal bovine serum (FBS). After sonication for 10 min, the suspended AgNPs were filtered (Nalgene, NY, USA, pore size 0.2 μm), and stored at −20 °C prior to use. The concentration of the AgNPs stock suspension after filtration was 15.3 ppm measured by ICP-MS. The stock suspension of AgNPs was re-sonicated for 3 to 5 min prior to tests,

Stability of AgNPs in the culture medium

TEM image of Silver nanoparticles (AgNPs) was shown in Fig. 1. According to the TEM image, AgNPs were well dispersed in culture media containing 10% FBS. The particle size measured by submicron particle sizer was 68.9 ± 30.3 nm in the culture media. Surface charge and conductance of AgNP are 0.91 mV and 5954 μS, respectively. The average mobility was 0.07 (μ/s).

Cytotoxicity in cultured RAW264.7 cells

When cells were treated with AgNPs (0.2, 0.4, 0.8, and 1.6 ppm for 24 h, 48 h, 72 h, and 96 h, respectively), viability of the cultured RAW264.7

Discussion

Silver nanoparticles (AgNPs) have specific physico-chemical properties that lead to their aggregation and ionization in FBS-free medium, PBS (phosphate buffered saline), or DDW (deionized distilled water). AgNPs seemed to easily agglomerated or aggregated in the test media. So, many researchers used various detergents to disperse them in the test solution, but these detergents may alter the toxicity of AgNPs. In this study, we prevented agglomeration of AgNPs using FBS, essential component in

Acknowledgement

This work was supported by National Institute of Environmental Research, Ministry of Environment.

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