Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

Abstract

Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7–20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds.

Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 μg/mL and 100 μg/mL SNP, respectively, although with minor decrease in confluence. IC₅₀ values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 μg/mL and 449 μg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC₅₀ SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with ∼ 1/2 IC₅₀ concentration of SNP (i.e. 30 μg/mL and 225 μg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (∼ 1.2 fold) and depletion of lipid peroxidation (∼ 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD (∼ 1.4 fold) and GSH (∼ 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 μg/mL in primary fibroblasts, 12.5 μg/mL in primary liver cells) than the necrotic concentration (100 μg/mL in primary fibroblasts, 500 μg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC₅₀ SNP (resulting in apoptosis) and 2× IC₅₀) cells (resulting in necrosis).

These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (∼ 20 μg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.

Introduction

Silver nanoparticles are emerging as one of the fastest growing product categories in the nanotechnology industry with focus on anti-microbial activity. This has led to increasing number of medical applications of silver nanoparticles. Some of the products which are already available in the market include wound dressings, contraceptive devices, surgical instruments and bone prostheses (Cheng et al., 2004, Chen et al., 2006, Cohen et al., 2007, Zhang et al., 2007, Lee et al., 2007). Apart from these applications, silver nanoparticles are being used for water purification, indoor air quality management (Cheng et al., 2004, Jain and Pradeep, 2005, Zhang and Sun, 2007). Thus, use of nanosilver is becoming more and more widespread in medicine and related applications. Such increasing exposure poses toxicological and environmental issues which need to be addressed (Chen and Schluesener, 2008).

As a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections, silver nanoparticles (SNP) were prepared by a proprietary process and were demonstrated to possess broad spectrum antimicrobial activities at concentrations ranging between 0.78–6.12 μg/mL. The intended use of SNP is in the form of water soluble gel (prepared using a polymer like Carbopol^®, containing 20 μg SNP per gram gel) for topical applications in treatment of burn wounds. For in vitro studies with SNP (intended for topical applications), the selection of cell types representing the target tissue is important. In the wound healing process, dermal fibroblasts are the main cell types implicated in extracellular matrix production (Hunt and Hopt, 1997). In earlier studies Takenaka et al. (2001) reported that liver appears to be a major accumulation site of circulatory silver nanoparticles. A recent clinical report also described absorption of nanosilver into the circulation following the use of nanosilver coated dressings for burns (Trop et al., 2006). Established cell lines are easy to maintain and are preferred in most toxicological studies for better reproducibility of data. The question whether the established cell lines are adequate target cells has often been raised with regard to clinical relevance of data derived from in vitro studies (Hanks, 1996). In such cases, primary cells isolated from target tissues are desirable for cytotoxicity testing to simulate the in vivo situation more closely. Further, primary cultured liver cells (rodent or human origin) also represent a useful tool for studying toxicity, drug metabolism and enzyme induction (Davila and Acosta, 1993, Zurlo and Arterburn, 1996).

Thus, bearing in mind the potential target organs, primary cells viz. primary mouse fibroblasts and primary mouse liver cells were selected for in vitro toxicity studies of SNP. An attempt was made to study intracellular localization of SNP and elucidate the biochemical changes in the intracellular milieu at 1/2 IC₅₀ concentration of SNP. Another purpose of this study was to carry out detailed investigation on the type of cell death (apoptotic/necrosis) after exposure to SNP by performing caspase-3 activity and visualization by fluorescence microscopy and CLSM upon acridine orange/ethidium bromide (AO/EB) double staining.