Toxicology Letters 179 (2008) 93–100 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Cellular responses induced by silver nanoparticles: In vitro studies S. Arora, J. Jain, J.M. Rajwade, K.M. Paknikar ∗ Centre for Nanobioscience, Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India article info Article history: Received 14 February 2008 Received in revised form 16 April 2008 Accepted 16 April 2008 Available online 25 April 2008 Keywords: Silver nanoparticles Oxidative stress Apoptosis A431 Ht-1080 abstract A systematic study on the in vitro interactions of 7–20nm spherical silver nanoparticles (SNP) with HT- 1080 and A431 cells was undertaken as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections. Upon exposure to SNP (up to 6.25 g/mL), morphology of both the cell types remained unaltered. How- ever, at higher concentrations (6.25–50 g/mL) cells became less polyhedral, more fusiform, shrunken and rounded. IC 50 values for HT-1080 and A431 as revealed by XTT assay were 10.6 and 11.6 g/mL, respec- tively. When the cells were challenged with ∼1/2 IC 50 concentration of SNP (6.25 g/mL), clear signs of oxidative stress, i.e. decreased GSH (∼2.5-folds in HT-1080, ∼2-folds in A431) and SOD (∼1.6-folds in HT- 1080, 3-folds in A431) as well as increased lipid peroxidation (∼2.5-folds in HT-1080, ∼2-folds in A431) were seen. Changes in the levels of catalase and GPx in A431 cells were statistically insignificant in both cell types. DNA fragmentation in SNP-exposed cells suggested apoptosis. When the apoptotic thresholds of SNP were monitored with caspase-3 assay the concentrations required for the onset of apoptosis were found to be much lower (0.78 g/mL in HT-1080, 1.56 g/mL in A431) than the necrotic concentration (12.5 g/mL in both cell types). These results can be used to define a safe range of SNP for the intended application as a topical antimicrobial agent after appropriate in vivo studies. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Since ages, silver has been known to possess excellent antibac- terial properties and it was used mainly because its toxicity to human cells is considerably lower as compared to bacteria (Volker et al., 2004). Monovalent silver compounds especially silver nitrate are used as chemoprophylactic agents in the treatment of burns to prevent bacterial infections, but their use is associated with a cosmetic disorder, viz., ‘Argyria’ (Rosenman et al., 1979). Further- more, the application of topical agents containing compounds such as silver sulfadiazine as a principal component, have been found to prevent bacterial infections but retard wound healing process, which is mediated by fibroblasts (McCauley et al., 1994, 1989; Schedle et al., 1995; Kuroyanagi et al., 1991; Leitch et al., 1993). The interest in use of silver based antimicrobial agents was primar- ily due to the emergence of antibiotic resistance among microbial populations and due to the fact that resistance to silver is not com- monly encountered. Considering the positive attributes of silver and few drawbacks of ionic silver and silver sulfadiazine espe- ∗ Corresponding author. Tel.: +91 20 25653680; fax: +91 20 25651542. E-mail address: paknikar@vsnl.com (K.M. Paknikar). cially in treatment of wounds, a search for new forms of silver was required. In 1990s, silver was introduced as a colloidal form (i.e. silver nanoparticles) in ointments that could be applied to open wounds to kill bacteria very effectively. Nanoparticulate silver containing bandages, wound dressings and ointments are readily available today (Margaret et al., 2006). Silver nanoparticles (SNP) used in the present study were pre- pared by a proprietary process as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections. The intended use of SNP is in the form of water soluble gel (prepared using a polymer like Carbopol ® ). Any substance that has to be used topically on human tissue should exert its action without interfering with the phys- iological status of target tissue. Assessment of human fibroblast cytotoxicity in vitro has been a useful tool for characterizing cell toxicity of topically applied antiseptics (Hidalgo et al., 1998). There- fore, in the present study a systematic study on interactions of synthesized SNP with HT-1080 (human fibrosarcoma) and A431 (human skin/carcinoma) cells in vitro was undertaken. An attempt was made to elucidate the biochemical mechanisms at ∼1/2 IC 50 concentration of SNP. Another purpose of this study was to inves- tigate apoptotic potential of SNP. 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.04.009