Cellular responses induced by silver nanoparticles: In vitro studies

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Toxicology Letters 179 (2008) 93–100

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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

a r t i c l e

i n f o

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

a b s t r a c t A systematic study on the in vitro interactions of 7–20 nm spherical silver nanoparticles (SNP) with HT1080 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. However, at higher concentrations (6.25–50 ␮g/mL) cells became less polyhedral, more fusiform, shrunken and rounded. IC50 values for HT-1080 and A431 as revealed by XTT assay were 10.6 and 11.6 ␮g/mL, respectively. When the cells were challenged with ∼1/2 IC50 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 HT1080, 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 antibacterial 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). Furthermore, 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 primarily due to the emergence of antibiotic resistance among microbial populations and due to the fact that resistance to silver is not commonly 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: [email protected] (K.M. Paknikar). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.04.009

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 prepared 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 physiological 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). Therefore, 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 IC50 concentration of SNP. Another purpose of this study was to investigate apoptotic potential of SNP.

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Fig. 1. Phase-contrast micrographs of HT-1080 cells: (A) unexposed cells; (B–F) 24 h after the cultures were exposed to 3.12, 6.25, 12.5, 25 and 50 ␮g/mL SNP, respectively (magniﬁcation 200×).

2. Materials and methods

2.2. Chemicals and glassware

2.1. Silver nanoparticles

The plasticware and glassware used for cell culturing were obtained from Nunc (Myriad industries, San Diego, California). Caspase-3 assay kit (colorimetric), Dulbecco’s modiﬁed Eagle’s medium (DMEM), fetal bovine serum (FBS), l-glutamine, penicillin and streptomycin were procured from Sigma Chemical Company (St. Louis, MO). Cell proliferation kit (XTT) was procured from Roche diagnostics GmbH (Mannheim, Germany). Glutathione reductase was purchased from Fluka chemika/biochemika (Fluka Chemie AG, Switzerland). All other analytical grade chemicals for biochemical studies were obtained from Sigma Chemical Company (St. Louis, MO).

Silver nanoparticles used in the present study were synthesized by a proprietary process (PCT patent applications pending). They were used as colloidal aqueous suspension and were found to retain their stability in the culture media. The nanoparticles were spherical (>90% particles in the size range of 7–20 nm, as revealed by high resolution TEM) exhibiting characteristic surface plasmon peak at 436 nm. The MIC values of the SNP were determined as per NCCLS standard (Anon, 2000). The observed MIC values for a variety of Gram positive and Gram negative bacteria were in the SNP concentration range of 1.56–6.25 ␮g/mL (unpublished data).

Fig. 2. Phase-contrast micrographs of A431 cells: (A) unexposed cells; (B–F) 24 h after the cultures were exposed to 3.12, 6.25, 12.5, 25 and 50 ␮g/mL SNP, respectively (magniﬁcation 200×).

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2.3. Cell lines, cell culture and maintenance The cell lines A431 (human skin carcinoma) and HT-1080 (human ﬁbrosarcoma) were obtained from National Center for Cell Sciences (NCCSs), Pune, India. Cell lines were cultured in Dulbecco’s modiﬁed Eagle’s medium supplemented with l-glutamine (4 mM), penicillin (100 units/mL), streptomycin (100 ␮g/mL) and 10% (v/v) heat inactivated fetal bovine serum (growth medium). Cells were maintained in 5% CO2 humidiﬁed incubator at 37 ◦ C. During subculture, cells were detached by trypsinization when they reached 80% conﬂuency and split (1:4). Growth medium was changed every 3 days. 2.4. Cell morphology Cells were plated into a 35 mm tissue culture plate at a density of 2 × 105 cells (in 2 mL growth medium). After overnight growth, supernatants from the culture plates were aspirated out and fresh aliquots of growth medium containing SNP in desired concentrations (0.76–50 ␮g/mL) were added. Silver concentration in the SNP preparation was determined using atomic absorption spectrometry (AAnalyst800, PerkinElmer, USA). Upon incubation, cells were washed with phosphate-buffered saline (PBS, pH 7.4) and the morphological changes were observed under an inverted phase-contrast microscope at 200× magniﬁcation. 2.5. In vitro cytotoxicity assay Cell viability was tested using XTT (sodium 3 -[1-(phenylaminocarbonyl)-3,4tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay (Gerlier and Thomasset, 1986) based on the cleavage of yellow tetrazolium salt XTT by metabolically active cells to form an orange formazan dye which was quantiﬁed using ELISA reader (Biorad, USA, model 680). XTT assay was performed according to manufacturer’s instructions with appropriate controls. Cells were seeded in 96 well microtitre plates (1 × 104 cells/200 ␮L growth medium/well) followed by overnight incubation. Supernatants from the wells were aspirated out and fresh aliquots of growth medium (containing SNP in desired concentrations in the range of 0.76–50 ␮g/mL) were added. After 24 h, supernatants were aspirated out and the cell monolayers in the wells were washed with 200 ␮L PBS (0.1 M, pH 7.4). Subsequently, XTT reagent (70 ␮L) was added in each well, incubated for 5 h and absorbance at two wavelengths (415 nm for soluble dye and 630 nm for cells) were recorded using ELISA reader. Concentrations of SNP showing 50% reduction in cell viability (i.e. IC50 values) were then calculated. 2.6. Preparation of cell extract and biochemical assays For the preparation of cell extract, 75 cm2 ﬂasks containing 15 mL of growth medium were seeded with ca. 1 × 107 cells as described by Christopher et al. (2004). After overnight growth the cells were challenged with ∼1/2 IC50 of SNP, viz. 6.25 ␮g/mL and incubated for 24 h. The cells (about 80% conﬂuent) were then trypsinized and pelleted by centrifugation at 500 × g for 5 min. The cell pellet was washed with PBS (0.1 M, pH 7.4), resuspended in 500 ␮L chilled homogenizing buffer (250 mM sucrose, 12 mM Tris–HCl, 0.1 mM DTT, pH 7.4) and lysed using Dounce homogenizer. The lysate was centrifuged (8000 × g, 10 min, 4 ◦ C) and the supernatant (cell extract) was used in various biochemical assays. Protein concentration in the cell extract was estimated by Bradford (1976) method. 2.6.1. Catalase Catalase activity was measured by the method described by Aebi (1984). In this, absorbance (240 nm) of 1 mL reaction mixture containing 0.8 mL H2 O2 phosphate buffer (H2 O2 diluted 500-folds with 0.1 M phosphate buffer of pH 7), 100 ␮L cell extract, and 100 ␮L distilled water was recorded for 4 min against blank (H2 O2 phosphate buffer). Change in absorbance per min (i.e. A240 ) was then calculated and used in the estimation of enzyme activity (extinction coefﬁcient value used was 39.4 L M−1 cm−1 ). 2.6.2. Superoxide dismutase Superoxide dismutase activity was measured according to the method described by Kono (1978). Brieﬂy, the reaction mixture (2.1 mL) contained 1924 ␮L sodium carbonate buffer (50 mM), 30 ␮L nitrobluetetrazolium (1.6 mM), 6 ␮L Triton X-100 (10%) and 20 ␮L hydroxylamine–HCl (100 mM). Subsequently 100 ␮L cell extract was added and absorbance (560 nm) was read for 5 min against blank (reaction mixture sans cell extract). In this experiment, a speciﬁc control containing reaction mixture with cell extract (of SNP-unexposed cells) and SNP (6.25 ␮g/mL) was also run. Change in absorbance per min (i.e. A560 ) was calculated and used in the estimation of enzyme activity (extinction coefﬁcient value used was 9920 L M−1 cm−1 ). 2.6.3. Glutathione peroxidase Glutathione peroxidase activity was measured as described by Flohe and Gunzler (1984). The reaction mixture contained 1.2 mL potassium phosphate buffer (0.1 M, pH 7), 200 ␮L GSH (10 mM), 10 ␮L sodium azide (200 mM), 200 ␮L cell extract and 200 ␮L glutathione reductase (2.4 U/mL). Following incubation at 37 ◦ C for 10 min, 200 ␮L NADPH (1.5 mM), 200 ␮L H2 O2 (1.5 mM) were added.

Fig. 3. (A and B) Percent cytotoxicity measured by XTT assay on HT-1080 cells and A-431 cells, respectively after treatment with SNP (1.56–50 ␮g/mL) for 24 h. The data are expressed as mean ± standard deviation (S.D.) of three-independent experiments. An OD value of control cells (Unexposed cells) was taken as 100% viability (0% cytotoxicity). *Denotes a statistically signiﬁcant difference compared to control (p < 0.05).

Absorbance (340 nm) was recorded for 4 min against blank (reaction mixture sans cell extract) and change in absorbance per min (i.e. A340 ) was calculated. The enzyme activity was calculated (extinction coefﬁcient value used was 6220 L M−1 cm−1 ).

2.6.4. Total glutathione (reduced) content Total GSH content was measured as described by Saldak and Lindsay (1968). The reaction mixture containing 1.2 mL EDTA (0.02 M), 1 mL distilled water, 250 ␮L 50% trichloroacetic acid, 50 ␮L Tris buffer (0.4 M, pH 8.9) was centrifuged at 300 × g for 15 min. Clear supernatant (500 ␮L) was mixed with 1 mL of 0.4 M Tris buffer (containing 0.02 M EDTA, pH 8.9), 100 ␮L of 0.01 M DTNB [5,5 -dithio-bis-(2-nitrobenzoic acid)] and 100 ␮L cell extract. The mixture was incubated at 37 ◦ C for 25 min and the yellow color developing was read at 412 nm against blank. The enzyme activity was calculated taking the extinction coefﬁcient as 14,150 L M−1 cm−1 .

2.6.5. Lipid peroxidation In this method described by Beuge and Aust (1978), a mixture of 100 ␮L Tris buffer (150 mM, pH 7.1), 10 ␮L ferrous sulfate (100 mM), 10 ␮L ascorbic acid (150 mM), 780 ␮L distilled water and 100 ␮L cell extract was incubated at 37 ◦ C for 15 min. Thiobarbituric acid (0.375%, 2 mL) was then added to the mixture and allowed to react at 100 ◦ C (in water bath) for 15 min. The reaction mixture was then centrifuged (800 × g for 10 min) and supernatant was read at 532 nm against blank. The enzyme activity was calculated (extinction coefﬁcient value used was 156,000 L M−1 cm−1 ).

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Fig. 4. Levels of catalase, SOD, GSH, lipid peroxidation and GPx in untreated (control) and treated (with SNP, 6.25 ␮g/mL for 24 h) HT-1080. The data are expressed as mean ± standard deviation (S.D.) of three-independent experiments. *Denotes a statistically signiﬁcant difference compared to control (p < 0.05). 2.7. Apoptosis DNA laddering experiments were initiated in 25 cm2 ﬂasks by seeding ca. 5 × 105 cells in 5 mL growth medium. After overnight growth, cells were treated with SNP (6.25 ␮g/mL) for 24 h. DNA was isolated using a commercially available kit (Bangalore Genei, India) by following the manufacturers instructions. DNA was resolved on 1.5% agarose gel (containing 3 ␮g/mL ethidium bromide in 1× TAE buffer of pH 8.5) at 90 V for 1.5 h, and bands visualized using a UV transilluminator. In another set of experiments, caspase-3 colorimetric assays were performed using a standard kit (Sigma, USA) to determine the concentrations of SNP inducing apoptosis and necrosis in HT-1080 and A431 cells. 2.8. Statistical analyses All the experiments were carried out three times, independently. The data obtained were expressed in terms of ‘mean ± standard deviation’ values. Wherever appropriate, the data were also subjected to unpaired two tailed Student’s t-test. A value of p < 0.05 was considered as signiﬁcant.

3. Results Under phase-contrast microscope, HT-1080 cells (control) appeared polyhedric or stellate showing slender lamellar expansions (Fig. 1A) that joined neighboring cells. With increasing concentration of SNP (from 6.25 to 50 ␮g/mL), cells were seen

as less polyhedric; and more fusiform, shrunken and rounded (Fig. 1C–F). Control A431 cells were polygonal with extensive and almost continuous intercellular contacts (Fig. 2A) while SNP treated cells exhibited bipolar, spindle-type cell morphology with lesser intercellular contacts. At concentrations >6.25 ␮g/mL (Fig. 2C–F), cell numbers decreased signiﬁcantly and the cells appeared rounded and refractile. The results of XTT assays (Fig. 3) showed a dose-dependent cytotoxicity for both the cell types with IC50 values working out as 10.6 and 11.6 ␮g/mL for HT-1080 and A431 cells, respectively (Fig. 3A and B). Based on these results, ∼1/2 IC50 dose of SNP (viz. 6.25 ␮g/mL) was selected for further biochemical and apoptosis studies. When the biochemical changes occurring in the SNP treated cells were monitored (with respect to enzyme levels indicative of oxidative stress), interesting data emerged. In HT-1080 (Fig. 4), treatment with SNP increased lipid peroxidation by ∼2.5-folds (0.17 ␮mol/mg protein in unexposed cells and 0.44 ␮mol/mg protein in SNP-exposed cells). In contradistinction, there was ∼2.5-folds decrease in GSH levels after SNP treatment (0.26 ␮mol/mg proteins as compared to 0.66 ␮mol/mg protein in control cells). SOD levels were found to be ∼1.6-folds lower for SNP treated cells (0.83 ␮mol/mg) as compared to unex-

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Fig. 5. Levels of catalase, SOD, GSH, lipid peroxidation and GPx in untreated (control) and treated A431 cells (with SNP, 6.25 ␮g/mL for 24 h). The data are expressed as mean ± standard deviation (S.D.) of three-independent experiments. *Denotes a statistically signiﬁcant difference compared to control (p < 0.05).

posed cells (1.4 ␮mol/mg protein). It was found that the difference in the SOD activities in (a) the reaction mixture with cell extract of SNP-unexposed cells and (b) the reaction mixture with cell extract of SNP-unexposed cells plus SNP, was statistically insignificant (p > 0.05). Changes observed in the levels of catalase and GPx were also not statistically signiﬁcant. In case of A431 cells lipid peroxidation levels in SNP-exposed cells were two times the unexposed cells (Fig. 5). In contrast, levels of GSH decreased ∼2-folds after treatment with SNP (0.89 ␮mol/mg protein as compared to 1.53 ␮mol/mg protein in control cells). SOD levels were found to be ∼3-folds lower in SNP treated cells (0.28 ␮mol/mg) as compared to unexposed cells (0.91 ␮mol/mg protein). Once again, the difference in the SOD activities in (a) the reaction mixture with cell extract of SNP-unexposed cells and (b) the reaction mixture with cell extract of SNP-unexposed cells plus SNP, was statistically insigniﬁcant (p > 0.05). Changes in the levels of catalase and GPx in A431 cells were also found to be statistically insigniﬁcant as was seen in HT-1080 cells. Fig. 6 depicts DNA fragmentation (ladder formation) in cells exposed to SNP suggestive of apoptosis. Next, the apoptotic thresholds of SNP were monitored. Caspase-3 assay was performed

on both cell lines after exposure to SNP for a range of doses (0.39–25 ␮g/mL). The results of a representative dose-range study are shown in Figs. 7 and 8. In case of A431 cells, SNP could induce apoptosis at concentrations in the range of 1.56–6.25 ␮g/mL. For HT-1080 cells this range changed to 0.78–6.25 ␮g/mL. At SNP concentrations 12.5 ␮g/mL, caspase-3 activity was not detected in both the cell types. It needs to be mentioned here that the possibility that SNP may directly inhibit caspase-3 activity was ruled out in this experiment by adding SNP to the positive control (caspase-3) provided with the Sigma (USA) kit. 4. Discussion In the present work, we have attempted a detailed investigation on the effects of silver nanoparticles in vitro. Such studies on toxic effects of nanomaterials are very far and few between and no clear guidelines are available to quantify these effects. Even though nanosilver-based wound dressings have received approval for clinical applications their possible dermal toxicity is reported to be a matter of concern (Chen and Schluesener, 2008). The intended use of SNP synthesized by us is also for the treatment of wounds in the form

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Fig. 8. Caspase-3 activity assay for A431 cells. SNP induce caspase-3 production (apoptosis) at concentrations in the range of 1.56–6.25 ␮g/mL. At SNP concentrations ≥12.5 ␮g/mL, lack of caspase-3 activity indicated necrosis. OD value of positive control was taken as 100%. *Denotes a statistically signiﬁcant difference compared to control (p < 0.05).

Fig. 6. Analysis of genomic DNA fragmentation. Lane M, standard DNA markers (100bp); lane 1, untreated control A431 cells; lane 2, SNP (6.25 ␮g/mL) treated A431 cells; lane 3, SNP (6.25 ␮g/mL) treated HT-1080 cells; lane 4, untreated control HT-1080 cells.

of a topical gel formulation. Therefore, toxicity evaluation (both in vitro and in vivo) of our SNP preparation becomes essential. Primary cells are a better representative of tissue and hence are ideal for in vitro toxicity studies. However, secondary cell lines are easy to maintain and are preferred in most toxicological studies for better reproducibility of data. In the wound healing process, dermal ﬁbroblasts are the main cell types implicated in extracellular matrix production (Hunt and Hopt, 1997) while epidermis provides a protective layer for underlying dermis. Considering this fact, in the present study we have selected HT-1080 (derived from human

Fig. 7. Caspase-3 activity assay for HT-1080 cells. SNP induce caspase-3 production (apoptosis) at concentrations in the range of 0.78–6.25 ␮g/mL. At SNP concentrations ≥12.5 ␮g/mL, caspase-3 activity was not detected indicating necrosis. OD value of positive control was taken as 100%. *Denotes a statistically signiﬁcant difference compared to control (p < 0.05).

dermis) and A431 (derived from human epidermis) cell lines for in vitro toxicity evaluation. Moreover, both these cell types have a consistent histological origin. The toxicity of SNP was evaluated by studying cellular morphology and mitochondrial function (XTT assay) under control and exposed conditions. To investigate the potential role of oxidative stress as a mechanism of SNP induced toxicity, the effects on catalase, SOD, GPx, GSH and lipid peroxidation were monitored. It is now well established that the generation or external addition of ROS can cause cell death by two distinct pathways, viz. apoptosis or necrosis. ROS are known to trigger the apoptotic cascade, via caspases, which are considered as the executioners of apoptosis (Fadeel et al., 1998). Caspase-3 activation is often considered as the point of no return in apoptosis (Cohen, 1997). Activation of caspase3 results in the cleavage of ICAD (inhibitor of caspase-activated DNAse) and translocation of CAD (caspase-activated DNAse) to the nucleus ultimately resulting in DNA fragmentation. The most prominent event in the early stages of apoptosis is internucleosomal DNA cleavage by endonuclease activities (Wyllie, 1980; McConkey et al., 1989). The connection between oxidative stress and apoptosis, as explained above prompted us to investigate the role of apoptosis in SNP toxicity. Results obtained by us clearly showed that cell morphology of both A431 and HT-1080 cells was unaltered in the presence of SNP up to a concentration of 6.25 ␮g/mL. However, abnormal size, shrinkage and rounded appearance of cells at higher dose suggested toxicity of SNP. In this study the effect of SNP on cell viability was estimated using XTT assay as the quantity of XTT reduced per unit time is proportional to the cell number (Harbell et al., 1997). Decreased mitochondrial function in cells exposed to SNP (3.12–50 ␮g/mL) in a dose-dependent manner as seen in the XTT assay is an expected result. This result is comparable to the one reported by (Hussain et al., 2005) where cytotoxicity due to decreased mitochondrial function was indicated in a rat liver derived cell line (BRL 3A) that was exposed to silver nanoparticles at concentrations in the range of 5–50 ␮g/mL. Nanosized particles of various chemistries preferentially mobilize to mitochondria (Foley et al., 2002; Li et al., 2003) thereby overloading or interfering with antioxidant defenses and creating ROS both in vitro and in vivo. ROS production has been reported by nanoparticles as diverse as C60 fullerenes (Oberdorster, 2004),

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single-walled carbon nanotubes (Shvedova et al., 2004), quantum dots (Derfus et al., 2004) and ultraﬁne particles (Brown et al., 2001). Studies on rat liver derived cell line (BRL 3A) by Hussain et al., 2005) showed that there was a signiﬁcant increase in ROS and decrease in GSH levels at 25 and 50 ␮g/mL of Ag (15, 100 nm). A signiﬁcant elevation of lipid peroxidation and marginal GSH depletion was demonstrated in a ﬁsh model upon exposure to fullerenes (Oberdorster, 2004). In the present study, when SNP induced oxidative stress as a mechanism(s) of toxicity was assessed depletion of GSH (∼2.5fold) was seen which probably results in the shift of overall redox balance towards oxidation, leading to functional damage of cells and enhanced lipid peroxidation (∼2.5-fold) in HT-1080 (human ﬁbrosarcoma) cells. In case of A431 (human skin/carcinoma) cells; decreased levels of SOD (∼3-fold), GSH (∼2-fold) and increased levels of lipid peroxidation (∼2-fold) as compared to unexposed cells were observed. These results also suggest oxidative damage to cells after exposure to SNP. Results obtained by us clearly indicated that SNP could not inhibit SOD activity by any direct mechanism. Therefore, the observed SOD inactivation might be due to generation of peroxy radicals after SNP exposure. Similar observations were made by (Tirkey et al., 2005) who reported inactivation of SOD due to carbon tetrachloride induced oxidative stress in rat liver and kidney cells. Statistically insigniﬁcant changes observed in the levels of catalase and GPx after treatment with SNP in this work is suggestive of a differential and less pronounced response by these cellular defense mechanisms as compared to GSH, SOD and lipid peroxidation. On the whole, data obtained by us clearly suggest that oxidative stress is the cause of ensuing cytotoxicity in case of SNP-exposed HT-1080 and A431 cells, at ∼1/2 IC50 concentrations. The role of apoptosis in SNP toxicity was evaluated by DNA fragmentation and caspase-3 assays. The presence of oligonucleosomal DNA fragments or DNA laddering at SNP concentration around 6.25 ␮g/mL indicates apoptotic cell death. Cytotoxicity (necrosis) at higher doses of SNP (≥12.5 ␮g/mL) is clearly demonstrated by total lack of caspase-3 activity. This SNP concentration matches well with the observed IC50 values and hence supports the ﬁndings of caspase-3 assay. Further, our data clearly show that at concentrations up to 6.25 ␮g/mL, cell death occurs due to apoptosis alone. The implication of this ﬁnding in a clinical application could be signiﬁcant as it could mean that SNP could provide a permissive environment that favors scar less wound repair. At higher doses of SNP, however, necrotic changes are seen. Therefore, at such doses SNP could elicit an inﬂammatory response which could adversely affect the wound healing process (Tian et al., 2007). In an interesting study by (Shin et al., 2007) it was shown that nano-silver at concentrations between 3 and 10 ␮g/mL modulated cytokine production and hence could be used to treat inﬂammatory diseases. We have not monitored any cytokine production in response to SNP in the present study. However, we envisage that around 1/2 IC50 values of SNP a mild anti-inﬂammatory response could manifest, adding immense value to the intended use of SNP, i.e. treatment of burn wounds. Such studies need to be undertaken in future. In summary, our studies show that at antimicrobial concentrations (1.56–6.25 ␮g/mL) SNP synthesized by us are safe as seen in the in vitro toxicity studies. Since the lowest concentration of SNP inducing apoptosis in the cell lines is several folds lower than the cytotoxic (necrotic) concentration, a safe range of SNP concentrations for the topical treatment of burn wound infections can be deﬁned after appropriate in vivo studies. In this study we have highlighted, probably for the ﬁrst time, the correlations among ` cytotoxicity, oxidative stress and apoptotic potential vis-a-vis silver nanoparticles.

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Cellular responses induced by silver nanoparticles: In vitro studies

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