Introduction

The development of green processes for the synthesis of nanopajmb_articles is evolving into an important branch of nanotechnology (1, 2).
Today, nanometal pajmb_articles, especially silver, have drawn the attention of scientists because of their extensive application in the development of new technologies in the areas of electronics, material sciences and medicine at the nanoscale (3-5). Silver nanopajmb_articles have many applications; for example, they might be used as spectrally selective coatings for solar energy absorption and intercalation material for electrical batteries, as optical receptors, as catalysts in chemical reactions, for biolabelling, and as antimicrobials. (3, 4, 6). Many reports have been published in the literature on the biogenesis of silver nano-pajmb_articles using several plant extracts, particularly Neem leaf broth (Azadirachta indica) and geranium leaves (P. graveolens) (7, 8). The reducing property of different plant constituents such as geraniol (Figure 1) may play a critical role in the reduction of Ag+ to silver nanopajmb_articles (8). However, the synthesis of silver nanopajmb_articles using plant constituents has not yet been studied for a large number of natural compounds. In this study, the synthe-sis of silver nanopajmb_articles using geraniol has been investigated. Also, in this study the cyto-toxicity of silver nanopajmb_articles prepared by geraniol was investigated using a cancer cell line (Fibrosarcoma-Wehi 164).

Materials and Methods

Synthesis and characterization of silver nano-pajmb_articles
Aqueous solution containing Ag+ ions (1 mM) are prepared by adding 100 µl of oily geraniol (Carol Roth GmbH+Co, Karlsruhe, Germany) and 10 ml of 1 %w/v aqueous solution of polyethylene glycol 4000 (Merck, Germany) to 90 ml of silver nitrate solution. This was then alkalized with 0.1 NaOH (20 µl) and treated in a microwave oven (850 W) for 40 sec for the reduction of metal ion. In a series of parallel experiments, the reaction takes place at room temperature. The reduction of the Ag+ ions by geraniol in the solutions was monitored by sampling the aqueous component (2 ml) and measuring the UV–visible spectrum of the solutions. UV–visible spectra of these aqueous colloid samples (1 mM) were measured on a Labo-med Model UVD-2950 UV-VIS Double Beam PC Scanning spectrophotometer, oper-ated at a resolution of 2 nm. Furthermore, silver nanopajmb_articles were characterized by transmission electron microscopy (CM 200 FEG, Philips) and energy-dispersive spectros-copy (EDS).


Cell culture and cytotoxicity assay
The Fibrosarcoma cell line (Wehi 164) was seeded in 96-well tissue culture plates. Cells were maintained in a RPMI-1640 medium that was supplemented with 5% fetal calf serum plus antibiotics at 5% CO2, 37 oC, and saturated humidity. The Fibrosarcoma-Wehi 164 cell line was obtained from the National Cell Bank of Iran (NCBI), Pasteur Institute of Iran, Tehran (Iran). Colloidal Ag-NPs solu-tion (10 mg/ml) was centrifuged at 18000 rpm for 1 hr and sediment was re-suspended in distilled water. Triplicate, different concentra-tions of silver nanopajmb_articles (1, 2, 3, 4 and 5 µg/ml) were transferred to overnight cultured cells. Non-treated cells were used as control. Cells were cultured overnight and were then subjected to Crystal Violet colori-metric assay. Cytotoxicity was expressed as the percentage of viable cells at different con-centrations of samples. IC 50 was calculated as the dose at which 50% cell death occurred relative to the untreated cells.
In the cytotoxicity assay, cells in the exponential phase of growth were incubated for 24 hr at 37 oC with 5% CO2 with different concentrations of silver nanopajmb_articles. The cell proliferation was evaluated by a modified Crystal Violet colorimetric assay (9). After each experiment, the cells were washed with ice-cold phosphate buffer solution and fixated in a 5% formaldehyde solution. Fixed cells were stained with 1% crystal violet. Stained cells were lysed and solubilized with a 33.3% acetic acid solution. The density of developed purple color was read at 580 nm. The differ-ences in cell cytotoxicity were compared using the Student’s t test. P values <0.05 were considered significant.

Result

Synthesis of silver nanopajmb_articles
The chemical reduction of aqueous solution of silver nitrate is one of the most widely used methods for the synthesis of silver colloids. In this study, the formation of silver nanopar-ticles by geraniol was investigated. The appearance of a yellowish brown color in the reaction vessels suggested the formation of silver nanopajmb_articles (10). Figure 2 shows the bottles containing the silver nitrate (1 mM) before (tube A) and after reaction with geraniol for 40 sec under heating in micro-wave oven (tube B). Also, no color change was observed when the procedure took place at room temperature or stayed for 24 hr in the same conditions (Figure not shown). These reaction mixtures were further characterized by UV-visible spectroscopy.
The technique outlined above proved to be very useful for the analysis of nanopajmb_articles (11, 12). As illustrated in Figure 3, a strong, broad absorption band with a maxima located at 440 nm was observed due to formation of silver nanopajmb_articles produced by the geraniol. This peak is assigned to a surface plasmon, phenomenon that is well-documented for various metal nanopajmb_articles with sizes ranging from 2 nm to 100 nm (11, 12).

Particle size and its chemical composition
Figure 4 shows representative TEM images recorded from the drop-coated film of the silver nanopajmb_articles synthesized by treating the silver nanopajmb_articles solution with the geraniol. The particle size histogram of silver pajmb_articles produced by geraniol (right illustra-tion in Figure 4) shows that the pajmb_articles range in size from 1 nm to 10 nm, and possess an average size of 6 nm. In the analysis of the silver nanopajmb_articles by Energy Dispersive Spectroscopy (EDS), the presence of elemen-tal silver signal was confirmed in the sample (Figure 5). The Ag nanocrystallites display an optical absorption band peaking at 3 keV which is typical of the absorption of metallic silver nanocrystallites (5).


Cytotoxicity of silver nanopajmb_articles
The cytotoxicity of the silver nanopajmb_articles was evaluated in vitro against Fibrosarcoma-Wehi 164 at different concentrations (1, 2, 3, 4, 5 µg/ml). Our cytotoxicity analysis of the sample shows a direct dose-response relation-ship; cytotoxicity increased at higher concen-trations (Figure 6). The samples demonstrated a considerable cytotoxicity against the Fibro-sarcoma-Wehi 164. The concentration neces-sary to produce 50% cell death was 2.6 µg/ml for the silver nanopajmb_articles. As shown in Figure 1, in the lowest tested concentration (1 µg/ml), silver nanopajmb_articles were able to inhibit the cell line’s growth by less than 30%. In contrast the presence of 5 µg/ml of silver nanopajmb_articles significantly inhibited the cell line’s growth (> 60%).

Discussion

The potential ability of geraniol for the reduction of Ag+ to silver nanopajmb_articles was investigated in different condition. Characteri-zation by UV-visible, TEM and EDS tech-niques confirmed the reduction of silver ions to silver nanopajmb_articles. To the best of our knowledge, and based on a thorough literature surveys, this is the first report on the synthesis of silver nanopajmb_articles using geraniol as a volatile compound from different plants such as Pelargonium graveolens (geranium).
Also, in this investigation, a cytotoxicity assay was used to assess the effect of silver nanopajmb_articles on the proliferation of a cancer cell line. No studies have been conducted, however, on the cytotoxicity of silver nano-pajmb_articles against Fibrosarcoma-Wehi 164. This is the first study of the cytotoxicity of silver nanopajmb_articles against Fibrosarcoma-Wehi 164. The silver nanopajmb_articles prepared by geraniol showed significant cytotoxicity against Fibrosarcoma-Wehi 164 cell line. Only 2.6 µg/ml silver nanopajmb_articles was necessary to decrease cell proliferation by 50%.

Acknowledgement

This work was financially supported by the Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran and by Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.