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 Labomed Model UVD-2950 UV-VIS Double Beam PC Scanning spectrophotometer, operated at a resolution of 2 nm. Furthermore, silver nanoparticles were characterized by transmission electron microscopy (CM 200 FEG, Philips) and energy-dispersive spectroscopy (EDS).

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 °C, 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 solution (10 mg/ml) was centrifuged at 18000 rpm for 1 hr and sediment was re-suspended in distilled water. Triplicate, different concentrations of silver nanoparticles (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 colorimetric assay. Cytotoxicity was expressed as the percentage of viable cells at different concentrations 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 °C with 5% CO₂ with different concentrations of silver nanoparticles. 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 differences in cell cytotoxicity were compared using the Student's t test. P values < 0.05 were considered significant.

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 nanoparticles by geraniol was investigated. The appearance of a yellowish brown color in the reaction vessels suggested the formation of silver nanoparticles (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 microwave 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 nanoparticles (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 nanoparticles produced by the geraniol. This peak is assigned to a surface plasmon, phenomenon that is well-documented for various metal nanoparticles with sizes ranging from 2 nm to 100 nm (11, 12).

Figure 4 shows representative TEM images recorded from the drop-coated film of the silver nanoparticles synthesized by treating the silver nanoparticles solution with the geraniol. The particle size histogram of silver particles produced by geraniol (right illustration in Figure 4) shows that the particles range in size from 1 nm to 10 nm, and possess an average size of 6 nm. In the analysis of the silver nanoparticles by Energy Dispersive Spectroscopy (EDS), the presence of elemental 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).

The cytotoxicity of the silver nanoparticles 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 relationship; cytotoxicity increased at higher concentrations (Figure 6). The samples demonstrated a considerable cytotoxicity against the Fibrosarcoma-Wehi 164. The concentration necessary to produce 50% cell death was 2.6 µg/ml for the silver nanoparticles. As shown in Figure 1, in the lowest tested concentration (1 µg/ml), silver nanoparticles were able to inhibit the cell line's growth by less than 30%. In contrast the presence of 5 µg/ml of silver nanoparticles significantly inhibited the cell line's growth (>60%).