A required weight (0.02 - 0.12 g) of pulverized algae was mixed with 50 mL of ultrapure water. The mixture was added with 3.45 µL of 1.4 M HAuCl₄ solution. Gold nanoparticles formation was carried out at different reaction temperature (25˚C - 80˚C) and time duration (60 - 120 min) with stirring condition. The solution was then filtered using filter paper, Whatman No. 5 to remove algae particles. The final concentration of algae in formulation was 0.04% - 0.24% w/v. The concentration of gold ion in final solution was 0.966 µM (20 ppm).

The characteristic peak of gold nanoparticle was observed using a UV-Visible spectrophotometer (UV-160A, Shimadzu, Japan). The morphology of nanoparticles was examined by using JEM-2100 Transmission Electron Microscopy (TEM) (JEOL, Japan). The gold diameter and size distribution were calculated using SemAfore software, JEOL (Japan). Energy-Dispersive X-ray spectroscopy (EDX) measurement was also studied for elemental analysis of the samples. The surface potential of gold nanoparticles was assessed using a ZEN 3600 zetasizer (Malvern Instrument, UK).

The MTT assay was used to evaluate cell viability of human non-small lung H460 cancer cells. The 2 × 10⁴ H-460 cells were grown in 96-well plate, 100 µL media was added, and the cells were cultured for 24 hours at 37˚C in a humidified 5% CO₂ atmosphere to allow cell growth. The cells were then incubated for 24 hours with either 100 µL of algae-stabilized gold nanoparticles or 100 µL of algae solution, while 100 µL of ultrapure water was used as a control. After incubation, the culture media was discarded and the cells were washed with phosphate buffered saline (PBS). Serum-free media (100 µl) was added along with 2 µL of 20 mg/mL of MTT in DMSO and the culture was incubated for another 4 hours at 37˚C. The culture media was discarded and the purple formazan was dissolved in 100 µL DMSO. The UV absorbance of the sample was measured using an Anthos HT II microplate reader (Anthos Labtec Instruments) at the wavelength of 570 nm.

The percentage of cell viability was expressed as average ± S.D. (n = 3) and statistically significant difference was determined using Student’s t-test at p-value ≤ 0.05.

The algae were able to reduce the gold solution into gold nanoparticles. The appearance of rather pink solution and the UV absorbance peak of around 530 - 535 nm represented the occurrence of gold nanoparticles [6]. It has been reported that reducing sugars and polysaccharides constituted in algae may facilitate the formation and stabilization of the gold particles [7].

The optimal condition for gold nanoparticle synthesis using wakame algae were studied in terms of algae concentration, reaction time and temperature. It was found that at higher algae concentration resulted in lower absorbance of gold nanoparticles (systems A1-A4 in Table 1). The higher in absorbance is related to an increase in a number of nanoparticles synthesized. The effect of reaction time was investigated at constant algae concentration (0.04% w/v) and reaction temperature of 80˚C. The result indicated the higher absorbance of gold nanoparticles with increasing time duration of reaction from 60 to 120 min (systems B1-B3 in Table 1). Moreover, at 0.04% w/v algae and reaction time of 60 min, the absorbance of nanoparticles was greater if the reaction temperature was raised from 25˚C to 80˚C (systems C1-C3 in Table 1).

The gold nanoparticles synthesized at 0.04% w/v algae and 80˚C were determined for morphology by TEM analysis. The overall shape of gold nanoparticles synthesized was spherical (Figure 1). The average particle size of gold nanoparticles formed in 60 min was 7.85 ± 1.43 nm (Figure 1), while that of 120 min was 10.15 ± 1.96 nm. It was found that when reaction time was increased, larger particles were formed. These results were expected since longer reaction time might allow gold nanoparticles to grow in size. The EDX spectrum of gold nanoparticles synthesized is illustrated in Figure 2. The spectrum possible indicated that the nanoparticles contained might be the gold (Au) element based on 2.8 KeV EDX peak and surface potential. The copper (Cu) spectrum at 7.8 KeV EDX peak and surface potential referred to the copper grid used in the TEM analysis. For the surface potential of nanoparticles, it was found that gold nanoparticles contained negative surface potential indicating that there were negatively charged molecules surround the particles (Table 2). It was likely that sulphated polysaccharides containing negative charges should be around the particle [8]. Hence, nanoparticles are stabilized by electrostatic repulsive force arising from the negative charges on the particle surface [9].

The effect of gold nanoparticles on viability of human non-small lung H460 cancer cells was determined using MTT assay. The algae-stabilized gold nanoparticles synthesized using 0.04% w/v algae, 80˚C and 120-min reaction caused the reduction in cell viability with increasing concentration of the nanoparticles (Figure 3). The toxicity of gold nanoparticles toward the cancer cells apparently was dependent on the synthetic conditions. At the same algae concentration and reaction temperature, an increase in reaction time caused less cellular toxicity (Figure 4(a)). The non-significant differences in cell viability of nanoparticles were found when the reaction temperature was changed from 25˚C to 80˚C at 120-min reaction and 0.04% w/v (Figure 4(b)). However, the nanoparticles seemed to be more toxic to the cancer cells than the corresponding algae solutions. The ability of gold nanoparticles to decrease the cell viability is considered to be arisen from the functional polysaccharides especially the sulfated-based polysaccharides in algae [10].

*mean ± S.D. (n = 3).