Gold-coated iron oxide nanoparticles as a potential photothermal therapy agent to enhance eradication of breast cancer cells

Iron oxide nanoparticles (Fe3O4 NPs) possess unique physicochemical properties which make them great for biomedical applications. This study reports the development of gold (Au) coated Fe3O4 (Au-Fe3O4 NPs) for photothermal therapy to eradicate breast cancer cells (MCF-7). The spherical shape of monodisperse Au-Fe3O4 NPs with an average size of 20.8 nm was confirmed by TEM. The cell viability evaluation of Au-Fe3O4 NPs showed negligible toxicity toward MCF-7 cells after 24 h. Significant cell reduction was observed for MCF-7 (73.9%) cells following photothermal therapy at highest concentration of NPs (50 μgFe/ml) for 10 minutes illumination when compared with other intervention groups. It can be concluded that, the synthesized Au-Fe3O4 NPs is an effective and promising photothermal therapy agent for breast cancer treatment.


Introduction
Recently, Fe 3 O 4 NPs have increasingly garnered interest owing to their extensive biomedical applications such as drug delivery, cancer treatment, hyperthermia therapy and magnetic resonance imaging (MRI) [1]. Fe 3 O 4 NPs possess excellent properties that involve non-toxicity, biocompatibility, superparamagnetic behavior and good magnetic susceptibility [2]. Also, Au NPs display the most potential to form a shell on the Fe 3 O 4 NPs surface due to its, chemically stability, biocompatible, resistant to oxidation and intrinsic optical properties [3]. The Au displays intense surface plasmon resonance absorption in near-infrared (NIR) region allowing it uses for photothermal therapy and thermal imaging [4]. For that reason, the productions of the biocompatible Au-Fe 3 O 4 NPs have emerged as a promising strategy over the recent years [5]. Regarding to these reports, the introduction of efficient photothermal therapy agent integrating magnetic Fe 3 O 4 core and Au shell NPs would be of great significance. Based on the literature, there are many works have done for photothermal therapy by using Au NPs [6] and silver (Ag) NPs [7]. However, there is a lack of study to see how the effect of Fe 3 O 4 (core) and Au (shell) as a photothermal therapy agent. In this study, we examined the efficacy of

Synthesis Au-Fe 3 O 4 NPs
The preparation of Au shell coated Fe 3 O 4 NPs involved the synthesis of Fe 3 O 4 NPs as seeds and following by reduction of HAuCl 4 in the presence of seeds using the sonication method. At 40 kHz (ultrasonic frequency), 5 mg of Fe 3 O 4 was ultrasonically dispersed in 20 ml of sodium citrate for 15 min. Afterwards, a freshly prepared HAuCl 4 solution (10 ml ,0.1 M) was added to reduce HAuCl 4 and form the shell located on Fe 3 O 4 surface. Sonicated process was then continued for 15 min. Au-Fe 3 O 4 NPs were collected by means of a permanent magnet and thoroughly rinsed three times with distilled water and re-dispersed in distilled water.

Cytotoxicity assay
The cytotoxicity of Au-Fe 3 O 4 NPs on human breast carcinomas (MCF-7 cell line) cultured in DMEM were determined by WST-1 assay.

In vitro photothermal ablation of MCF-7 cells
cells were illuminated by NIR (808 nm, at an output power 200 mW) for 1,5 and10 min. Cell viability was measured after illumination by NIR laser using WST-1assay.  [8]. The absence of Fe 3 O 4 peaks has been reported by others [9]. This is may be due to the heavy atom effect from Au [10] thus, the Fe 3 O 4 core was completely covered by the Au shell. More specific, the core signal was shielded by the Au shell and thus disappeared in the X-ray diffraction pattern.   Figure (d). The thickness of the Au shell was estimated to be 13.3 nm. the shell thickness could be changed by varying experimental parameters. To assess the biocompatibility and treatment efficacy in cancer therapy of Au-Fe 3 O 4 NPs (50, 25, 12.5 and 6.25 µgFe/ml), WST-1 cell assay was carried out on human breast carcinoma (MCF-7 cell lines), as shown in Figure 1(a). The cell viability evaluation of Au-Fe 3 O 4 NPs showed negligible toxicity toward MCF-7 cells lines after 24 h, even at their highest concentrations, which confirms their applicability for biomedical purposes.

Results and Discussion
In this experimental study, the treated MCF-7 cells with Au-Fe 3 O 4 NPs (50, 25, 12.5 and 6.25 μgFe/ml) overnight, were irradiated to NIR (808 nm, at a power of 200 mW, and spot size 2mm) for 1, 5 and 15 minutes times' period. Moreover, Control groups were determined as cells incubated with same concentrations of NPs without NIR irradiation. After NIR irradiation, reduction of MCF-7 cells treated with Au-Fe 3 O 4 NPs was observed more in compared to the control groups, a fact which resulted from photothermal effect of Au-Fe 3 O 4 NPs.   Table 1 contains mean values of cell viability ± standard deviations (STDs) in the different time durations of irradiation and different concentrations of nanoparticle. The results of the Kruskal-Wallis test showed that there were no statistically significant differences in cell viability after incubation with different concentrations of nanoparticle in control group (p-value > 0.05). But different concentrations of Fe significantly effect cell viability after one, five and ten minutes of irradiation (p-value<0.01 for all). From Table 1, in study groups (one, five and ten minutes after irradiation) the lowest cell viability percentage was observed in concentration of 50 gFe/ml of nanoparticle (78.069±18.189,