# Synthesis of Ag@Fe3O4 Nanoparticles for Photothermal Treatment of Ovarian Cancer

Authors: Bing Liu; Jian Zhou; Bin Zhang; Jing Qu
Journal: Journal of Nanomaterials
 (2019)

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## Abstract

Photothermal therapy is a promising approach for cancer treatment. In our study, we investigate the photothermal effect of different concentrations of the Ag@Fe3O4 nanoparticles on apoptosis and proliferation in the human epithelial ovarian cancer cells SKOV3. Ovarian cancer cells SKOV3 were treated with the Ag@Fe3O4 nanoparticles under an 808 nm near-infrared (NIR) laser irradiation at different concentrations. The cell proliferation was measured by the cell counting kit-8 (CCK-8) assay. The results show that the Ag@Fe3O4 nanoparticles with NIR laser irradiation could markedly inhibit the proliferation of the ovarian cancer cells SKOV3 independent of a concentration-time manner. Meanwhile, the cell morphology was also seriously damaged under the treatment of high-concentration nanoparticles. However, Ag@Fe3O4 nanoparticles have almost no obvious effect on the growth of SKOV3 cells without NIR laser illumination treatment. Therefore, it is reasonable to believe that the Ag@Fe3O4 nanoparticles have promising applications in photothermal treatment of cancer cells.


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## Body

## 1. Introduction

Ovarian cancer is the fourth leading cause of cancer-related deaths among women, with an estimated 200,000 new cases, and 125,000 women die of ovarian cancer annually worldwide [1, 2]. For the past decade, the main treatment for women with advanced ovarian cancer has been surgery and platinum-based chemotherapy. However, due to drug resistance and other reasons, the treatment effect and prognosis are not very good. Thus, development of new therapeutic methods and strategies is critical for increasing the survival of this devastating malignancy.Photothermal therapy has been widely applied as a high-efficiency treatment for varieties of cancer cells [3, 4]. The photothermal therapy has great potential in enhancing therapeutic efficacy and lowering adverse effects [5]. To date, several nanomaterials including Au-based nanocomposites [6–9], silver-based nanoparticles [10], palladium/silver nanosheets [11, 12], carbon-based nanomaterials [13–15], and polymeric nanoparticles [16–18] have been developed for the treatment of cancer under (near-infrared) NIR laser irradiation. For example, Liu et al. reported a novel TaOx@Cat hollow nanosphere as a bionanoreactor for effectively enhancing radiation therapy for cancer cells [19]. Song et al. have synthesized the Janus iron oxides @ semiconducting polymer nanoparticles, which were applied to cancer cell labeling and in vivo tracking by MPI and fluorescence imaging [20]. Among those nanomaterials, the Ag@Fe3O4 nanoparticles, a novel photothermal agent, are expected be a good candidate for photothermal therapy application due to their strong SPR absorption property, high photothermal conversion efficacy, and outstanding photothermal stability [21].In this study, we have synthesized the Ag@Fe3O4 nanoparticles through a sample one-pot hydrothermal method. By TEM, SEM, XRD, UV-Vis-NIR, and photothermal effect analysis, the synthesized Ag@Fe3O4 nanoparticles show a high dispersion and photothermal effect. Finally, their efficacy in photothermal treatment for cancer under NIR laser irradiation is also investigated by SKOV3 cells. Our results have shown that the Ag@Fe3O4 nanoparticles could markedly inhibit the proliferation of the ovarian cancer cells SKOV3 under NIR laser irradiation and are exhibited independent of a concentration-time manner. However, Ag@Fe3O4 nanoparticles have almost no obvious effect on the growth of SKOV3 cells without NIR laser illumination treatment.


## 2. Materials and Methods

### 2.1. Materials

Ferric nitrate nonahydrate (Fe(NO3)3·9H2O), sodium acetate (NaOAc), and ethylene glycol (EG) were obtained from Sinopharm Chemical Reagent Co. Ltd. (China). Silver nitrate (AgNO3) was purchased from Aladdin (China).


### 2.2. Synthesis of Ag@Fe3O4 Nanoparticles

Ag@Fe3O4 nanoparticles were synthesized through the one-pot method reported by Fang et al. [21]. Briefly, Fe(NO3)3·9H2O (404 mg, 1.0 mmol) and NaOAc (328 mg, 4.0 mmol) were dissolved in 16 mL of EG. Then, 42.5 mg of AgNO3 (0.25 mmol) was added to the solution. The obtained mixture solution was sealed in Teflon-lined stainless-steel autoclaves, heated from 30°C to 200°C for 0.5 h, and maintained at 200°C for 8 h. The resulting Ag@Fe3O4 nanoparticles were collected by a magnet and washed with ethanol and water.


### 2.3. Photothermal Effect Measurement of the Ag@Fe3O4 Nanoparticles

To measure the photothermal effect of the Ag@Fe3O4 nanoparticles under the irradiation from an 808 nm laser, 1.0 mL of aqueous solution containing various concentrations of Ag@Fe3O4 nanoparticles (0 ppm, 10.0 ppm, 20.0 ppm, and 40.0 ppm) was irradiated for 12 min by a near-infrared laser (808 nm, 2.0 W/cm2). The temperature of the solution was recorded by an online-type thermocouple thermometer every 15 s.


### 2.4. NIR Heating of Ag@Fe3O4 Nanoparticles in Solution

Ag@Fe3O4 nanoparticles with different concentrations were prepared, and 500 μL of aliquots was deposited into the wells of a 24-well cell culture plate. Wells were illuminated with an 808 nm continuous-wave NIR laser illumination (fluence: 2 W/cm2 and exposure duration: 600 s).


### 2.5. Cell Cultures

The human ovarian cancer cells SKOV3 were cultured in Dulbecco’s modified Eagle medium with high glucose (DMEM, HG, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C with 5% CO2.


### 2.6. Cell Proliferation Assay

SKOV3 cells were seeded into 24-well plates in octuplicate at a starting density of5×103 cells/well. After overnight culture, Ag@Fe3O4 nanoparticles were added at different concentrations. Then, the MTT was performed after 24 h treated with Ag@Fe3O4 nanoparticles with or without NIR laser illumination (2 W/cm2, 600 s). The cells were incubated with 0.5 mg/mL MTT in DMEM for 1 h and then mixed with dimethyl sulfoxide after the supernatant was removed. The OD value at 570 nm was read using the microplate reader. Cell viability was determined by the percentage of OD value of the study group over the control group.


### 2.7. Statistical Analysis

Statistical analyses were used by the Studentt-test (two-tailed) using GraphPad Prism software. Differences with P<0.05 were considered statistically significant. Results were expressed as mean±SEM.


## 2.1. Materials

Ferric nitrate nonahydrate (Fe(NO3)3·9H2O), sodium acetate (NaOAc), and ethylene glycol (EG) were obtained from Sinopharm Chemical Reagent Co. Ltd. (China). Silver nitrate (AgNO3) was purchased from Aladdin (China).


## 2.2. Synthesis of Ag@Fe3O4 Nanoparticles

Ag@Fe3O4 nanoparticles were synthesized through the one-pot method reported by Fang et al. [21]. Briefly, Fe(NO3)3·9H2O (404 mg, 1.0 mmol) and NaOAc (328 mg, 4.0 mmol) were dissolved in 16 mL of EG. Then, 42.5 mg of AgNO3 (0.25 mmol) was added to the solution. The obtained mixture solution was sealed in Teflon-lined stainless-steel autoclaves, heated from 30°C to 200°C for 0.5 h, and maintained at 200°C for 8 h. The resulting Ag@Fe3O4 nanoparticles were collected by a magnet and washed with ethanol and water.


## 2.3. Photothermal Effect Measurement of the Ag@Fe3O4 Nanoparticles

To measure the photothermal effect of the Ag@Fe3O4 nanoparticles under the irradiation from an 808 nm laser, 1.0 mL of aqueous solution containing various concentrations of Ag@Fe3O4 nanoparticles (0 ppm, 10.0 ppm, 20.0 ppm, and 40.0 ppm) was irradiated for 12 min by a near-infrared laser (808 nm, 2.0 W/cm2). The temperature of the solution was recorded by an online-type thermocouple thermometer every 15 s.


## 2.4. NIR Heating of Ag@Fe3O4 Nanoparticles in Solution

Ag@Fe3O4 nanoparticles with different concentrations were prepared, and 500 μL of aliquots was deposited into the wells of a 24-well cell culture plate. Wells were illuminated with an 808 nm continuous-wave NIR laser illumination (fluence: 2 W/cm2 and exposure duration: 600 s).


## 2.5. Cell Cultures

The human ovarian cancer cells SKOV3 were cultured in Dulbecco’s modified Eagle medium with high glucose (DMEM, HG, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C with 5% CO2.


## 2.6. Cell Proliferation Assay

SKOV3 cells were seeded into 24-well plates in octuplicate at a starting density of5×103 cells/well. After overnight culture, Ag@Fe3O4 nanoparticles were added at different concentrations. Then, the MTT was performed after 24 h treated with Ag@Fe3O4 nanoparticles with or without NIR laser illumination (2 W/cm2, 600 s). The cells were incubated with 0.5 mg/mL MTT in DMEM for 1 h and then mixed with dimethyl sulfoxide after the supernatant was removed. The OD value at 570 nm was read using the microplate reader. Cell viability was determined by the percentage of OD value of the study group over the control group.


## 2.7. Statistical Analysis

Statistical analyses were used by the Studentt-test (two-tailed) using GraphPad Prism software. Differences with P<0.05 were considered statistically significant. Results were expressed as mean±SEM.


## 3. Results and Discussion

The synthesized Ag@Fe3O4 nanoparticles were investigated first by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The TEM images in Figures 1(a) and 1(b) confirm that the Ag@Fe3O4 nanoparticles have a well-defined core-shell structure with a silver core diameter of ~90 nm and a Fe3O4 shell thickness of ~80 nm. The SEM image in Figure 1(c) shows that the Ag@Fe3O4 nanoparticles are spherical in shape with an average diameter about 250 nm. A closer look (Figure 1(d)) shows that the Fe3O4 shell is very rough and is composed of many tiny magnetite nanocrystals. The dynamic light scattering (DLS) measurement (Figure S1) also shows that the Ag@Fe3O4 nanoparticles have a unimodal size distribution with an average size of ~225.8 nm. These results agree well with the previous reports [15, 22].Figure 1
TEM images (a, b) and SEM images (c, d) of Ag@Fe3O4 nanoparticles.
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(b)
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(d)The crystal structure and composition of the Ag@Fe3O4 nanoparticles were identified by X-ray diffraction (XRD). As illustrated in Figure 2(a), the diffraction peaks centered at 2θ angles of 38.18, 44.36, and 64.58 can be indexed from the (111), (200), and (220) faces of face-centered cubic silver, respectively. The other diffraction peaks centered at 30.14, 35.5, 35.4, 43.22, 53.58, 56.86, and 62.64 came from the (220), (311), (400), (422), (511), and (440) faces of the inverse spinel iron oxide, respectively. The M-H curve for the as-synthesized products is reversible (Figure 2(b)), which suggests that the Ag@Fe3O4 nanoparticles exhibit superparamagnetic characteristics at room temperature. The saturation magnetization of the Ag@Fe3O4 nanoparticles is about 37.2 emu/g at 15,000 Oe. This low saturation magnetization could be attributed to having no magnetic silver core.Figure 2
(a) X-ray diffraction pattern of Ag@Fe3O4 nanoparticles; (b) the M-H curve of Ag@Fe3O4 nanoparticles.
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(b)Figure3(a) presents the UV-Vis-NIR spectrum of the Ag@Fe3O4 nanoparticles. There are two optical absorption maxima for the nanocomposites, one at 406 nm and the other at 829 nm. The strong SPR absorption in the near-infrared region motivated us to study their photothermal effects. To prove the potential of the Ag@Fe3O4 nanoparticles as the photothermal agents, the Ag@Fe3O4 nanoparticles’ solutions at different concentrations were exposed under an 808 nm NIR laser at a power density of 2.0 W/cm2 (Figure 3(b)). As shown in Figure 3(b), the temperature of the nanoparticles’ solutions was increased to 34.4°C, 42.3°C, and 53.7°C at the concentrations of 10 ppm, 20 ppm, and 40 ppm, respectively. However, insignificant temperature increase was observed in the PBS buffer in the absence of nanoparticles. These data indicate that the Ag@Fe3O4 nanoparticles can effectively convert NIR light into heat. In addition, no morphological changes of the Ag@Fe3O4 nanoparticles are observed from the TEM images (Figure S2) after irradiation by the 808 nm laser (15 min, 5.0 W/cm2), suggesting that this core-shell nanoparticle possesses excellent photothermal stability.Figure 3
(a) UV-Vis-NIR spectra of Ag@Fe3O4 nanoparticles in PBS solution; (b) photothermal effect of Ag@Fe3O4 nanoparticles at various concentrations with irradiation of an 808 nm laser.
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(b)In order to evaluate the roles of the Ag@Fe3O4 nanoparticles on the growth of ovarian cancer cell lines, we performed the MTT assays in ovarian cancer cell line SKOV3 cells. Our results show that only the Ag@Fe3O4 nanoparticles have no significant effect on cell growth, even at concentrations up to 80 ppm. The results showed that the Ag@Fe3O4 nanoparticles were almost nontoxic to SKOV3 cells within a certain concentration range. Furthermore, the photothermal therapy on SKOV3 cells was studied with various concentrations of Ag@Fe3O4 nanoparticles. Our data clearly revealed that Ag@Fe3O4 nanoparticles exhibited better anticancer properties under NIR laser; moreover, the inhibition efficiency of Ag@Fe3O4 nanoparticles was enhanced with the increase of concentration after being treated with the NIR laser.In order to determine the effect of the Ag@Fe3O4 nanoparticles on ovarian cancer cell growth, we cultured SKOV3 cells for 24 h in medium containing various concentrations of Ag@Fe3O4 nanoparticles (0–80 ppm) and examined the cell toxicity of Ag@Fe3O4 nanoparticles. The Ag@Fe3O4 nanoparticles without NIR laser illumination had no significant effect on cell viability up to 80 ppm (Figure 4). In order to further examine the photothermal destruction of cancer cells, SKOV3 cells were treated with NIR laser illumination under different concentrations of the Ag@Fe3O4 nanoparticles. Our MTT assay showed that the Ag@Fe3O4 nanoparticles stimulated apoptosis and morphological changes of SKOV3 cells in a dose-dependent manner (Figures 4 and 5). Ag@Fe3O4 nanoparticles significantly inhibited the proliferation of SKOV3 cells under the higher concentrations accompanied with NIR laser illumination treatment (Figure 4). However, Ag@Fe3O4 nanoparticles have almost no effect on the growth of SKOV3 cells without NIR laser illumination treatment, indicating that it mainly depends on heating effect rather than on free radical effect.Figure 4
MTT assay of viability effect of Ag@Fe3O4 nanoparticles on SKOV3 cells. SKOV3 cells were incubated in the absence or in the presence of various concentrations of the Ag@Fe3O4 nanoparticles for 24 h, then treatment of NIR irradiation (IR) for 10 min. ∗∗P<0.001 and ∗∗∗P<0.001 versus the control experiments (without the Ag@Fe3O4 nanoparticle treatment and laser irradiation).Figure 5
The morphological changes of SKOV3 cells treated with various concentrations of the Ag@Fe3O4 nanoparticles and laser treatment of NIR laser illumination (2 W/cm2) for 10 min. Original magnification, ×200. Scale bar, 50 μm.Previous studies have showed that cancer cells incubated under the temperature at 40-60°C can be killed effectively within several minutes [23, 24]. Our result demonstrated that the photothermal effect of Ag@Fe3O4 nanoparticles can be a therapeutic route to eliminate cancer cells through NIR laser irradiation.


## 4. Conclusion

In conclusion, we firstly demonstrate that the Ag@Fe3O4 nanoparticles can be used as an efficient photothermal agent for killing cancer cells. The Ag@Fe3O4 nanoparticles are easily synthesized by a one-pot hydrothermal method and possess a strong SPR absorption property, high photothermal conversion efficacy, and photothermal stability. These features could make them promising for biomedical applications.



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*Source: 6457968-2019-01-28.xml*