Decorated of Au NPs over L-arginine-modified Fe₃O₄ nanoparticles as a novel nanomagnetic composite for the treatment of human ovarian cancer

2.1 Preparations of Au NPs/L-arginine/Fe₃O₄ nanocomposite

Fe₃O₄ NPs were prepared through typical co-precipitation method as published earlier (Abdoli et al., 2020).

For the preparation of L-arginine/Fe₃O₄ nanocomposite, initially 0.5 g of the Fe₃O₄ NPs were uniformly dispersed in 100 mL water by sonication for 30 min and then the L-arginine (0.1 g) was added into it. The mixture was stirred for 12 h under reflux conditions for the possible surface modifications of Fe₃O₄ NPs. The as synthesized L-arginine/Fe₃O₄ nanocomposite was retrieved magnetically and washed thoroughly with deionized water (Amaria et al., 2017).

In order to support the L-arginine/Fe₃O₄ composite with Au NPs, 0.5 gm of it was spread over 100 mL DI water and sonicated for 30 min. The precursor, a solution of HAuCl₄ (20 mg) in 20 mL H₂O was then added dropwise and stirred for 1 h to afford the coordinated gold ions on the surface of magnetic nanocomposite. Next, 5 mL (NaBH₄, 0.25 mmol) was added and stirred for 10 min. The resulted Au NPs/L-arginine/Fe₃O₄ nanocomposite was isolated by magnetic decantation, rinsed with DI-H₂O and treated in vacuum at 40 °C. Finally, ICP-AES analysis was performed to assess the Au content, as being 0.08 mmol/g.

2.2 Anti-human ovarian cancer properties of Au NPs/L-arginine/Fe₃O₄ nanocomposite

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) is a colorimetric technique. Based on the fact that living cells can do oxidative metabolism, as a result, oxidation, breaks down the MTT dye and produces a dye ranging from yellow to blue. This test determines the number of living cells (Arunachalam, 2003). In this research, we used the following Cell lines to evaluating anti-human ovarian cancer and cytotoxicity effects of Au NPs/L-arginine/Fe₃O₄ nanocomposite using an MTT method.

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  Normal cell line: Human umbilical vein endothelial cells (HUVECs).

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  Human ovarian cancer cell lines: SK-OV-3, SW-626, and PA-1.

These cell lines were cultured in appropriate numbers (10,000 cells/well) in a 96-well microplate. They were then incubated at 37 °C and 5% CO₂ for 24 h to form a cell monolayer. Then, out of the greenhouse and under the hood, different concentrations of Au NPs/L-arginine/Fe₃O₄ nanocomposite were added to the cells. Control wells also included cell control containing complete cell and culture medium and blank control containing no cell and complete culture medium, and the microplates were again placed in the oven at 37 °C and 5% CO₂ until the required time (24–48–72 h). After the test times, the cells were washed with PBS saline phosphate, MTT dye was added to the wells and the microplates were incubated for 3 h. Finally, the optical absorption of cells at 570 nm was read by the ELISA reader (model: Tek Bio Elx800). The cell viability percentage was calculated by the following formula (Arunachalam, 2003): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

2.3 Antioxidant activities of Au NPs/L-arginine/Fe₃O₄ nanocomposite

The free radical scavenging test was first performed by Blois in 1958, and after some modification by numerous studies in its current form. 2,2-diphenyl-1-picrylhydrazyl (DPPH) method is one of the most widely used methods for estimating antioxidant content. DPPH is a stable radical that reacts with hydrogen atom compounds. This test is based on the inhibition of DPPH, which causes the decolorization of DPPH solution by adding radical species or antioxidants. DPPH changes color from purple to yellow by taking an electron from the antioxidant compound. The free radicals in DPPH are adsorbed at 517 nm, which follows Beer Lambert's law, and decreased absorption is linearly related to the amount of antioxidants; the higher the amount of antioxidants, the more DPPH is consumed and the more purple turns yellow (Shaneza, 2018; Amaria et al., 2017). In the recent study, the degree of inhibition of DPPH radicals was evaluated by Shaneza et al. (Shaneza, 2018; Amaria et al., 2017). For this purpose, solutions with different samples of the Au NPs/L-arginine/Fe₃O₄ nanocomposite of variable concentrations (0–1000 µg/mL) as well as synthetic antioxidant butylated hydroxytoluene (BHT) in methanol solvent were prepared. The test method was that one mL of DPPH methanolic solution (at a concentration of 1 mM) was added to 4 mL of the extract and the resulting mixture was stirred vigorously. The test tubes were placed in a dark place for 60 min. After this period, the absorbance was read at 517 nm. Finally, the DPPH radicals’ inhibition percentage of the Au NPs/L-arginine/Fe₃O₄ nanocomposite was calculated by the below formula (Shaneza, 2018; Amaria et al., 2017): $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

IC50 factor was used to evaluate better the antioxidant activity, which indicates the concentration of the Au NPs/L-arginine/Fe₃O₄ nanocomposite that can reduce the concentration of free radical DPPH. The initial is 50% of the initial value, and the lower the amount, the greater the antioxidant activity (Shaneza, 2018; Amaria et al., 2017).

2.4 Qualitative measurement

After collecting data, Minitab statistical software was used for statistical analysis. Evaluation of antioxidant results in a completely randomized design and comparison of means was Duncan post-hoc test with a maximum error of 5%. To measure the percentage of cell survival in factorial experiments with the original design of completely randomized blocks and compare the means, Duncan post-hoc test with a maximum error of 5% was used. The 50% cytotoxicity (IC50) and 50% free radical scavenging (IC50)) was estimated with ED50 plus software (INER, V: 1.0). Measurements were reported as mean ± standard deviation.

3.1 Chemical characterization of Au NPs/L-arginine/Fe₃O₄ nanocomposite

A post-synthetic modification pathway was followed to prepare Au NPs/L-arginine/Fe₃O₄ nanocomposite (Scheme 1). The L-arginine acted as good coordinating agent for the anchored Au nanoparticles as well as stabilizer for the immobilized Au NPs. The material was physicochemically characterized through field emission scanning electron microscopy (FESEM), energy dispersive X-ray analysis (EDX), transmission electron microscopes (TEM), vibrating-sample magnetometer (VSM), X-ray diffraction (XRD) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis.

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Scheme 1 Preparation of Au NPs/L-arginine/Fe₃O₄ nanocomposite.

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FT-IR spectra of the Au NPs/L-arginine/Fe₃O₄ nanocomposite were compared with the spectra of its precursor components such as Fe₃O₄ and L-arginine/Fe₃O₄ to justify the stepwise architecture (Fig. 1). Fig. 1a displays the representative peaks of bare Fe₃O₄ NPs, where the sharp bands at 580–640 cm⁻¹ correspond to Fe–O–Fe stretching vibrations. The broadband in the 3400 cm⁻¹ regions is attributed to the surface hydroxyl groups over ferrite NPs. FT-IR spectra of L-arginine/Fe₃O₄ (Fig. 1b) represents all the individual peaks from Fe₃O₄ and L-arginine indicating successful assembling of them. Only, the corresponding peaks are seen to be slightly moved to higher or lower regions. An additional peak is observed at 1635 cm⁻¹, attributed to the imine functionality. Finally, in the analysis of Au NPs/L-arginine/Fe₃O₄ nanocomposite we observed almost the same fashion as Fig. 1b except for very slight peak shifts of the imine, alcoholic C–O, C–N stretching vibrations to 1623, 1430, 1045 cm⁻¹ due to strong complexation of the corresponding functions to the Au NPs. The Fe-O bond vibration was also found moved to 566 cm⁻¹.

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Fig. 1 FT-IR spectra of a) Fe₃O₄, b) L-arginine/Fe₃O₄ and c) Au NPs/L-arginine/Fe₃O₄ nanocomposite.

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FESEM and TEM analysis were used to ascertain the comprehensive morphology, size and shape of the Au NPs/L-arginine/Fe₃O₄ nanocomposite (Figs. 2 and 3). The thin continuous layer over the core surface signifies the L-arginine functionalization. It is evident from Fig. 2 that the ligand-coating uniformly covers the surface ferrite particles. OH and NH groups around the surface of Fe₃O₄ NPs facilitate the covalent linkage for gold nanoparticles. The black dots correspond to the Au NPs formed in the mid-region. Moreover, Fig. 2 determines the particle size distribution histograms for Fe₃O₄ NPs and Au NPs whose average size are approximately 33 and 13 nm respectively. Fig. 3 represents the FESEM image of the nanocomposite. The particle is texturally globular with some sign of aggregation in quite a few sections. This is attributed to manual sampling. The particles are almost homo-morphic and are sized between 30 and 40 nm. Fig. 3, also displays the SEM image and the corresponding elemental maps of the hybrid nanomaterial. Elemental analysis on the selected area of the nanocomposite demonstrates that Fe, O, C, N and Au atoms are homogeneously distributed over the surface.

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Fig. 2 TEM images of (a) Fe₃O₄ and (b) Au NPs/L-arginine/Fe₃O₄ nanocomposite particle size distribution histograms for (c) Fe₃O₄ NPs and (d) Au NPs.

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Fig. 3 FE-SEM image of Au NPs/L-arginine/Fe₃O₄ nanocomposite and its elemental mapping.

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EDX analysis of the Au NPs/L-arginine/Fe₃O₄ nanocomposite was executed to have a sheer knowledge of constituent elements and it displays the occurrence of C, O, Fe and Au elements (Fig. 4).

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Fig. 4 EDX spectrum of the Au NPs/L-arginine/Fe₃O₄ nanocomposite.

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The saturation magnetization (Ms) value obtained from magnetic hysteresis loops of Fe₃O₄ and Au NPs/L-arginine/Fe₃O₄ nanocomposite was 43.1 and 17.7 emu/g respectively (Fig. 5).

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Fig. 5 VSM analysis of (a) Fe₃O₄ and (b) Au NPs/L-arginine/Fe₃O₄ nanocomposite.

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Fig. 6 exhibits the crystalline phases of Fe₃O₄ and Au NPs/L-arginine/Fe₃O₄ being determined over XRD. The said profile demonstrates the typical peaks of cubic spinel Fe₃O₄ NPs. The characteristic peaks observed at 2θ = 30.2°, 35.8°, 43.2°, 54. 1°, 57.2° and 63.2° closely resembles to ferrite NPs that are corroborated to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) diffraction planes (JCPDS No. 19–0629). This suggest that the inner core structure remained undisturbed even after post-synthetic modification. The extra peaks appeared at 2θ = 38.8°, 44.3°, 64.5° and 77.6° can be allocated to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of Au fcc crystalline phases.

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Fig. 6 X-ray diffraction study of (a) Fe₃O₄ and (b) Au NPs/L-arginine/Fe₃O₄ nanocomposite.

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3.2 Antioxidant properties of Au NPs/L-arginine/Fe₃O₄ nanocomposite

In this study, we assessed the antioxidant properties of Au NPs/L-arginine/Fe₃O₄ nanocomposite by using the DPPH test as a common free radical. Free radicals are atoms, molecules, or ions with unpaired electrons and are therefore very active, unstable, and highly reactive. Free radicals are formed by breaking a bond of a stable molecule. Free radicals collide with other molecules to achieve stability and can separate electrons from them, as a result, they form a chain of more unstable molecules. A free radical can have a positive, negative or neutral charge (Shaneza, 2018; Amaria et al., 2017). During the body's natural metabolism or under conditions such as smoking, pollution, the entry of unnecessary chemicals into the body in any way, radiation and stress in the body produce free radicals. The most important free radical in the human body is oxygen, which can damage DNA and other molecules. Oxidative stress is the victory of free radicals over the body's antioxidant defense and is a biological attack on the body (Abdoli et al., 2020; Mahdavi et al., 2020; Mahdavi et al., 2019). Antioxidants are molecules that can donate an electron to a free radical without destabilizing themselves. This stabilizes the free radical and makes it less reactive. The result of oxidative stress in the body is various degeneration, eye damage, premature aging, muscle problems, brain damage, heart failure, diabetes, cancer, and overall weakness of the immune system (Li et al., 2020; Shu and Tang, 2020; Yu et al., 2021; Lv et al., 2020; Zhang et al., 2020; Zeng et al., 2020). Oxygen radicals are continuously produced in all living organisms and with destructive effects, lead to cell damage and death. The production of oxidant species under physiological conditions has a controlled rate, but this production increases under oxidative conditions (Shaneza, 2018; Amaria et al., 2017). Various studies have shown that antioxidant compounds have very significant anti-cancer effects with omitting the free radicals.

The scavenging capacity of Au NPs/L-arginine/Fe₃O₄ nanocomposite and BHT at different concentrations expressed as percentage inhibition has been indicated in Fig. 7 and Table 1. In the antioxidant test, the IC50 of Au NPs/L-arginine/Fe₃O₄ nanocomposite and BHT against DPPH free radicals were 180 and 125 µg/mL, respectively (Fig. 7 and Table 1).

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Fig. 7 The antioxidant properties of Au NPs/L-arginine/Fe₃O₄ nanocomposite (A) and BHT (B) against DPPH.

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The numbers indicate the percent of free radical (DPPH) inhibition at the concentrations of 0–1000 μg/mL of Au NPs/L-arginine/Fe₃O₄ nanocomposite (A) and BHT (B).

3.3 Cytotoxicity and anti-human ovarian cancer activities of Au NPs/L-arginine/Fe₃O₄ nanocomposite

One of the cytotoxicity test methods to measure the rate of cell death is the MTT method, which is based on the formation of formazan dye by reducing the substance MTT (dimethyl thiazole 2 and 5 diphenyltetrazolium bromide) or other tetrazolium salts (Tahvilian et al., 2019; You et al., 2012; Mao, 2016). By breaking the MTT tetrazolium ring by mitochondrial enzymes in living cells, insoluble purple formazan crystals are formed. The formation of these crystals indicates the activity of respiratory chain enzymes and is a measure of cell viability. By measuring the amount of absorption by spectrophotometer at specific wavelengths, the number of living cells can be determined. This test is performed according to ISO 10993-5 and its purpose is in vitro evaluation of cytotoxicity. Cytotoxicity test is performed according to ISO10993-5 standard and in three ways: NRU test, CFU test, MTT test and XTT test. The most common method for assessing cytotoxicity is to measure cell survival by MTT (You et al., 2012; Mao, 2016; Namvar et al., 2014).

The basis of MTT method is based on the intensity of dye produced by the mitochondrial activity of cells, that measured at a wavelength of 540 to 630 nm and directly proportional to the number of living cells, the increase or decrease in the number of living cells is linearly related to the activity of cell mitochondria. MTT tetrazolium dye is revived in active (metabolically) cells. Mitochondrial dehydrogenases in living cells produce NADH and NADPH, leading to an insoluble purple precipitate called formazan. This precipitate can be dissolved by isopropanol or dimethyl sulfoxide (You et al., 2012; Mao, 2016; Namvar et al., 2014; Sankar et al., 2014). Dead cells, on the other hand, are unable to perform this conversion due to the inactivity of their mitochondria and therefore do not show a signal. In this method, dye formation is used as a marker for the presence of living cells (Sankar et al., 2014; Katata-Seru et al., 2018). In recent years, MTT testing has been the most important measurement method to evaluate the toxicity and anti-cancer effects of metal nanoparticles (Arunachalam, 2003; Sankar et al., 2014; Katata-Seru et al., 2018).

In this investigation, the treated cells with different concentrations of the present Au NPs/L-arginine/Fe₃O₄ nanocomposite were assessed by MTT assay for 48 h about the cytotoxicity properties on normal (HUVEC) and ovarian malignancy cell lines i.e. SK-OV-3, SW-626, and PA-1.

The viability of malignant ovarian cell line reduced dose-dependently in the presence of Au NPs/L-arginine/Fe₃O₄ nanocomposite. The IC50 of Au NPs/L-arginine/Fe₃O₄ nanocomposite were 149, 162, and 213 µg/mL against SK-OV-3, SW-626, and PA-1 cell lines, respectively (Fig. 8 and Table 2).

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Fig. 8 The anti-human ovarian cancer properties (Cell viability (%)) of Au NPs/L-arginine/Fe₃O₄ nanocomposite (Concentrations of 0–1000 µg/mL) against normal (HUVEC: A) and human ovarian cancer (PA-1 (B), SW-626 (C), and SK-OV-3 (D)) cell lines.

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The absorbance rate was evaluated at 570 nm, which represented viability on normal cell line (HUVEC) even up to 1000 μg/mL for Au NPs/L-arginine/Fe₃O₄ nanocomposite (Fig. 8 and Table 2).

It seems that the anti-human ovarian cancer effect of recent nanoparticles is due to their antioxidant effects. Because tumor progression is so closely linked to inflammation and oxidative stress, a compound with anti-inflammatory or antioxidant properties can be an anticarcinogenic agent (Sangami and Manu, 2017; Radini et al., 2018).

Many nanoparticles have pharmacological and biochemical properties, including antioxidant and anti-inflammatory properties, which appear to be involved in anticarcinogenic and antimutagenic activities (Shaneza, 2018; Amaria et al., 2017; Arunachalam, 2003). Today, nanoparticles synthesized by biological methods play a vital role in treating many diseases, including cancer (Beheshtkhoo et al., 2018; Radini et al., 2018). Nanoparticles synthesized by biological methods are no longer the only ones in traditional medicine, in addition, they have been able to adopt an industrial line of natural products for treating various cancers. Various cell lines from cancers of the prostate, ovary, lung, liver, and pancreas have been treated with herbal nanoparticles synthesized (Sangami and Manu, 2017; Beheshtkhoo et al., 2018; Radini et al., 2018).

The numbers indicate the percent of cell viability at the concentrations of 0–1000 μg/mL of Au NPs/L-arginine/Fe₃O₄ nanocomposite against several human ovarian cancer cell lines.