Investigation of anti-human ovarian cancer effects of decorated Au nanoparticles on Thymbra spicata extract modified Fe₃O₄ nanoparticles

2.1 Materials and apparatus

All the reagents were purchased from Aldrich and Merck and were used without any purification. In instrumental section, the particle size, morphology and atomic mapping analysis of the nanomaterial was investigated using a FESEM-TESCAN MIRA3 microscope equipped with EDX. The crystalline structures of the samples were evaluated by X-ray diffraction (XRD) analysis on a Bruker D8 Advance diffractometer with CuKα radiation at 40 kV and 20 mA. Fourier transform infrared (FT-IR) spectra were recorded with a Perkin Elmer 65 spectrometer in the range of 400–4000 cm⁻¹. Transmission electron microscopy (TEM) analysis was performed on a Phillips CM10 microscope at an accelerating voltage of 200 kV. Magnetization measurements were carried out on a BHV-55 vibrating sample magnetometer (VSM).

2.2 Preparation of Thymbra spicata leaf extract

One hundred grams of Thymbra spicata dried leaves were ground and heated at 70 °C with 500 mL of sterile distilled water for 2 h. Afterward, the mixture was cooled down to room temperature. The aqueous extract of Thymbra spicata leaves was centrifuged at 6500 rpm. Moreover, the supernatant was separated by filtration and was kept in a refrigerator at 4^οC for later investigations.

2.3 Preparation of the biologically active magnetic Fe₃O₄@Thymbra spicata/Au nanocomposite

The magnetic Fe₃O₄ nanoparticles were prepared by the chemical co-precipitation method according to the literature [18b]. In the first step, magnetite nanoparticle (0.5 g) was dispersed in 100 mL water and sonicated for 20 min at 60 °C. Next, the Thymbra spicata extract was added to the mixture. Afterward, the solution was sonicated again at this temperature for 60 min, and subsequently, a solution of HAuCl₄ in water was added dropwise using a dropping funnel in the reaction media (0.02 g, 10 mL, the drip rate was 1 mL/min) under sonication. After complete addition, the reaction continued for another 60 min under ultrasonic conditions. Then, the mixture was cooled to room temperature and the magnetic nanoparticles Fe₃O₄@Thymbra spicata/Au as a black reddish solid were isolated from the solution by magnetic separation and washed several times by DI water and ethanol. The final nanocomposite was dried in a vacuum at 40 °C. The concentration of gold was 0.12 mmol/g, which was determined by ICP-OES.

2.4 Determination of anti-human ovarian cancer properties of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite

In this assay, following human ovarian cell lines and the normal cell line (HUVEC) were used to study the cytotoxicity and anticancer potential of human ovarian over the Fe₃O₄@Thymbra spicata/Au NPs nanocomposite using the common cytotoxicity test i.e., MTT assay in vitro condition:

1. Normal cell line: HUVEC.

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

The cells were cultured as a monolayer culture in 90% RPMI-1640 medium and 10% fetal serum and were immediately after supplemented with 200 mg/mL streptomycin, 125 mg/mL penicillin, and 8 mg/mL amphotericin B. The culture was then exposed to 0.5 atmospheric carbon dioxide at 37 °C, on which the tests were performed after at least ten successful passages. MTT was an assay used to investigate the toxic effects of various materials on various cell lines, including non-cancer and cancer cells. To evaluate the cell toxicity effects of the compounds used in this research, the cells were transferred from the T25 flask to the 96-well flasks. In each cell of the 96-cell flasks, 7000 cells of cancer and fibroblast cell lines were cultured, and the volume of each cell was eventually increased to 100 µL. Before treating the cells in the 96-well flak, the density of cells was increased to 70%, so the 96-well flasks were incubated for 24 h to obtain the cell density of 7 × 10³. Thereafter, the initial culture medium was discarded, and variable concentrations (0–1000 µg/mL) of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite were incubated at 37 °C and 5% CO₂ for 24, 48, and 72 h. Then, 20 µL MTT was added to each well. This step was followed by the addition of 100 µL of DMSO solvent to each well. The treated wells were then kept at room temperature for 25 min and read at 490 and 630 nm by a microtitre plate reader. The cell lines were also treated with the hydroalcoholic extract (1.25 mg/mL), which inhibited about 20% of the cell growth. Annexin/PI method was used to determine the apoptosis level in the treated and control cell lines using a flow cytometry machine. Under the optimized experimental conditions, the cell lines were treated with variable concentrations (0–1000 µg/mL) of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite for 24 h. Cells were irrigated with phosphate-buffered saline (PBS). After centrifugation, buffer binding was added to the obtained precipitate. Then, 5 µL Annexin V dye was added and incubated for 15 min at 25 °C. Cells were washed with the binding solution, following by the addition of 10 µL of PI dye. Finally, cell analysis was conducted by a flow cytometry machine according to the below formula (Abdoli et al., 2020; Mahdavi et al., 2020): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{Control}A.}\times 100$$

2.5 Determination of the antioxidant activities of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite

To study the radical scavenging antioxidant property of our Fe₃O₄@Thymbra spicata/Au NPs, DPPH (2,2-diphenyl-1-picrylhydrazyl) is being used (Ghashghaii et al., 2017; Abdoli et al., 2020; Mahdavi et al., 2020).

A 39.4% DPPH solution (w/V) was prepared in 1:1 aqueous MeOH. Simultaneously, different samples of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite of variable concentrations (0–1000 µg/mL) were prepared. The DPPH solution was then added to Fe₃O₄@Thymbra spicata/Au NPs nanocomposite samples and incubated at 37 °C. After 30 min of incubation, the absorbances of the mixtures were measured at 570 nm. MeOH (50%) and butylated hydroxytoluene (BHT) were considered as negative and positive controls respectively in the study. The antioxidant properties of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite were determined in terms of % inhibition and expressed as $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

Calculation of half-maximal inhibitory concentration (IC₅₀) is a suitable method for comprising the activity of pharmaceutical-based materials. In this method, the measurement and comparison criterion is the concentration in which 50% of the final activity of the drug occurs. In this experiment, the IC₅₀ of various repeats is estimated and compared with the IC₅₀ of BHT, which is introduced as the antioxidant activity index. The closer is the obtained value to the IC₅₀ of BHT, the stronger is the antioxidant activity of the material. The graph of the IC₅₀ of the extract was produced by drawing the percent inhibition curve versus the extract concentration. First, three stock samples with variable concentrations (0–1000 µg/mL) of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite were prepared. Then, a serial dilution was prepared from each sample, and IC₅₀ of the above samples was measured separately, and finally their mean was calculated. BHT, with different concentrations, was considered positive control. All experiments were performed in triplicate (Ghashghaii et al., 2017; Abdoli et al., 2020; Mahdavi et al., 2020).

2.6 Qualitative measurement

The obtained results were loaded into the “SPSS-22” program and evaluated by “one-way ANOVA”, accompanied by a “Duncan post-hoc” check (p ≤ 0.01).

3.1 Chemical characterization of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite

To rationalize the stepwise synthesis of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite, FT-IR spectra of all the corresponding intermediates have been depicted in Fig. 1. Bare unmodified Fe₃O₄ NPs show a typical absorption at 588 and 638 cm⁻¹ due to Fe-O stretching (Fig. 1a). The characteristic absorption bands of Thymbra spicata extract are represented in Fig. 1b as, 3370 (OH), 2924 (C-H), 1619 (CO), 1400 to 2600 (C⚌C), 1286 (C—O), 1071 (C—O—C) and 865 cm⁻¹ (O—H) and represent polyols, carbonyls and the C⚌C bonds of flavonoids and terpenoids (Baghayeri et al., 2018). Fig. 1c, representing the FT-IR spectrum of Fe₃O₄@Thymbra spicata/Au NPs is an amalgamation of both individual component peaks with the slight shifting of characteristic peaks due to strong complexation of Au atoms with the surface functions like OH, CHOH, CH₂OH, C⚌O etc, which is a clear sign of successful functionalization of the biomolecules and Au NPs over Fe₃O₄ NPs. Interestingly, the strong peak due to Fe—O stretching has also been moved to 572 cm⁻¹ which means that the Au chelation affects the center core (Baghayeri et al., 2018).

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Fig. 1

FT-IR spectra of a) Fe₃O₄, b) Thymbra spicata extract and c) Fe₃O₄@Thymbra spicata/Au NPs.

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The detailed morphological structure, shape and size of the Fe₃O₄@Thymbra spicata/Au NPs nanocomposite were ascertained by SEM and TEM studies. The globular shape nanoparticles can be observed from the SEM images having a mean diameter of a nanometer (Fig. 2). The surface modification over Fe₃O₄ NPs by extract biomolecules can be adjudged from its appearances. There occurs a homogeneous growth of extract over it. The agglomeration of nanocomposite particles can be understood due to manual sample preparation. The bright spots in the image correspond to the Au nanoparticles are easily detectable.

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Fig. 2

FESEM image of Fe₃O₄@Thymbra spicata/Au NPs.

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To have the information about the elemental composition of the nanocomposite EDX analysis was carried out. The corresponding profile exhibits Fe, O, C, N and Au atoms (Fig. 3). It demonstrates the successful fabrication of the desired nanocomposite. Carbon and nitrogen atoms originated from the extract that coated on the surface of magnetic nanoparticles.

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Fig. 3

EDX pattern of Fe₃O₄@Thymbra spicata/Au NPs.

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The inherent structural features are demonstrated via TEM study of the nanocomposite. The thin layer of the extract surrounding the black-colored Fe₃O₄ NPs can be visible (Fig. 4). The size of the composite particles was by the SEM study and is around 10–15 nm. The tiny black dots correspond to the Au NPs, being ∼30 nm in size. In addition, the presence of the extract layer is found effective on in situ reductions and immobilization of Au onto the surface of the Fe₃O₄/extract composite. The biomolecular conjugate favors the binding of Au NPs by electrostastic force of attraction and thereby gets stabilized. These data demonstrate the proposed architecture of the material. Moreover, Fig. 4 determines the particle size distribution histograms for Au NPs and Fe3O₄ NPs whose average size are approximately 32.54 and 9.21 nm respectively.

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Fig. 4

TEM image of Fe₃O₄@Thymbra spicata/Au NPs and particle size distribution histograms for Au NPs and Fe3O₄ NPs.

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The crystalline phase of Fe₃O₄@Thymbra spicata/Au NPs nanocomposite was ascertained by XRD and the comparative study is presented in Fig. 5. It demonstrates the typical cubic spinel phase of Fe₃O₄ NPs, the characteristic diffraction peaks are attributed to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) crystal planes (JCPDS No. 19–0629). This also documents that the functionalization over Fe₃O₄ could not change its crystal structure. The additional peaks due to crystalline Au NPs. The peaks appeared at 2Θ = 38.6°, 44.4°, 64.4° and 77.3° corresponds to the face-centered cubic (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes Au crystalline phases (Hamelian et al., 2018). However, the peak intensities are relatively low due to extracting coating over the Fe₃O₄ NPs core.

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Fig. 5

XRD pattern of Fe₃O₄@Thymbra spicata/Au NPs.

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Magnetic properties of unmodified Fe₃O₄ NPs and Fe₃O₄@Thymbra spicata/Au NPs nanocomposites were determined by VSM analysis at room temperature (Fig. 6). The patterns indicate that they are superparamagnetic nanostructures. The materials exhibit the saturation magnetization (M_s) values of 61.5 and 23.9 emu/g respectively. The gradual decrease in magnetization values is obviously due to the incorporation of extract composites and diamagnetic Au respectively. Still, the final material behaves like a superparamagnet and can be easily attracted by external magnets.

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Fig. 6

VSM analysis for a) Fe₃O₄ NPs and b) Fe₃O₄@Thymbra spicata/Au NPs.

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3.2 Cytotoxicity and anti-ovarian cancer potentials of Fe₃O₄@Thymbra spicata/Au nanocomposite

In the recent research, the treated cells with several concentrations of the present Fe₃O₄@ Thymbra spicata/Au nanocomposite were examined by MTT test for 48 h regarding the cytotoxicity properties on normal (HUVEC) and common ovarian cancer cell lines i.e., SK-OV-3, SW-626, and PA-1.

The absorbance rate was determined at 570 nm, which indicated extraordinary viability on normal cell line (HUVEC) even up to 1000 μg/mL for Fe₃O₄@Thymbra spicata/Au nanocomposite (Fig. 7).

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Fig. 7

The anti-human ovarian cancer properties (Cell viability (%)) of Fe₃O₄@Thymbra spicata/AuNPs (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. The numbers indicate the percent of cell viability at the concentrations of 0–1000 μg/mL of Fe₃O₄@Thymbra spicata/AuNPs against several human ovarian cancer cell lines.

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In the case of ovarian cancer cell lines, the viability of them reduced dose-dependently in the presence of Fe₃O₄@Thymbra spicata/Au nanocomposite. The IC50 of Fe₃O₄@Thymbra spicata/Au nanocomposite were 185, 251, and 407 µg/mL against PA-1, SW-626, and SK-OV-3 cell lines, respectively (Table 1).

Among the different parameters of metallic nanoparticles such as, size, texture and nature of surface functions, the size effect is most essential in the anticancer assay using standard cancer cell lines. Previous reports revealed that the anticancer activity increases with a decrease in particle size based on their better penetration ability over the cell lines. It has been surveyed that particle size lower than 50 nm displays better activity in the corresponding cancer cell lines (Abdoli et al., 2020; Mahdavi et al., 2020).

As shown in Figs. 3 and 4 of our study, the average size of Fe₃O₄@Thymbra spicata/Au nanocomposite is lower than 50 nm.

In the anticancer effects of Au nanoparticles, they have been used to treat several cancers including human lung cancer, mammary carcinoma, uterus cancer, lung epithelial cancer, Lewis lung carcinoma, colon cancer, and human glioma (Abdoli et al., 20202; Mahdavi et al., 2020).

3.3 Antioxidant capacities of Fe₃O₄@Thymbra spicata/AuNPs nanocomposite

Now, turning our attention to investigate the bioactivity of Fe₃O₄@Thymbra spicata/AuNPs nanocomposite a concentration-dependent DPPH radical scavenging effect of nanomaterial was observed against BHT as a reference. DPPH process is widely applied to determine the free radical scavenging effect of different antioxidant materials. The DPPH scavenging abilities are known to be because of the hydrogen donating activities of antioxidant materials. When DPPH results are examined, it is observed that it has increased in a dose-dependent manner (Ghashghaii et al., 2017; Abdoli et al., 2020; Mahdavi et al., 2020).

The interaction between Fe₃O₄@Thymbra spicata/AuNPs nanocomposite and DPPH might have occurred by transferring electrons and hydrogen ions (Abdoli et al., 2020; Mahdavi et al., 2020). The scavenging capacity of the Fe₃O₄@Thymbra spicata/AuNPs nanocomposite and BHT at different concentrations, expressed in terms of percentage inhibition, has been shown in Fig. 8.

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Fig. 8

The antioxidant properties of Fe₃O₄@Thymbra spicata/AuNPs (A) and BHT (B) against DPPH. The numbers indicate the percent of free radical (DPPH) inhibition at the concentrations of 0–1000 μg/mL of Fe₃O₄@Thymbra spicata/AuNPs (A) and BHT (B).

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In the antioxidant test, the IC50 of butylated hydroxytoluene and Fe₃O₄@Thymbra spicata/AuNPs nanocomposite were 199 and 107 µg/mL, respectively (Table 2).

Au nanoparticles green-mediated show higher antioxidant effects against free radicals formation into the living system (Ghashghaii et al., 2017; Abdoli et al., 2020; Mahdavi et al., 2020). The Au nanoparticles green-formulated have excellent redox properties and have a significant role in free radicals deactivating (Mahdavi et al., 2020). Previous researches have indicated that flavonoids and phenolic compounds attached to the metallic nanoparticles have significant antioxidant properties (Abdoli et al., 2020; Mahdavi et al., 2020).

Likely significant anti-human ovarian cancer potentials of Fe₃O₄@Thymbra spicata/AuNPs nanocomposite against common ovarian cancer cell lines i.e., SK-OV-3, SW-626, and PA-1 are linked to their antioxidant effects. Similar reports have indicated the antioxidant materials such as metallic nanoparticles especially Au nanoparticles reduce the volume of tumors by removing free radicals (Katata-Seru et al., 2018).

In detail, the high presence of free radicals in the normal cells make many mutations in their DNA and RNA, destroy their gene expression and then accelerate the proliferation and growth of abnormal cells or cancerous cells (Katata-Seru et al., 2018; Sangami and Manu, 2017).

The free radicals high presences in all cancers such as breast, gallbladder, stomach, rectal, liver, gastrointestinal stromal, esophageal, bile duct, small intestine, pancreatic, colon, parathyroid, thyroid, bladder, prostate, testicular, fallopian tube, vaginal, ovarian, hypopharyngeal, throat, lung, and skin cancers indicate the significant role of these molecules in making angiogenesis and tumorigenesis (Sangami and Manu, 2017; Beheshtkhoo et al., 2018).

Many researchers reported that Au nanoparticles green-synthesized have a remarkable role in removing free radicals and growth inhibition of all cancerous cells (Radini et al., 2018; Oganesvan et al., 1991).