Efficiency of the green formulated iron nanoparticles as a new tool in lung cancer therapy

2.1 Synthesis of the nanoparticles

The biosynthesis of FeNPs was carried out according to the previous studies with some modification (Radini et al., 2018). A 10 mL of aqueous extract solution (20 mg/mL) was added to 30 mL of FeCl₃·6H₂O in the concentration of 0.02 M (deionized water was used for the all steps of this section). The mixture was refluxed for 90 min at 50 °C. The color-changing from yellow to black indicated the formation of iron nanoparticles. The precipitate was triplet washed with water and centrifuged at 12000 rpm for 15 min subsequently. The obtained black powder was kept in a vial for the chemical characterization and evaluation of its biological activity.

2.2 Antioxidant activities of nanoparticles

In this method, 1 mL of different concentrations of the nanoparticles (0–1000 µg/ml) (with 1 mL of DPPH (300 µmol/l) combined and then the final volume of the combination with methanol reached 4000 µl. The falcons were then vertexed and kept in the dark for 60 min. The absorbance was read at 517 nm. The DPPH radical inhibition percentage was calculated using the following equation (Hummers and Offeman, 1958; Kooti et al., 2017; Thema et al., 2015; Shaneza, 2018): $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}\times 100$$

2.3 Anticancer properties of nanoparticles

Cell culture is the process of culturing eukaryotes in a culture medium where the culture conditions are different for each cell type and these conditions must be provided for its better growth; But in general, artificial environments should contain nutrients such as vitamins, minerals, sugars, amino acids, hormones and gases. In this environment, physical and chemical conditions must also be adjusted for the cells to grow better. Due to the differences in cell types, some of these cells need a surface to attach to grow, and others can grow floating in the culture medium. In the laboratory, there is a specific cell culture medium for specific cells. The culture medium suitable for hepatocytes is different from the culture medium for neurons. For the better proliferation of cells in the culture medium, the cell density in the culture medium should not be high. The cells should be transferred to a new medium (passage) every few days. If the density and volume of the cells increase, the proliferation decreases due to lack of contact and the cells differentiate (Arunachalam, 2003; Dou et al., 2020; You et al., 2012; Mao, 2016; Radini et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018). Evaluation of cells in terms of growth, nutritional requirements and growth arrest, changes in cell morphology under a microscope are the goals of cell culture. To study the cell growth cycle, control the growth of cancer cells and study the expression of genes, cell culture must be performed outside the body to better study the type of growth and growth rate of cells. The study of animal evolution is one of the applications of cell culture. How can a fertilized cell become a percellular organism, and how does each cell differ in morphology under a microscope? To answer this question, it is better to culture the fertilized a cell in the laboratory and evaluate the stages of cell culture based on evolution. In cell culture medium, cells are prepared that are in the process of differentiation and differentiate into new cells with hormones and growth factors (Arunachalam, 2003; Dou et al., 2020; You et al., 2012; Mao, 2016). By cell culture, identical cells are produced and intracellular functions such as DNA replication, DNA transcription, RNA and protein synthesis, and cell metabolism are examined. After the molecule binds to its membrane receptor, intracellular reactions such as complexes, intracellular messages, and message transmission are evaluated. The cultured cells can be stored at a very low temperature. The low temperature maintained the growth rate or genetic composition of the cells. Cells can be used when needed, for example, a month later. This method prevents cell aging. In studies with animals such as rats and rabbits, animal homeostasis and experimental stress should be considered. However, this is not the case in cell culture. Standardizing laboratory tests is easier than studying animals; in the laboratory, it is easier to control physicochemical factors, cell environments such as acidity, heat, osmotic pressure, and oxygen and carbon dioxide pressures (Mao, 2016; Radini et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018).

In this research, we used the following cell lines to evaluating anticancer and cytotoxicity effects of nanoparticles using an MTT method.

Human lung cancer cell lines: HT144, SKMEL2, and WM266-4.

Normal cell line: HUVEC.

These cells in DMEM culture medium (Gibco, USA) with 10% FBS (Gibco, USA) and penicillin/streptomycin (100 μl / 100 μg / ml) in an incubator containing 5 % Carbon dioxide with 90% humidity was stored at 37 °C. Then, when about 80% of the flask was filled, cell passage was performed and about 5 × 10⁴ cells (per square centimeter) were placed in 24 house bacterial petri dishes in the usual environment. The cells were treated with different concentrations of nanoparticles 24 h later and kept in this condition for 3 days. The survival rate of cultured cells was prepared with different concentrations. In this experiment, cells were cultured at 3 × 10⁴ cells/well in 24-well plates and kept in an incubator at 37° C for 24 h. Then the old culture medium was taken out of the wells and the cells were treated with different concentrations of nanoxidro. This test was performed on the first, second and third days after exposing the cells to the compounds; thus, at the appropriate time after culturing the cells in plates of 24 cells, the culture medium was removed and about 300 μl of fresh medium containing 30 μl of MTT solution was added to each cell. After 3–4 h of incubation at 37° C, MTT solution is removed and 200 μl (Dimethyl Sulfoxide, Merck, USA, 100%) DMSO is added to each house. Then the sample absorption was read at 570 wavelengths using ELISA rider (Expert 96, Asys Hitch, Ec Austria). This experiment was repeated 3 times and each time, four wells were considered for each nano oxide concentration. Cell survival percentage was evaluated by the following formula (Arunachalam, 2003): $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{ControlA}.}\times 100$$

The results were evaluated as Mean ± SE using SPSS software version 12 and statistical tests of variance of completely randomized block design. Drawing graphs in Excel software was performed and the significance level of the differences was considered p < 0.05.

3.1 Chemical characterization of nanopaticles

Fig. 1 demonstrates the superposition of FT-IR spectrum of the nanoparticles. The characteristic strong absorption peaks of Fe₃O₄ can be seen at 469, 546, and 599 cm⁻¹ due to octahedral bending and tetrahedral stretching vibrations of Fe—O—Fe and ferrite spinel structure. In the spectrum of nanoparticles, all the peaks of Fe₃O₄ are present with the additional peaks due to extract, being appeared at 3414, 2927 and 1659 cm⁻¹ related to the O—H, C—H and C⚌O stretching vibrations. All the aromatic bond vibrations due to guaiacyl and syringyl scaffolds appear in the wavenumber region of 1600 cm⁻¹ to 1000 cm⁻¹ respectively.

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Fig. 1

FT-IR spectra of nanoparticles.

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In order to have the particle morphology and size of nanoparticles, FE-SEM analysis was provided (Fig. 2). 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 10 and 60 nm.

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Fig. 2

FE-SEM image of nanoparticles.

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The EDX analysis of the synthesized nanoparticles is shown in Fig. 3. The spectrum clearly shows the elemental composition profile of the FeNPs. the signals around 0.7 Kev (for FeLα) 6.4 Kev (for FeKα), and 7.1 Kev (for FeKβ) belong to iron in nanoparticles. These signals are as well as match to the biosynthesized FeNPs that reported previously (Radini et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018). A single at 0.52 Kev shows the presence of oxygen and another one at 0.28 Kev belongs to carbon. These signal can be ascribed to the oxygen a carbon of iron oxide nanoparticles and to the molecules of natural compounds in extract that linked to the surface of nanoparticles.

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Fig. 3

EDX of nanoparticles.

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In the recent study, Fig. 4 exhibits the UV–Vis. spectrum of silver nanoparticles. The band at 291 nm approves the formation of the iron nanoparticles.

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Fig. 4

UV–Vis analysis of nanoparticles.

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More detailed structural information are obtained by TEM. Fig. 5 describes the corresponding image of nanoparticles. The spherical morphology with a range size of 10–60 nm is obtained for synthesized nanoparticles. The images show an aggregation for nanoparticles that is one of common properties of green synthesized for the metallic nanoparticles.

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Fig. 5

TEM image of nanoparicles.

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The XRD diffraction patterns of nanoparticles evaluated its crystallinity. The pattern of the diffractogram is shown in Fig. 6. The formation of nanoparticles was approved to this result. Despite the small size of nanoparticles, the pattern of XRD indicated well crystallizing. The achieved data were compared with the standard database of ICDD PDF card no. 96–900-5813. The signals with 2θ values of 35.2, 37.3, 54, 61.3, and 75.3 are indexed as (3 1 1), (2 2 2), (4 2 2), (4 0 4), and (5 3 3) planes. The peaks at different degrees are also reported previously (Radini et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018). A 34.11 nm was measured for the crystal size of nanoparticles that was calculated using X-ray diffraction and according to Scherer’s equation.

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Fig. 6

XRD profile of nanoparticles.

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3.2 Cytotoxicity and anticancer activities of nanoparticles

Cancer is one of the leading causes of death, characterized by uncontrolled growth and abnormal cell proliferation. Cancer is caused by external factors such as chemicals and tobacco, as well as internal factors such as genetic mutations and hormones (You et al., 2012; Mao, 2016; Radini et al., 2018; Sangami and Manu, 2017). Cancer is usually diagnosed 10 years or more after exposure to chemicals. Cancer is treated with various methods such as surgery, radiation therapy and chemotherapy. One of the main causes of high cancer mortality is the inability to deliver targeted drugs to cancer cells without side effects on healthy cells. It seems that nanotechnology can be a good solution to this problem (Sangami and Manu, 2017; Beheshtkhoo et al., 2018; Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Jiang et al., 2021). Unique properties of nanoparticles such as particle size, high surface-to-volume ratio, their targeting ability, loading ability of water-insoluble drugs and controlled release, and responsiveness to the stimulant drug can make them a good candidate for cancer treatment. When the nanoparticles are injected into the bloodstream, they need to pass through the walls of the arteries to reach the target site and then release the drug. Unlike small molecules, nanoparticles, because of their relatively large size, cannot pass through the tight connections between the endothelial cells of healthy blood vessels (Sankar et al., 2014; Katata-Seru et al., 2018; Jiang et al., 2021). Tumor blood vessels, due to their leaky walls, allow nanoparticles to pass well. Targeted drug delivery to the tumor by nanoparticles is done in both passive and active targeting. In passive targeting, tumor tissue properties are used for drug delivery. In tumor tissue, due to rapid cell growth, rapid angiogenesis occurs, but the distance between vascular endothelial cells is too large, causing macromolecules and nanoparticles to leak from blood vessels into tumor tissue. On the other hand, the lymphatic system of tumor tissue is not complete and is not able to collect nanoparticles and insert them into the bloodstream; As a result, nanoparticles accumulate in the tumor tissue and release their drug. For passive targeting, the nanoparticle size should usually be below 200 nm. Active targeting of the tumor is achieved by binding a ligand such as antibodies, peptides, aptamers and some small molecules such as folic acid to nanoparticles (Mahdavi et al., 2020; Ghashghaii et al., 2017).

In this study, the treated cells with different concentrations of the present nanoparticles were assessed by MTT assay for 48 h about the cytotoxicity properties on normal (HUVEC) and lung malignancy cell lines. The absorbance rate was evaluated at 570 nm, which represented viability on normal cell line (HUVEC) even up to 1000 μg/mL for nanoparticles (Table 1 and Figs. 7 and 8). The viability of malignant cell lines reduced dose-dependently in the presence of nanoparticles. The IC50 of nanoparticles were 273, 216, and 250 µg/mL against HT144, SKMEL2, and WM266-4 cell lines, respectively (Table 1 and Figs. 7 and 8).

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Fig. 7

The anticancer properties of nanoparticles against HT144 (A), SKMEL2 (B), and WM266-4 (C) cell lines.

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Fig. 8

The cytotoxicity effects of nanoparticles against normal (HUVEC) cell line.

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It seems that the anticancer 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 (Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Jiang et al., 2021). Many nanoparticles have pharmacological and biochemical properties, including antioxidant and anti-inflammatory properties, which appear to be involved in anticarcinogenic and antimutagenic activities (Sankar et al., 2014; Katata-Seru et al., 2018; Jiang et al., 2021).

3.3 Antioxidant activities of nanoparticles

In this study, we assessed the antioxidant properties of nanoparticles by the DPPH test as a common free radical. Plants have several compounds, each with a different structure. The extraction of these compounds depends on several factors, the most important of which are the type of solvent and the extraction method. It will be very difficult to choose a solvent for any group of plant compounds, because with these compounds, there are other substances that affect the degree of solubility of these substances. When extracting plant extracts, it should be noted that always use a method that has the best performance in the survival of antioxidant compounds (Abdoli et al., 2020; Mahdavi et al., 2020; Ghashghaii et al., 2017). Usually plant extracts have unique antioxidant effects. Recent studies have shown that when these plant extracts are placed in metal nanoparticles as stabilizing and reducing compounds, they form nanocomposites with extraordinary antioxidant effects. Antioxidants are generally referred to as substances that can delay, slow down, and even stop oxidation processes. These compounds can optimally prevent changes in the color and taste of food because of oxidation reactions (Hummers and Offeman, 1958; Kooti et al., 2017; Thema et al., 2015; Shaneza, 2018; Arunachalam, 2003). The antidote to the mechanism of oxidants is that they prevent the spread of oxidation chain reactions by giving hydrogen atoms to free radicals. In recent years, the synthetic antioxidants use such as BHT, BHA, TBHQ as well as other chemical additives has been limited due to their potential toxicity and carcinogenicity. Today, most research in this area focuses on the use of new and safe antioxidants from plant, animal, microbial and food sources (Abdoli et al., 2020; Mahdavi et al., 2020; Ghashghaii et al., 2017; Hummers and Offeman, 1958).

Secondary metabolites in plants include alkaloids, terpenoids, steroids, saponins, phenols, flavonoids, and amino acids that are used to make various drugs (Abdoli et al., 2020; Mahdavi et al., 2020; Ghashghaii et al., 2017). This trend has paved the way for successful efforts to produce some valuable drugs. Flavonoids have the structural skeleton of polyphenols or diphenylpropane and consist of two aromatic benzene rings. These compounds are secondary metabolites large family and so far 6,000 various flavonoids have been identified. Major flavonoids include compferrol, quercetin, and myristin. The two main flavonoids are luteolin and apigenin, which contain 62 different types found in a variety of vegetables, fruits and edible tropical plants. Flavonoids have known properties including: removal of free radicals, inhibitors of some enzymes and anti-inflammatory properties. There is some evidence that the biological effects of these compounds are related to their antioxidant activity. Antioxidants work through various mechanisms in the body such as inducing apoptosis, anti-inflammatory effect, immune-boosting effect, cell cycle stopping and cell differentiation (Mahdavi et al., 2020; Ghashghaii et al., 2017; Hummers and Offeman, 1958).

The scavenging capacity of nanoparticles and BHT at different concentrations expressed as percentage inhibition has been indicated in Table 2 and Fig. 9. In the antioxidant test, the IC50 of nanoparticles and BHT against DPPH free radicals were 165 and 62 µg/mL, respectively (Table 2 and Fig. 9).

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Fig. 9

The antioxidant properties of nanoparticles and BHT against DPPH.

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