Physico-chemical characterization and anti-laryngeal cancer effects of the gold nanoparticles

2.1 Preparation of the Au NPs

After collecting the Ocimum basilicum extract, in a 50 ml 1 (mM) aqueous solution of HAuCl₄ and 10 ml of the flower extract were added in stirring condition at room temperature. After 20 min, the light yellow colored mixture changed to wine red, an indication for the synthesis of Au NPs. Then the solution containing nanoparticles was centrifuged at 4000 rpm for 20 min and the upper transparent layer was decanted off. The residues obtained were washed for several times with deionized water and finally dried in an oven at 50˚C.

2.2 Anti-cancer properties of Au nanoparticles

Cell culture should be performed under aseptic (disinfected) conditions because the growth of these cells is much slower than the growth of bacteria and yeasts and there is a possibility of contamination of the culture medium. Antibiotics such as penicillin, streptomycin, or gentamicin are sometimes used to stop the growth of bacteria. In order for cells to proliferate well in culture medium, their density in culture medium must be low. For this purpose, the cells should be passed to the fresh culture medium from time to time. One of the goals of cell culture is to study cells in terms of how they grow, their nutritional needs, and the reasons they stop growing, each of which can have a profound effect on the morphology of the cells we see under a microscope. Therefore, to study the cell growth cycle, develop methods to control the growth of cancer cells and modulate the expression of genes, it is necessary to cultivate these cells in the external environment (Li and G f., 2014). With the help of cell culture, cells can be prepared that are in different stages of differentiation and can be differentiated into other cells with the help of hormones and growth factors. With the help of cell culture, homogenous cells can be prepared and intracellular activities such as DNA replication, DNA transcription synthesis, RNA and protein synthesis and other details related to metabolism can be studied. It is also possible to examine the subsequent events and intracellular currents, such as the displacement of these complexes, the type of intracellular messages, and how the messages are transmitted, after connecting different molecules to the corresponding membrane receptor. The cultured cells can be stored frozen at very low temperatures. Such conditions will maintain the growth rate or genetic composition of these cells and can be thawed and used again at the appropriate time. This prevents the aging of cells, while it is currently not possible to prevent the aging of animals. When working with laboratory animals, systemic changes due to the effect of the animal's natural homeostasis or the stress of the experiments on the results should be considered. While the use of cell culture eliminates this problem. In addition, standardizing laboratory tests is easier and more practical than tests on living organisms. In laboratory environments, it is much easier to control the physical and chemical factors in the living environment of cells, including acidity, heat, osmotic pressure, and the pressure of gases such as oxygen and carbon dioxide. Cells that are taken directly from the individual are known as primer cells and have a limited lifespan. Most cells have a limited lifespan, except for those taken from a tumor. An immortal cell line can proliferate indefinitely by creating a random or targeted mutation (such as artificial expression of the genus and be established as a representative of specific cell types (Li and G f., 2014).

In this research, we used the following cell lines to evaluating anti-laryngeal cancer and cytotoxicity effects of Au nanoparticles using an MTT method.

a) Normal cell line: HUVEC.

b) Cancer cell lines: HEp-2, TU212, KB, UM-SCC-5, UM-SCC-11A and UM-SCC-11B.

In the recent study, the cells were cultured in medium (RPMI1640 = Roswell Park MemoryL Institute1640) with 10 % FBS combined with penicillin and streptomycin antibiotics in an incubator containing 5 % CO₂ in a flask (T25). After three passages for purification, the cells were used to perform the next steps. Cell count and the number of viable cells were performed with a homocytometer slide using trypan blue. Evaluation of the cytotoxic effect of the Au nanoparticles was performed by the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric test. In this method, MTT, which is yellow, is converted to insoluble and formazan purple dye by the dehydrogenase enzymes in the mitochondria of active cells. The adsorption of this compound can be measured after dissolving at 570–540 nm. After two days and covering the flask bottom with cell, the cell layer adhering to the flask bottom was isolated enzymatically using trypsin-EDTA (5 %) (Tetraacetic acid ethylenediamine), after transfer to sterile test tubes, it was centrifuged at 2000 rpm for 10 min. The cells were then resuspended in a fresh culture medium with the help of a pasteur pipette and cell suspension (10⁶ ml/μg) was prepared from them. 40 μl of this cell suspension (equivalent to 10⁴ × 4 cells) was poured into 96-well plate flat-bottomed wells (for cell culture). Then the final volume of each well with 10 % FBS medium reached 200 μl. The first-row containing cell suspension was considered as negative control (control). After incubation for 18–24 h to remove cells from the stress caused by trypsinization, the supernatant was removed slowly and carefully, A new medium was added to all rows with different concentrations of the Au nanoparticles (only a new medium was added to the negative and positive control rows), so that the diluted Au nanoparticles with concentrations of 1–1000 μg/ml was added to the third to sixth rows, respectively, the plate was incubated in CO2 for 48, 24 and 72 h. After the incubation time, the plate was taken out of the incubator and 20 μl of MTT (Sigma) was added to all wells, and incubated for 3 h. The supernatant was then gently removed and 100 μl of DMSO was added to the wells and pipetted to dissolve the formazan crystals. The amount of light absorption (OD) according to the intensity of the blue color of formazan at 540 nm was read by Eliza reader. To convert OD to the percentage of living cells, the following formula was used and the percentage of cell life at each concentration was calculated after 48, 24 and 72 h (Li and G f., 2014). $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{ControlA}.}x100$$

The concentration of the tested compounds that reduced the percentage of cell life by half was considered as IC50 (The half maximal inhibitory concentration) (Li and G f., 2014).

2.3 Antioxidant activities of Au nanoparticles

In this method, first pour 0.3 ml of the sample solution into a test tube, then add 9 ml of the DPPH methanolic solution. The contents of each tube were thoroughly mixed with the vortex. After 30 min, at room temperature and in the dark, their absorbance was read at 517 nm using a UV/Vis spectrophotometer against a methanol-containing blank. In this method, BHT was used as a positive control. According to the mentioned mechanism, the higher the antioxidant power of the sample, the yellower the color of the resulting solution will be (Yang et al., 2011).

The following formula was used to determine the antioxidant properties of Au nanoparticles: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}x100$$

Excel and Bio Data Fit 1.02: Data Fit For Biologists software were used to calculate the IC50 and to compare the antioxidant effects of these extracts with the standards and negative control and plotting.

2.4 Qualitative measurement

At least three independent replications were performed for each data and the result was presented as mean ± SD. Data statistical analysis was done with SPSS software version 19 and Anova Way One and Duncan’s test. Significance was considered at the level of P ≤ 0.05.

3.1 Structural characterization of synthesized Au nanoparticles

A post-synthetic modification approach was followed in the strategically developed Au nanoparticles. The final Au nanoparticles was subsequently analyzed through several techniques, such as, FT-IR, SEM, TEM, EDX, XRD and UV–vis.

Fourier transform infrared spectroscopy (FTIR) uses the mathematical process (Fourier transform) to translate the raw data (interferogram) into the actual spectrum. FTIR method is used to obtain the infrared spectrum of transmission or absorption of a fuel sample. FTIR identifies the presence of organic and inorganic compounds in the sample (Lu et al., 2021; Shaneza, 2018; Gao et al., 2015). Fig. 1 exhibits the FT-IR spectrum of AuNPs. The peaks at 464, 536 and 607 cm⁻¹ that belong to the vibration of Au-O. These peak with a little difference in wavenumber have been reported for gold nanoparticles (Lu et al., 2021; Shaneza, 2018; Gao et al., 2015). The others in the other regions such as peaks at 3419, 2925, 1621, 1214, and 1003 cm⁻¹ belong to functional groups of organic compound in plant extract that have linked to the surface of AuNPs phenolic, flavonoid, saponins, Quinones, terpenoids compounds are the main organic compound in the plant extract (Shaneza, 2018; Gao et al., 2015; Mohammed et al., 2016).

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Fig. 1

FT-IR spectrum of Au NPs.

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SEM, field emission SEM, TEM and X-ray diffraction (XRD) are the most common techniques used to characterize surface morphology, microstructure, and the phase composition of conversion coatings (Lu et al., 2021; Shaneza, 2018; Gao et al., 2015). Shape, size and structural morphology of the as-synthesized Au nanoparticles were investigated by FE-SEM and TEM analysis. Fig. 2 displays the FE-SEM image with pseudo-spherical shaped particles. A homogeneous layer of the hydrogel polymers over the surface can be seen on close observation. This makes the particles fluffy. Due to a high surface energy, the particles have a high tendency to agglomerate forming lump-like appearances. Some more detailed structural features are obtained from TEM images (Fig. 3). They are absolutely spherical and well separated from each other. The particle sizes are mono dispersed with an average particle diameter of 19–44 nm.

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Fig. 2

FE-SEM image of the Au nanoparticles.

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Fig. 3

TEM images of the Au nanoparticles.

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Energy Dispersive X-ray Analysis (EDX), referred to as EDS or EDAX, is an X-ray technique used to identify the elemental composition of materials. Applications include materials and product research, troubleshooting, deformulation, and more. EDX systems are attachments to Electron Microscopy instruments (Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM)) instruments where the imaging capability of the microscope identifies the specimen of interest. The data generated by EDX analysis consist of spectra showing peaks corresponding to the elements making up the true composition of the sample being analysed. Elemental mapping of a sample and image analysis are also possible (Lu et al., 2021; Shaneza, 2018; Gao et al., 2015). The prepared Au nanoparticles was validated from EDX analysis, as depicted in Fig. 4. The profile shows a major and distinct signal of Au appeared at 2.2 keV. However, some other peaks of C, N and O as constitutional elements are also observed. They can be corresponded to the plant extract, being associated with the Au NPs surfaces.

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Fig. 4

EDX spectrum of the Au nanoparticles.

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Fig. 5 shows the UV–Vis. Spectrum of AuNPs. The formation of AuNPs was approved by the band at 537 nm. The bands are very similar to that of a previous studies on the synthesis of gold nanoparticles using medicinal plant extracts (Wang et al., 2010).(See Fig. 6).

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Fig. 5

UV–vis pattern of the Au nanoparticles.

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Fig. 6

XRD pattern of the Au nanoparticles.

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Finally, the phase structure and crystallinity of the prepared Au nanoparticles was ascertained from XRD analysis. The diffraction peaks at 2θ = 38.2°, 44.3°, 64.6° and 77.6° confirms the Au crystalline phases. These correspond to the diffraction on fcc gold (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes (JCPDS No.65–2870).

3.2 Cytotoxicity and anti-cancer activities of Au nanoparticles

Gold nanoparticles (GNPs) have received much attention in nanobiotechnology and biomedicine due to their excellent adsorption with molecular probes and remarkable optical and immunological properties. Recent examples include applications of gold nanoparticles in genomics, biosensory, immunoassay, clinical chemistry, detection and control of microorganisms, photothermolysis of cancer cells, targeted delivery of drugs or other substances, and optical imaging and monitoring of biological cells and tissues (Tsai et al., 2007; Brown et al., 2008; Paciotti et al., 2004; Paciotti et al., 2006; Paciotti et al., 2006). Gold nanoparticles are increasingly used not only in diagnostic and photometric tests of cells but also in therapeutic purposes. Researchers at the University of Maryland used a colloidal gold vector to deliver TNF to solid tumors in mice. After intravenous injection, gold nanoparticles with TNF accumulate rapidly in tumor cells, and cells in the liver, spleen, and other organs are diagnosed as healthy (Park et al., 2013; Erik, 2012; Remant Bahadur, 2014; Mendes, 2017; Huang et al., 2008). Gold-INF vector had lower toxicity and higher effect in reducing tumor size compared to TNF alone, because the maximum antitumor response was obtained using lower doses of the drug. Gold nanoparticles are one of the most widely used nanoparticles (Remant Bahadur, 2014; Mendes, 2017; Huang et al., 2008; Cruz et al., 2012). One of the reasons for the great attention paid to gold nanoparticles and their use for biological and medical purposes is their quick and easy synthesis method. Various methods for the preparation of gold nanoparticles, all of which are based on the reduction of salts (Au (III), the most important of which is the hydrogen tetrachloroate salt with the chemical formula (HAuC1₄) (Andersson et al., 2014; Namvar et al., 2014; Sankar et al., 2014). In fact, to make gold nanoparticles, we must have Au ions suspended in the solvent. Various laboratory and industrial methods for preparing gold nanoparticles; these include chemical reduction methods, electrochemical methods, chemical sound methods and biochemical light methods. In all the mentioned methods, soluble polymer, surfactants and various ligands are used as stabilizing agents (Paciotti et al., 2006; Park et al., 2013; Erik, 2012; Remant Bahadur, 2014; Mendes, 2017). The inertia of gold and its resistance to surface oxidation are important properties of this metal. The optical properties of gold at the nanoscale are also very significant. It has now been proven that gold nanoparticles have been used as catalysts in a number of important commercial reactions and have excellent surface chemistry. Based on this unique feature, new applications of nanotechnology using gold are expanding (Andersson et al., 2014; Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017). At the nanoscale, gold exhibits properties that make it an important metal for future nanotechnology products. This nanoparticle has applications in pharmacy, medicine, agriculture, electronics, coating, paints, and catalysts. It is widely used in biosensors today to detect nucleic acids and proteins (Paciotti et al., 2004; Paciotti et al., 2006; Paciotti et al., 2006; Park et al., 2013; Erik, 2012; Remant Bahadur, 2014).

Another advantage of gold nanoparticles is their ability to detect microorganisms, cancerous tissues, etc., both in vivo and in vitro. Gold nanoparticles coated with anti-cancer antibodies can effectively bind to cancer cells (Andersson et al., 2014; Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018). Many cancer cells have a protein on their surface called the epidermal growth factor receptor (EFGR). This protein is not found mainly in healthy cells of the body. By attaching gold nanoparticles to the EFGR antibody (known as -Anti EFGR), researchers have attached these nanoparticles to cancer cells. Gold nanoparticles are an excellent carrier for immunotherapy because, like other nanoparticles, they can easily accumulate inside immune cells (Erik, 2012; Remant Bahadur, 2014; Mendes, 2017; Huang et al., 2008; Cruz et al., 2012; Andersson et al., 2014). Gold nanoparticles can transport several drug molecules, recombinant proteins, vaccines or nucleotides into the target cell. It can also control the release or release of drugs. Conjugation of gold nanoparticles with drug molecules plays an important role in intracellular diseases (Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017). Antibiotics and other drugs can bind directly to gold nanoparticles with the help of ion or covalent bonds. The surface of gold nanoparticles can be easily changed to bind drug molecules. Changing the level of gold nanoparticles with polymers plays an important role in its conjugation with the drug. With this strategy, many therapeutic drugs can be successfully transferred, such as doxorubicin and tamoxifen (Andersson et al., 2014; Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018). An important challenge in cancer is the delivery of water-soluble drugs, which used hydrophobic polymers to coat the nanoparticles; the drug is encapsulated by a hydrophobic coating. Methotrexate is used as an anti-cancer drug. It is a folic acid analog that can inhibit the growth and proliferation of cancer cells (Paciotti et al., 2006; Park et al., 2013; Erik, 2012; Remant Bahadur, 2014; Mendes, 2017). The carboxylic group on the drug can bind to the surface of gold nanoparticles and exert its effect on cancer cells. Polyethylene glycol increases the adsorption of gold nanoparticles. The hydrophilic properties of PEG prevent the opsonization and purification of nanoparticles in the body. It can also be used as conjugation of gold nanoparticles and drug molecules (Namvar et al., 2014; Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017).

In this experiment, the treated cells with different concentrations of the present Au nanoparticles were assessed by MTT assay for 48 h about the cytotoxicity properties on normal (HUVEC) and laryngeal malignancy cell lines i.e. HEp-2, TU212, KB, UM-SCC-5, UM-SCC-11A and UM-SCC-11B.

The absorbance rate was evaluated at 570 nm, which represented viability on normal cell line (HUVEC) even up to 1000 μg/mL for Au nanoparticles (Fig. 7).

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Fig. 7

The cytotoxicity properties (Cell viability (%)) of Au nanoparticles (Concentrations of 0–1000 µg/mL) against human normal (HUVEC) cell line.

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The viability of malignant laryngeal cell lines reduced dose-dependently in the presence of Au nanoparticles. The IC50 of Au nanoparticles were 174, 231, 179, 143, 230, and 216 µg/mL against HEp-2, TU212, KB, UM-SCC-5, UM-SCC-11A and UM-SCC-11B cell lines, respectively (Figs. 8-10).

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Fig. 8

The anti-laryngeal cancer properties (Cell viability (%)) of Au nanoparticles (Concentrations of 0–1000 µg/mL) against HEp-2 (a) and TU212 (b) cell lines.

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Fig. 9

The anti-laryngeal cancer (Cell viability (%)) of Au nanoparticles (Concentrations of 0–1000 µg/mL) against KB (a) and UM-SCC-5 (b) cell lines.

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Fig. 10

The anti-laryngeal cancer properties (Cell viability (%)) of Au nanoparticles (Concentrations of 0–1000 µg/mL) against UM-SCC-11A (a) and UM-SCC-11B (b) cell lines.

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It seems that the anti-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 (You et al., 2012; Mao, 2016; Namvar et al., 2014). 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). Today, nanoparticles synthesized by biological methods play a vital role in treating many diseases, including cancer (Sangami and Manu, 2017; Beheshtkhoo et al., 2018; Radini et al., 2018).

3.3 Antioxidant properties of Au nanoparticles

In this study, we assessed the antioxidant properties of Au nanoparticles by the DPPH test as a common free radical. Free radicals are molecules with a free electron ready to react, and oxygen is produced with some molecules. If many of them are suddenly produced in the body, they react with some parts of the cell, such as DNA and cell membranes, and cause cell damage or even death (Sankar et al., 2014; Katata-Seru et al., 2018). Normally, the body's defense system neutralizes these harmless free radicals. Antioxidants prevent the spread of oxidation chain reactions. Thus, the strength of an antioxidant formed by the contact of an H atom with a free radical is due to the effect of an antioxidant on the ease with which this H atom separates from it. Thus, antioxidants can protect cell membranes and various living compounds against oxidants in small amounts (Sangami and Manu, 2017; Beheshtkhoo et al., 2018). Numerous biochemical and physiological processes may cause the production of free radicals. Reactive oxygen species (ROS) include free radicals and radical-free forms. Free radicals include hydrogen peroxide (H₂O₂), hydroxyl radical (.OH), and superoxide anion radical (O₂). When the concentration of ROS increases, it can oxidize macromolecules such as proteins, nucleic acids, and membrane lipids, resulting in cell damage and possibly “cell and tissue destruction” (Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018). Natural compounds and molecules have two main mechanisms for reducing the concentration of ROS, in other words, natural compounds and molecules reduce the concentration of ROS by producing antioxidants and thus prevent cell damage (Beheshtkhoo et al., 2018; Radini et al., 2018). Recently, many researchers have paid close attention to natural compounds and molecules and their relationship to their antioxidant properties, and many natural compounds and molecules have been studied for their antioxidant activity (Sankar et al., 2014; Katata-Seru et al., 2018; Sangami and Manu, 2017; Beheshtkhoo et al., 2018; Radini et al., 2018).

The scavenging capacity of Au nanoparticles and BHT at different concentrations expressed as percentage inhibition has been indicated in Fig. 11. In the antioxidant test, the IC50 of Au nanoparticles and BHT against DPPH free radicals were 228 and 208 µg/mL, respectively (Fig. 11).

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Fig. 11

The antioxidant properties of Au nanoparticles and BHT against DPPH.

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