Green synthesis of Ag nanoparticles in oil-in-water nano-emulsion and evaluation of their antibacterial and cytotoxic properties as well as molecular docking

2.1 Materials

Whole chemical materials which had analytical purity applied for this research without extra purification process. Silver nitrate (AgNO₃, 99%) was purchased from Merck Company (Pvt. Ltd.), Germany. Reagents and standard laboratory chemicals were prepared from the Sigma-Aldrich Company, USA that includes penicillin–streptomycin (100 μg/ml), Dulbecco's modified eagle's medium, fetal bovine serum (FBS), phosphate-buffered saline (PBS), trypsin- EDTA solution, dimethyl sulfoxide, trypan blue dye solution, iodonitrotetrazolium (INT), Ciprofloxacin. 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium (WST-1 reagent) was prepared from Roche Holding AG. All eukaryote cells were prepared from Pasteur institute of Iran.

2.2 Essential oil extraction

Dark place and inert atmosphere used for drying herbal samples at room temperature for 15 days. Aerial parts and hydro-methanol distilled in a Clevenger-type apparatus for 3 h in order to extract the essential oil. All experiments were extracted two times. Anhydrous sodium sulfate (Na₂SO₄) was used to dry essential oils and sealed vials used to store and to protect from light during analysis processes at 4 °C.

2.3 Green synthesis processes of silver nanoparticles

The syntheses were carried out in oil-in-water (O/W) nano-emulsions by mixing the right amounts of the reagents to obtain the desired composition. Typically, about 5 ml of various extracted oil solutions gradually added to 30 ml of 1.75 mM AgNO₃ solution in separated beakers, while stirring at 5000 rpm speed via a homogenizer at 40 °C. The color of reaction solutions were changed to light brown rapidly and turn into turbid. Then standing for overnight, the color of solution states dark brown. After the synthesis was finished, the nanoparticles were separated by centrifugation and subjected to several washings with acetone, ethanol and water and dried at 40 ^◦C in a vacuum oven finally. The small droplet size of nano-emulsions confers stability against sedimentation (or creaming) because the Brownian motion and consequently the diffusion rate are higher than the sedimentation (or creaming) rate induced by the gravity force. Ostwald ripening or molecular diffusion, which a rises from emulsion polydispersity and the difference in solubility between small and large droplets, is the main mechanism for nano-emulsion destabilization (Tadros et al., 2004).

In order to optimize the AgNPs synthesis parameters, effect of some parameters like, different AgNO₃ concentrations (0.75, 1, 1.25, 1.5, 1.75 mM), extract volume (1, 2, 3, 4, 5 ml), temperature (27, 40, 60, 80 °C), and pH (8, 10 and 12) in the presence of different extracts was observed as well.

2.4 Microbial culture and antimicrobial effect of AgNPs

In order to investigate antimicrobial effect of nanostructures microbial cultures of Bacillus subtilis (ATCC 6051), Micrococcus luteus (ATCC 4698), Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Proteus mirabilis (ATCC 21100) and Pseudomonas aeruginosa (ATCC 27853) were chosen carefully. In order to obtain the aimed fresh cultures, the Mueller-Hinton agar was used to prepare them and overnighted at 37 °C in medium. A modified micro-dilution assay protocol was applied to determine the antibacterial effect (Elshikh et al., 2016). At first all microbial inoculums were prepared (0.5 McFarland) then preparation of the serial dilution (0.3–1000 µg/ml) of the silver nanoparticles were done in a 96-well round-bottom microplate. After that, microbial strains cultured and incubated at 37 °C for 24 h to desire well. After passing one day, 40 µl INT solution (0.6 mg/ml) was added to every well and incubated 20 min at 37 °C and the absorbance of each solution was read at 490 nm. Ciprofloxacin as positive control at 0.5–8 µg/ml concentration was used and blank was containing of all reagents except the bacteria, culture medium was used as the negative control. Below equation was applied to calculate the mortality rate (%) (Eslaminejad et al., 2016): Mortality rate (%) = ((A − C)/(B − C)) × 100; Where A = Test sample; B = positive control; C = negative control.

2.5 Computational method

Protein Data Bank (PDB) (www.rcsb.org) used for preparing crystal structures of bacteria. Amber force field selected for minimizing the docking process. Molecular docking experiment was employed by AutoDock4. The grid box parameters values were used to as center 33.4238, center 9.4981, center 28.8764 in x, y, and z direction respectively. Then binding energy of docking calculated by low RMSD evaluation by The Lamarckian genetic algorithm and graphical presentation has been done by using Discovery Studio (2.1.0) (Accelrys Software Inc., 2007).

2.6 Cell culture and in vitro cytotoxicity

In vitro cytotoxicity investigated as our previous papers (Amiri et al., 2018). Briefly Human breast cancer (MCF-7) cells and 3T3 fibroblasts as a normal cell line was used and was incubated in 5% CO₂ at 37 °C and until flaunted the 75% of the culture plate. DMEM was used to grow cell lines that accompanied with 10% fetal bovine serum, penicillin–streptomycin (100 U/ml) and the medium replaced every other day. 24 h applied for incubating the 10,000 cells/well of each cell line on the 96 well plates. After one day the medium aspirated. 100 µl of various concentrations (4, 8, 16, 32, 64, 125, 250, 500 and 1000 µg/ml) poured to wells and the plates were kept overnight at 37 °C under 5% CO₂. After one day, 10 µl of solution of WST-1 was added to each well and was incubated for 4 h. The absorbance of solution read at 420 nm by ELISA reader (BioTeks Elx 800). Viability of cell was stated as 100% for untreated control cells. The survival rate (%) was calculated by below equation (Eslaminejad et al., 2016) and All samples were performed in triplicates: Survival rate (%) = (OD in treatment group/OD in control group) × 100. The inhibitory concentration required for 50% cytotoxicity (IC₅₀) value was determined. The optical density (MTT assay) was determined at 420 nm using a microplate reader (BioTeks Elx 800).

2.7 Characterization of Ag nanostructures

The range of 300–600 nm wavelength was used to measure UV–Vis spectra with a wavelength step size of 2 nm at room temperature by Optizen 3220 UV (Corea). A series of technologies for characterization of synthesized samples were applied. Fourier transform infrared (FT-IR) spectra were verified by Bruker, FT-IR alpha model. X-ray diffraction (XRD) were detailed by X-ray diffractometer using Ni-filtered Cu Kα radiation (Philips-X’pertpro). SEM (Scanning electron microscopy (LEO instrument model 1455VP)) and TEM (transmission electron microscope (Philips EM208, accelerating voltage of 200 kV)) were applied to recognize the morphologies of the made-up NPs. Vasco and Wallis model of Nanosizer cordouan (France) was applied to determination of particle size and zeta potential. pH measurements were done by an 827 lab pHmeter made by Metrohm Company.

2.8 Statistical analysis

Student's t-test and analysis of variances were applied to evaluated significance among groups. All data are represented as mean ± SD. The probability level of p < 0.05 was considered as statistical significance.

3.1 Ag nanostructures characterization

Generally, FT-IR spectrum provides structural and functional groups information and molecular bonds nature in the substances. FT-IR spectroscopy applied to classify the functional groups that have controllable role for the bioreduction and capping of noble ion (Ag⁺ to Ag⁰) in this work, Fig. 1 exhibits the FT-IR spectra of synthesized AgNPs in herbal extract. The spectrum demonstrates bands at 3442, 2923, 1635, 1157, 1057 and 795 cm⁻¹. Mostly, expand peak in 3307–3325 cm⁻¹ shows the presence of the OH stretching of phenolic and flavonoids mixtures, although AgNPs synthesized using the extract shows the same band at higher frequency (Dubey et al., 2010). Further, AgNPs displays the weak band at the 2923 cm⁻¹ attributed to the alkenes C-H stretching vibration (Varadavenkatesan et al., 2017). The peak with sharp absorption intensity that was detected at the 1635 cm⁻¹ can be pointed to the amide-I bond because of carbonyl-amide linkages stretching that is representing which proteins were interrelating with green synthesized AgNPs and their secondary structure has not affected during or afterward the reaction with Ag⁺ ions. Strong binding ability was produced with amino acid residues (carbonyl function group) and metal (capping agent) which avoid agglomeration and provide medium stability (Boopathi et al., 2012). Moreover, the existence of aliphatic amines in proteins were represented by two bands between 1157 and 1057 cm⁻¹ vibration of C-N stretching. The band peak at the 795 cm⁻¹ states heterocyclic compounds that related to present flavonoids (Kacurakova et al., 2000). Observed data verifies action of proteins as coating/stabilizing agent and antioxidants reducing agent like as flavonoids/phenolic compounds. XRD technique has applied to analyze the crystal structure nature of the synthesized AgNPs. Multiple strong peaks recognized at (1 1 1), (2 0 0) and (2 2 0) of the face centered cubic lattic that resulted crystal structure of Ag metal (Gogoi et al., 2015) (Fig. 2) which are consistent with Ag⁰ standard (JCPDS Card no. 04–0783) (Coseri et al., 2015). The plane (1 1 1) was the major element of Ag planes. The average of crystallite size D can be obtained from the (1 1 1) peak in XRD patterns by driving in the Scherrer Equation (Amiri et al., 2016): D = kλ/Bcosθ; where B is the full-width at half maximum (FWHM) of a diffraction peak, k is the Scherrer constant (0.89), and θ is the Bragg‘s angle. The calculated crystal size by the XRD reflections is about 38 ± 2 nm. Nanosize range was indicated from the XRD lines according to sharper reflexes with improved intensity emerge, signifying the presence of a smaller nanocrystalline phase. Generally, Electrokinetic potential can be taken from zeta potential in colloidal dispersions scientifically. Measurements of Zeta potential were done and confirmed that the nanoparticles surface have a negative electric charge which let them to stay in colloidal suspension manner. Results established that the zeta potential of the AgNPs increases by increasing of extract content, therefore the higher content of extracted biomolecules (the herbal extract/AgNO₃ ratio of 5) present in the system, nanoparticles indicated the most negatively charged of −56 mV. The stability of the nanoparticles provided by negative charges which preventing aggregation and agglomeration.

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Fig. 1

FT-IR spectrum of green synthesized Ag nanostructures.

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Fig. 2

X-ray diffraction pattern of green synthesized Ag nanostructures.

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The SEM images of synthesized NPs at two different pH are presented in Fig. 3(A-D). SEM image (Fig. 3A and B) evidently demonstrated that AgNPs were not agglomerated and are spherically shaped with a diameter ranging from 40 to 50 nm. By increasing the pH (the pH increased by gradually addition of 0.20 M NaOH solution) from 10 to 12 (Fig. 3C and D), AgNPs indicated spherical- to oval-shape with a larger average diameter and more agglomeration. It is possibly owing to the growth rate of AgNPs become faster than the nucleation rate with increasing the pH (Li et al., 2017). Extracted Biomolecules like proteins, enzymes, terpenoids and flavonoids cofactors play both capping and reducing role in the nanoparticles synthesis (Raja et al., 2017), Furthermore due to strong binding ability with amino acid residues (carbonyl group) with metal as capping agent, agglomeration behavior was prevented and stability of medium was provided (Boopathi et al., 2012).

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Fig. 3

Scanning electron microscopy (SEM) of AgNPs synthesized in the presence of Artemisia herba-alba extract at pH of 10 (A, B) and pH of 12 (C, D).

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Transmission electron microscopy images of AgNPs prepared at herbal extract, is shown in Fig. 4A and the size distribution from TEM images is presenrted. As shown, TEM images confirmed a spherical shaped and well-dispersion of nanostructures. The results were in good accommodation with the observation of SEM and XRD (scherrer equation) that approve the synthesis of AgNPs by a natural material (Arokiyaraj et al., 2014). For better understanding of the real size of nanoparticles, Nanosizer used for calculating particle size and stated by SBL (Statistical Bin Limits) analyze. For this propose, reduction of agglomeration fault to state actual particles size were done by omitting hydrodynamic radius. Fig. 4B reported the histogram of SBL nanosizer of NPs in which showed the mean diameter of size of particle is ∼51.39 nm. Reported results demonstrated narrow size distribution and homogenous dispersity of NPs.

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Fig. 4

TEM image of Ag nanostructures synthesized at pH 10 at different magnitude as well as size distribution of Ag NPs based TEM images (A), The SBL (Statistical Bin Limits) nanosizer histogram of NPs (B).

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3.2 Ag nanoparticles synthesis investigation

For Ag nanoparticles synthesis, the reaction mixture (AgNO₃ solution) entirely turned to brown after Artemisia extract addition at room temperature (Fig. 5A). The brown color of the reaction mixture corresponds to silver nanoparticles surface plasmonace that made in the reaction mixture. Mostly, UV–Vis spectroscopy can be employed for the detection of the appropriate size and distribution of AgNPs due to the surface plasmon resonance excitation (SPR) (Lee and Mooney, 2012). The UV–Vis spectra of the AgNPs prepared at different pH levels in the presence of Artemisia is revealed in Fig. 5B. The strongest symmetric absorbance band is positioned at approximately 420 and 415 nm (pH 8 and 10) and 395 nm (pH 12) in the UV–Vis spectra, demonstrative to the formation of well-dispersed Ag NPs. With increasing pH value, the absorption peak intensity steadily increased, once the pH value reached 12, the band obviously shifted toward lower wavelengths. The higher pH value would make the alkaline hydrolysis to produce smaller molecular masses of fragments with higher reducibility and affinity, which are essential for the well-dispersed and stable AgNPs synthesis. Additionally, the silver salt concentration and extract volume are another factors that can excellently modify the silver nanoparticles shape. Fig. 6A and 6B demonstrate the UV–Vis spectra of silver nanoparticles prepared by different concentrations of AgNO₃ and herbal extracts volume respectively. When the concentration of Ag salt increases to 1.75 mM, a very identical style in the nanoparticles synthesis was detected for all investigated media. The adsorption peak strength increases with the increasing the content of metallic precursors and similarly by increasing the herbal extracts amount, consequently a displacement to longer wavelengths is observed in the UV–Vis spectrum of the nanoparticles synthesized with a higher metallic precursor content (figure not shown). The results demonstrate that by increasing AgNO₃ concentration, the peak intensity increased for all samples as well. No significant effect of increasing the extract was observed in Mentha while silver nanostructures synthesized in the presence of other extracts designated more adsorption peak intensity. In addition, the effect of varying temperature (Fig. 6C) was more significant for AgNPs synthesized in the presence of Rosmarinus and Mentha that by increasing temperature reduction in adsorption peaks revealed that possibly is due to the AgNPs aggregation. However, at 80 °C Ranunculus presented relatively (1.4 folds) higher synthesis, while Artemisia exhibited the highest one. These results suggested better efficacy of Rosmarinus particularly, even at lower temperature in comparison with other extracts.

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Fig. 5

Photographic image of reduction of Ag⁺ to Ag⁰ before and after addition of herbal extract (A), UV–Vis spectra of pH dependent synthesis of silver nanoparticles in the presence of Artemisia herba-alba extract (B).

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Fig. 6

Various AgNO₃ concentration effect (A) extract volume (B) and temperature (C) on production of AgNPs using various aqueous extracts respectively.

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3.3 Ag nanoparticles antimicrobial evaluation

Antibiotic resistance is one of the main public health concerns of this century. This resistance is also associated with oxidative stress, which could contribute to the selection of resistant bacterial strains. Antimicrobial evaluation of the green synthesized silver nanoparticles was investigated on the four gram-negative, and three gram-positive including E. coli, K. pneumonia, P. mirabilis, P. aeruginosa, M. luteus, B. subtilis, and S. aureus. The bactericidal properties of AgNPs depends on their stability in the growth medium, the capping agents and the shape/size of AgNPs which plays an important role in improving their antimicrobial properties (Pal et al., 2007). Overall, acceptable antimicrobial activity of Ag NPs is due to smaller size and higher surface to volume ratio of NPs which permits the presence of active Ag atoms on their surface, leading to enhanced antimicrobial activity (Ocsoy et al., 2017). Obtained results presented that the antimicrobial activity varied between the microbial strains. Both of the gram positive and negative bacteria were sensitive to the test samples. The MIC₅₀ values for this synthesized nanoparticle were 8, 4, 4, 2, 2, 1 and 4 ppm, for the investigated bacteria; E. coli, K. pneumonia, P. mirabilis, P. aeruginosa, M. luteus, B. subtilis, and S. aureus. respectively as seen in the following Fig. 7A and B. Although there is a distinct variance between the activities of the AgNPs in the case of the 7 bacteria strains, the antimicrobial activity in the case of B. subtilis is noticeably higher compared to the other ones. The effect of silver particles against bacteria is due to their interaction with thiol group compounds, which are found in the bacterial cells respiratory enzymes. It was reported earlier that, Silver binds to the bacterial cell wall and cell membrane that hinders the respiration process, due to the much larger surface area, AgNPs demonstrate increased antimicrobial activity, AgNPs get attached to the bacterium cell membrane and also penetrate the bacteria inside. The bacterial membrane covers sulfur-containing proteins and the silver nanoparticles act together with these proteins in the cell as well as with the phosphorus containing compounds like DNA. AgNPs preferably attack the respiratory chain (Rai et al., 2009; Singh et al., 2015).

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Fig. 7

Mortality (%) of the green synthesized silver nanoparticles on the gram-negative bacteria (A) and on the gram-positive bacteria (B).

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3.4 Ag nanoparticles cytotoxicity studies

WST-1 assay was performed for calculating of AgNPs cytotoxicity against the MCF-7 cells and 3T3 cell lines (Fig. 8B). In cultured cells, viability was meaningfully reduced in the presence of AgNPs at several concentrations that suggested a dose-dependent manner. The silver NPs revealed a potent index of cytotoxicity effect on cancerous and normal cell line as verified by the low IC₅₀ value of 1 ppm (Fig. 8B). The inhibitory effects of AgNPs on the breast cancer cells may be due to the characteristics of this disease, so that the cells of this cancer illustrate a higher rate of metabolism, quick cell division, and consequently exhibit improved internalization of AgNPs, causing in a higher cell death rate. Also, other studies established AgNPs at the concentrations above 2 μg Ag/mL displayed to be harmful/cytotoxic for the MCF-7 cells (Roszak et al., 2017). The decreased cell viability against AgNPs is because of not only generation of reactive oxygen species, but also decreased cellular ATP content as well as reduced dehydrogenase activity that does damage to mitochondrial respiratory chain and cellular components resulting in cell death (Justin Packia Jacob et al., 2012). Hence, cytotoxicity of phytogenic AgNPs is dependent on cell type that demonstrates a specific intracellular mechanism for proliferation inhibition rather than an unspecific disturbance of cell membrane functionality. However, cytotoxicity of NPs is exclusively based on their shape, size, surface chemistry, interaction time and agglomeration state (Rajkuberan et al., 2015).

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Fig. 8

The cytotoxicity investigation of AgNPs on MCF and 3T3 cell lines.

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3.5 Ag nanoparticles docking finding

Protein Data Bank (PDB) (www.rcsb.org) used for preparing crystal structures of bacteria. Amber force field selected for minimizing the docking process. Molecular docking experiment was employed by AutoDock4 (Rizwan, 2012). The grid box parameters values was used to as center 33.4238, center 9.4981, center 28.8764 in x, y, and z direction respectively. Then binding energy of docking calculated by low RMSD evaluation by the Lamarckian genetic algorithm and graphical presentation has been done by using Discovery Studio (2.1.0) (Kyte and Doolittle, 1982).

Hydrophobicity of proteins, amino acids and microbiological structure such as bacteria cause the ability of water proofing in biochemistry. This effect indicate the hydrogen bonding in structure (Morris et al., 2009). A hydrophilicity plots Fig. 9(A-G) are a quantitative analysis of the degree of hydrophobicity or hydrophilicity of amino acids of bacteria’s that applied in this study. The figures indicated all bacteria have interaction by polar solvents with special amino acids. All plots showed that parts of alpha helix with positive hydrophobicity are contained amino acids. Therefore amino acids with high hydrophilicity indicate which these residues are in contact with solvent and they are therefore likely to reside on the outer surface of the protein (Accelrys Software Inc., 2007). Electron density surface is a real place which chemical compounds have ability to accept the other structures. In Fig. 10(A-G) surface of acceptor and donor electron of amino acids in bacteria indicated red color shows the negative place and blue color shows the positive place. Green box shows active site of bacteria which Ag has interaction with. Accessible pose for getting dock and binding site recognized by green box. In fact, for evaluating and predicting the accuracy of binding ability between ligands and target protein, the binding free energies (ΔG) for the docking models and the crystal structures were calculated. The lower binding energy value specify the binding strength of the ligands and docking model. Therefore, to calculate the binding strength of Ag, the Ag-bacteria docked complexes were analyzed based on minimum binding energy values and Ag interaction (hydrogen/hydrophobic) pattern. Docking results reported in Table 1 recognized that AgNPs had good interaction by bacteria and indicated antibacterial manner in solvent. According to the results lower binding energy is belonged to Klebsiella pneumoniae and Escherichia coli. Therefore strong interaction related to Ag with Klebsiella pneumoniae and Escherichia coli. Fig. 11 illustrates the structure of all of applied bacteria as well.

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Fig. 9

Hydrophobicity plot of (A) Staphylococcus aureus, (B) Escherichia coli, (C) Pseudomonas aeruginosa, (D) Bacillus subtilis, (E) Klebsiella pneumoniae, (F) Micrococcus luteus, (G) Proteus mirabilis.

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Fig. 10

Electron density surface and pose of binding with Ag nanostructures of (A) Staphylococcus aureus, (B) Escherichia coli, (C) Pseudomonas aeruginosa, (D) Bacillus subtilis, (E) Klebsiella pneumoniae, (F) Micrococcus luteus, (G) Proteus mirabilis.

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Fig. 11

Structure of all of applied bacteria.

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