# Investigation of Antioxidant and Cytotoxicity Effects of Silver Nanoparticles Produced by Biosynthesis Using Lactobacillus gasseri

Document Type : Original Articles

Authors

Departments of Applied Sciences, University of Technology, Iraq

Abstract

The biosynthesis of silver nanoparticles (AgNPs) is a new approach in nanotechnology which was optimistically implemented in medicine, food control, and pharmacology. The present study aimed to investigate the antioxidant and cytotoxicity effects of AgNPs produced by Lactobacillus gasseri filtrate. Also, changing color from yellow to brown confirmed the production of AgNPs. AgNPs were characterized using ultraviolet-visible spectroscopy, FE-SEM, and Fourier Transform Infrared Spectroscopy (FTIR). The antioxidant activity of AgNPs was tested using the DPPH assay. The scavenging test for DPPH showed 19.3%, 32.6%, 47.6%, 72%, 85.3% at concentrations (6.25, 12.5, 25, 52, 100) µg/ml, respectively, which proved that the scavenging percentage increased with increasing concentration. The effect of AgNPs on the chromosomal pattern was also studied. The results of the experiment of AgNPs against SK-GT-4 cancer cells showed the toxic activity of the used particles against the strains of these human esophageal cancer cells and failed to affect normal cells.

Keywords

Main Subjects

## 1. Introduction

The term "probiotic" was first used in 1965 by Lilly and Stillwell to describe substances secreted by one organism which stimulate the growth of another. Probiotics encourage the multiplication of other bacteria, often known as the normal flora, or types of beneficial bacteria that are cultivated in the laboratory and kept alive in special packages to reach millions or billions of bacterial cells in a single dose. Probiotics were discovered in 1953 and have been particularly effective in treating gastrointestinal diseases ( 1 ). Probiotics are completely absorbing foods and preventing inflammatory bowel diseases, colon cancer, and lactose intolerance, especially in infants, reducing flatulence, treating constipation and colic, as well as nutritional deficiencies by producing several vitamins in the gastrointestinal tract, reducing fungal infections in the vagina, especially those caused by C. albicans, which endanger the health of pregnant women. Lactobacillus is one of the most important species of bacteria used in manufacturing probiotics ( 2 ).

Lactobacillus spp. are on-spore-forming, rod-shaped, facultative anaerobic, catalase-negative, (Gram +ve) which grow better under microaerophilic conditions. Their Gram stain morphology can vary, including short, plump rods, long, thin rods, in chains or palisades. Also, their colonial morphology ranges from small to larger gray colonies with alpha hemolysis on blood agar. Lactobacillus can also be grown on other media, such as MRS (Man, Rogosa, and Sharpe) agar, where they appear as white, mucoid colonies. Lactobacillus consists of about 170 species and 17 subspecies, all of which have been formally described and have a solid nomenclatural standing. They are naturally found in the gastrointestinal system and vaginal canal in humans, however can also be opportunistic pathogens ( 3 ).

Silver metal has been known since 4000 B.C. which was used in many medical uses, even before it was realized that microorganisms are the main cause of infection ( 4 ). Also, producing nanoscale silver became possible with the emergence of nanotechnology ( 5 , 6 ). Silver nanoparticles have been used against reactive oxygen species (ROS), which gave excellent results. Antioxidants are known to have many benefits in scavenging free radicals and eliminating many cardiovascular and cancers of the body ( 7 ). Silver nanoparticles have also been used in various types of cancer lines, and it has been observed that tumor progression and the disease are inhibited by them without causing toxicity to normal cells ( 8 ). Silver nanoparticles can be synthesized using traditional or unconventional methods, with two different approaches of top-down and down-top. Many traditional methods are used to obtain silver nanoparticles, such as chemical/photochemical reactions, thermal decomposition of various silver compounds, electrochemical, radiation, and microwave-assisted methods ( 9 ). Unconventional methods of creating these particles depend on using microorganisms such as bacteria, fungi, marine algae, and yeasts or various extracts of alcoholic or aqueous plants, as they are considered as reducing or inhibitory agents ( 10 ). Green synthesis methods are the best due to their many advantages in preparing silver nanoparticles: they are low cost, environment friendly, and do not require high pressure, energy, or the use of chemical reagents ( 11 ). The present study aims to perform the biosynthesis of AgNPs using L. gasseri bacterial filtrate with silver nitrate solution and to investigate the cytotoxicity of silver nanoparticles on cancer cells.

## 2. Material and Methods

### 2.1. Collection of Bacterial Isolates

Twenty isolates for Lactobacillus sp. were diagnosed with a VITEK device from the Microbiology Laboratory at Al-Olwiya Teaching Hospital for Children in Baghdad. The isolates then were planted on the surface of the slanted bed medium formed from De Man, Rogosa, Sharpe agar (MRS) agar, and then incubated and kept at 4°C until use.

### 2.2. Morphological and Microscopic Examination

Isolates were grown on MRS agar to determine the color and size of colonies and then stained with Gram stain.

### 2.3. Maintenance of Cell Cultures

SK-GT-4 cells were maintained in RPMI-1640 supplemented with 10% Fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were passaged using Trypsin-EDTA reseeded at 80% confluence twice a week and incubated at 37 °C ( 12 ).

### 2.4. Biosynthesis and Purification of Silver Nanoparticles Using L. gasseri Bacteria

The bacteria were seeded in an MRS agar medium for 48 hours, and then the growth medium was discarded by a 4000 rpm centrifuge for 10 minutes. The precipitate was removed and the filtrate sterilized with filters of 0.22 µm pore and preserved until use ( 13 ). Silver nanoparticles were synthesized using a filtrate of L.gasseri bacteria; 10 ml of culture filtrate was mixed with 90 ml of 5 mM silver nitrate solution and incubated at room temperature for 48 hours. The synthesized silver nanoparticles were discovered in the reaction mixture by detecting the color alteration of the medium from pale yellow to brown as well as observed optical density ( 13 ). The aqueous solution containing AgNPs was placed in test tubes for purifying AgNPs and then centrifuged at 10,000 rpm, for 10 minutes. One minute for 10 minutes and the process was repeated 3 times until the filtrate became colorless, and the concentrated precipitate containing silver nanoparticles was placed in an hour bottle and dried in an electric oven at 50° C (the thermal drying method) so that excess water was removed and then the precipitate was collected after drying and preserving until use ( 14 ).

### 2.5. Characterization of Silver Nanoparticles

Take 1 ml of a solution containing silver nanoparticles and add 9 ml of deionized water. The aqueous solution was then measured to reveal the nanoparticles formed at a wavelength ranging from 300 to 600 nm. The highest absorption (λ max) appeared after the wavelength of 400 nm, indicating the formation of silver nanoparticles ( 15 ).

FTIR was used to characterize nanoparticles. The bio-produced silver solution was mixed with potassium bromide at a ratio of 1: 100 and then tested using FTIR Infrared Spectrometer within the range of 4000-400 cm-1 ( 15 ).

A scanning electron microscope (FE-SEM) was used to determine the particles size and morphology of AgNPs. The sample was dispersed on a smooth surface of carbon base and then coated with a thin layer of gold and subjected to examination. EDX equipped included with FE-SEM was used to determine the chemical elements of AgNPs.

### 2.6. Antioxidant Activity of AgNPs

Antioxidant activity of AgNPs was measured using stable 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radicals with minor adjustments according to ( 16 , 17 ). Silver nanoparticles were used in concentrations (6.25µg/ml, 12.5 µg/ml, 25 µg/ml, 25 µg/ml, 100 µg/ml) to investigate the scavenging activity. The samples were mixed with 450 µl of DPPH solution and then the volume of the mixture was completed to 1 ml using absolute ethanol. Ascorbic acid was used as a positive control at a concentration of 100 µg/ml. The samples and control are kept in dark at room temperature for 30 minutes. The absorbance was measured at 517 nm. Scavenging activity was measured according to the equation formula:

$\mathrm{Scavengingi}%=\frac{\mathrm{Absorbanceiofi control}-\mathrm{Absorbanceiofi sample}}{\mathrm{Absorbanceiofi control}}\mathrm{Xi}100%$

### 2.7. Cytotoxicity Assays of AgNPs

MTT assay was performed using 96-well plates to determine the cytotoxic effect of AgNPs ( 18 ). Cell lines were seeded at 1 × 104 cells/well. After 24 h the attached monolayer was obtained. Furthermore, the cells were treated with the tested compounds at different concentrations. Cell viability was measured after 72 h of treatment by removing the medium, adding 28 µL of 2 mg/mL solution of MTT, and incubating the cells for 2.5 h at 37 °C. After removing the MTT solution, the crystals remaining in the wells were solubilized by adding 130 µL of DMSO (Dimethyl Sulphoxide) and then incubating at 37 ° C for 15 minutes by shaking ( 19 ). The absorbency was determined on a microplate reader at 492 nm; the assay was performed in triplicate. The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as the following equation ( 20 ).

Inhibition rate = A- B/A*100; where A is the optical density of control, and B is that of the samples ( 21 ).

The cell was seeded into 24-well micro-titration plates at a density of 1×105 cells mL−1 and incubated for 24 h at 37 °C to visualize the shape of the cells under an inverted microscope. Then, cells were exposed to AgNPs at IC50 for 24 h after which the plates were stained with crystal violet and incubated at 37 °C for 10–15 min ( 22 ). The stain was washed off gently with tap water until the dye was completely removed. The cells were observed under an inverted microscope at 40× magnification and the images were captured with a digital camera attached to the microscope ( 23 ).

### 2.8. Study Effect of Silver Nanoparticles on the Chromosomal Pattern

Transplantation was performed to obtain chromosomes according to the method mentioned in ( 24 ) by adding 0.5 ml of peripheral blood to 4.5 ml of complete culture medium (RPMI-1640) prepared with 10 µg/ml of phytohemagglutinin (PHA) stimulator and two concentrations. Then two different concentrations of silver nanoparticles were biologically prepared using L.gasseri filtrate (0.0, 27, 54) µg/ml, and a positive control tube containing 0.65 µg/ml of methotrexate (MTX) was counted as a positive control. The tubes were incubated tilted at 37 °C for 70 hours, and colchicine solution was added at a final concentration of 10 μg/ml. The tubes were then returned to the incubator at 37 °C for 2 hours, after which the implant was centrifuged at a speed of 2000 rpm for 10 minutes and the filtrate was neglected. The precipitate was suspended with 5 ml of hypotonic calcium chloride solution, which was gradually added by continuous shaking. Then the tubes were returned to the incubator for another 45 minutes to detonate blood cells in the (KCL) solution, and swelling of lymphocytes preparesto create its chromosomes. Then the contents of the tubes were centrifuged at 2000 rpm for 10 minutes to remove the filtrate and suspend the precipitate with a cold fixative solution which was immediately prepared by mixing three parts of absolute methanol with one part of glacial acetic acid. This solution is in the form of drops on the wall of the tube with continuous shaking to reach a volume of approximately 5 ml, after which the centrifugation process is carried out at a speed of 2000 rpm. This process was repeated several times to decolorize the solution, then the precipitated cells were suspended with (1.5 - 1 ml) of the fixative solution, mixed by a clean, dry Pasteur pipette, ready for installation on cool, wet slides. A certain size of the prepared cells was taken from above and 7 spaced drops were poured on the glass slide at a height of about 30 cm to obtain a good spread of chromosomes for easy observation of chromosomal changes.

The slides were completely air-dried in the previous paragraph, and they were stained with Giemsa stain for two minutes, then washed with distilled water, during which Blast Index (BI) and Mitotic Index (MI) were calculated according to the following equations.

$\mathrm{MI}=\frac{\mathrm{Mitotic cells}}{1000\mathrm{cells}}×100%$

$\mathrm{BI}=\frac{\mathrm{Stimulated cells}}{1000\mathrm{cells}}×100%$

Chromosomal aberrations= summation of total abnormalities in 25 mitotic cells.

### 2.9. Statistical Analysis

The obtained data were statically analyzed using an unpaired t-test with GraphPad Prism 6. The values were presented as the mean ± SD of triplicate measurements ( 25 ).

## 3. Results

### 3.1. Morphological and microscopic examination

L.gasseri bacteria grew on De Man, Rogosa, Sharpe agar (MRS) by streaking method and incubated for 24 hours at 37 ° C. and the results are as shown in (Figure 1A). Microscopic examination was performed using a Gram stain and the cells were examined with a light microscope to identify the shape, color, and size of the cells. The results are shown in (Figure 1B).

### 3.2. Biosynthesis of Silver Nanoparticle

The results of the biosynthesis process using L.gasseri bacteria filtrate with AgNO3 solution at a concentration of 5 mmol, at pH of 6.5 and room temperature of 37°C for 48 hours were shown on the synthesis of AgNPs, and the change in color of the mixture to brown was evidence of a positive synthesis process (Figure 2A and Figure 2B).

### 3.3. Characterization of AgNPs Using UV-Visible Spectrophotometer

The characterization of the silver nanoparticles created using a UV visible spectrophotometer showed the highest absorbance (λmax) at the wavelength of 424 nm (Figure 3).

### 3.4. Characterization of AgNPs Using Fourier

Transform Infrared (FTIR) Spectrometer

The samples were examined by FTIR device. The spectral scanning of the samples was conducted within the range of (400-4000) cm-1. The spectral absorption results of silver nanoparticles showed the presence of bands at the range of (3368.79), (2958.69), (1632.3), (1399.38) and (1060.21) on the presence of the O–H, C–H, C=O, C–H and C–N bonds, respectively, which is consistent with the results of ( 26 , 27 ) as shown in (Figure 4).

FTIR assay is one of the precise laboratory tests used to identify the chemical elements in the compounds, in which the identity of the chemical compounds is determined based on how chemical bonds in the compounds absorb infrared radiation, as each compound has its absorption ( 28 ).

### 3.5. Characterization of AgNPs Using FE-SEM Analysis

The FE-SEM analysis was conducted to evaluate the size, surface morphology, and uniformity of nanoparticles. It is a technique used for obtaining qualitative and quantitative information and provides further approaches to the morphology and size of the nanoparticles ( 29 ). Most AgNPs displayed a more obvious structural arrangement, having spherical and smooth surfaces with an observation of particles agglomeration and aggregation with a size range between (58.06 to72.91) nm, as shown in figure 5A and figure 5B.

EDX results showed that silver atoms contain 71.3 % of the total sample components. Also, a small percentage of Cl, O, C, S, Si noted that the peak of Au is present due to coating sample with gold in FE-SEM, as shown in figure 6.

### 3.6. Antioxidant Activity Using DPPH Radicals Scavenging Assay

DPPH is a free radical that is stable at room temperature, produces a dark violet color when dissolved

in organic solvents, and is responsible for absorbance at the wavelength of 517 nm. DPPH reduces and turns yellow when silver nanoparticles are present due to the presence of a phenolic hydroxyl group ( 30 ). Figure 7 shows the antioxidant activity of silver nanoparticles using different concentrations, and the results of table 1 showed that the antioxidant activity of silver nanoparticles at a concentration of 100 μg/ml and 6.25 μg/ml were 85.3% and 19.3%, respectively.

Silver nanoparticles
Concentration μg/ml Antioxidant Activity %
6.25 19.3
12.5 32.6
25 47.6
50 72
100 85.3
Table 1. Antioxidant activity using DPPH assay.

### 3.7. Cytotoxicity Effect of Silver Nanoparticles against Cancer Cell Line

The cytotoxic effect of AgNPs against SK-GT-4 cells was studied. The antitumor activity of the AgNPs was tested by studying their ability to inhibit the proliferation of tested cells. The results of this study showed highly significant cytotoxic activity of AgNPs against the cancer cell line, however, not normal cell lines as shown in figure 8, figure 9 and figure 10. The results suggest that AgNPs suppress the growth of cell lines with a concentration-dependent effect (Table 2).

Concentration μg/ml Mean±SEM
6.25 2.33 ±0.882
12.5 11 ±1.527
25 30.66 ±2.404
50 52 ±2.886
100 79 ±2.309
Standard Error Mean = SEM
Table 2. The toxic activity of AgNPs against SK-GT-4 cells at different concentrations

### 3.8. Effect of Silver Nanoparticles on the Chromosomal Pattern

Table 3 shows that the effect of chromosomal aberrations (MI) is inversely proportional to the increase in AgNPs concentrations, which leads to chromosomal abnormalities through the appearance of diploid and ring chromosomes after treatment with concentrations of silver nanoparticles. The decrease in the indicators of MI and BI occurred as a result of cell death or stopping at interphase and the effect of silver nanoparticles on its division.

Silver nanoparticles
Concentration µg/mL BI MI TCA
0.0 66.22 1.25 0.24
27 62.12 1.12 0.29
54 60.51 0.92 0.31
MTX0.65 16.55 0.11 0.52
BI=Blast Index, MI= Mitotic Index, TCA= Total Chromosomal Aberrations
Table 3. Chromosomal aberrations induced in peripheral blood lymphocytes (PBLs) treated with AgNPs

## 4. Discussion

In the biosynthesis of silver nanoparticles, discoloration of the solution explains the formation of the silver nanoparticles by existing enzymes, such as the nitrate reductase, which the bacteria secrete from the cell and reduces silver nitrate to nanoparticles ( 31 ).

The characterization of AgNPs using a UV visible spectrophotometer is one of the most important tests used to verify the formation of silver nanoparticles which depends on the extent of the optical absorption between wavelengths (300-900) nm to investigate the size of the nanoparticles, which ranges between (2-100) nm, as metals of nanoscale have free electrons which give (SPR) depending on their size, which results from the vibrations of the metal's electrons compared to light waves. By showing (SPR) after the wavelength of 400 nanometers in the process of synthesizing silver nanoparticles using L.gasseri filtrate, the results were in line with ( 32 ).

The antioxidant activity of silver nanoparticles revealed that this effect increases with increasing concentration but remains lower compared to ascorbic acid, this agrees with ( 33 ). Plant-mediated AgNPs production has also been investigated, and researchers have shown that antioxidant activity gradually increases by increasing therapeutic doses ( 34 ). Antioxidants are beneficial in the treatment of illnesses such as neurological disorders, cancers, and AIDS due to their scavenging capability ( 35 ).

The unique ability of silver nanoparticles to kill cancer cells has been proven by targeting the structure and function of mitochondria, in which silver accumulates and affects the respiratory chain of the cell, causing its programmed death (Apoptosis) ( 36 ).

Some studies have shown that small silver nanoparticles enter the nucleus, affecting its chromatin and compressing the cell, which leads to cell shrinkage and death ( 37 ). Several studies indicate that the toxic effect on cancer cells results from the active Physico-chemical interaction of silver atoms with cellular proteins for functional groups in cells, as well as with nitrogenous bases and phosphate groups in DNA, which causes cell death induced by DNA damage ( 38 ).

It has been observed that the toxicity of cancer cells is greater than that of normal uninfected cells. Therefore, using silver nanoparticles is a typical therapeutic strategy ( 39 ).

Decreased MI and BI indices occur as a result of cell death or cessation at an interphase stage and the effect of silver nanoparticles on its division, which may be due to the effect of the toxin that binds to cell receptors located in the plasma membrane and leads to the sensitization process and responds to these substances and stimulates the systems responsible for the detoxification process ( 40 ). The effect of any cleaved material is occurred by activating the genetic material of the cell, as growth genes are activated to command the entry into the mitotic cycle and within a specific period that depends on the cell type and the efficiency of the stimulating material in the presence of silver nanoparticles, the cell will not be able to go through its four phases during the normal division period and may have exceeded the first mitotic cycle (M1), however, will not be able to pass in the second and third ones ( 41 ).

## 5. Conclusion

The present study demonstrated the synthesis of silver nanoparticles using locally L. gasseri. The synthesized silver nanoparticles were characterized by using UV-visible spectrophotometer, FE-SEM, and Fourier Transform Infrared Measurement (FTIR). The antioxidant activity of AgNPs was observed. Silver nanoparticles exhibited cytotoxic activity against SK-GT-4 cancer cells and do not affect normal cells.

## Authors' Contribution

Study concept and design: R. A. J.

Acquisition of data: N. N. H.

Analysis and interpretation of data: R. A. J.

Drafting of the manuscript: R. A. J.

Critical revision of the manuscript for important intellectual content: R. A. J. and N. N. H.

Statistical analysis: N. N. H.

Administrative, technical, and material support: N. N. H.

## Conflict of Interest

The authors declare that they have no conflict of interest.

## Grant Support

No funding has been provided by the institution and the authors are responsible for their financial support.

## Acknowledgement

The authors would like to extend their sincere appreciation to the Department of Applied Science Laboratories, University of Technology, Baghdad, Iraq for their experimental assistance.

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