Mycosynthesis of Silver Nanoparticles by Candida albicans Yeast and its Biological Applications

1. Introduction

Nanotechnology is a new domain of science that agrees with the synthesis and application of nanoparticles (NPs). Their size ranged from 1 to 100 nanometers. NPs have been studied extensively due to their special physicochemical features, including antibacterial properties, catalytic activity, anticancer properties, electronic properties, and antioxidant characteristics ( 1 ). Using multiple physical, chemical, and biological processes, various metals such as silver (Ag), gold (Au), zinc (Zn), and copper (Cu) have been converted to the nano formula for a wide range of useful uses. Biosynthesis of NPs can be performed using organisms, such as bacteria, fungi, and plants or their products, which act as reducing and stabilizing agents. Biosynthesis is relatively simple, clean, sustainable, and economical that provides excellent biocompatibility in NPs. The fungi, primarily yeast, represent a suitable option for large-scale green nano production. The mechanism of biosynthesis of NPs by using fungi may be intracellular or extracellular. Several studies demonstrated the biosynthesis of extracellular AgNPs utilizing many fungal species. The second method is most widely used because the releasing of NPs from the cells is simpler and easier than the intracellular mechanism. Moreover, extracellular biosynthesis required no complex procedures for separation and purification steps. However, to reject fungal remnants and contaminants, the NPs dispersion must be filtered, which can be accomplished using simple filtration, membrane filtration, gel filtration, dialysis, and ultracentrifugation ( 2 - 4 ).

Many studies reported the fungi to represent a good source for the production of AgNPs with cytotoxic activity ( 5 - 7 ). Antioxidants are helpful in the treatment of hypertension and ischemic heart disease. Therefore, the interest in metal NPs in therapies to prevent oxidative damage has grown, and accordingly, they are considered to reduce the apoptotic pathway. The antioxidant properties of AgNPs make them useful in curing diseases ( 8 , 9 ). This study aimed to conduct an extracellular mycosynthesis of AgNPs by C. albicans supernatant and their biological applications.

2. Material and Methods

2.1. Collection and Identification of C. albicans Yeast

In total, 50 isolates of C. albicans were collected from the patients at Al-Elweya children's teaching hospital in Iraq. Following that, the isolates were diagnosed with Gram stain, microscopic examination, and VITIC2 system. Subsequently, the suitable strain of C. albicans was employed for the biosynthesis of AgNPs.

2.2 Preparation of C. albicans Supernatant

The fresh characterized colonies of C. albicans were cultured in 50 ml of Potato Dextrose Broth medium placed in a falcon tube. 250 ml can ensure aerated circumference for C. albicans growth through a clean and sterilized loop inside the hood. In the next stage, the culture was incubated for 48 h in a shaker incubator at 37°C and 150 rpm. After the incubation period, 4000 rpm centrifuge centrifuged the growth medium for 30 min, the sediment was removed, and the supernatant was filtrate by a syringe filter with a diameter of 0.22 µm, and the supernatant was stored at 4°C until it was used. This preparation method was used as previously described with some modifications ( 10 ).

2.3 Preparation of Silver Nitrate Solution

To prepare 100 ml of 2mM silver nitrate (AgNO₃) solution, 0.0339 grams of AgNO₃ was dissolved in 100 mL of deionized water. After preparation, the solution was kept in an opaque condition, away from light, to prevent oxidation, and it was used later ( 11 ).

2.4 Extracellular Mycosynthesis of Silver Nanoparticles through C. albicans Supernatant

10 ml of C. albicans supernatant was mixed with 90ml of 2mM silver nitrate solution in a falcon tube and incubated in a shaking incubator at 37°C and 150 rpm for 48 h in dark conditions. After 48 h, the color alteration of the medium should be noticed to detect the formation of AgNPs, which is the first indication of the synthesis of the silver nanoparticles. This mycosynthesis method was used as previously described with some modifications ( 12 ).

2.5 Purification of Mycosynthesized Silver Nanoparticles

The reaction mixture that contained AgNPs was put in a centrifuge at a speed of 4000 rpm for 30 min three times with Milli-Q water to reject the medium remains and other impurities. This procedure was recurred at least three times to assure a better separation of AgNPs. This purification method was used as previously described with some modifications ( 10 ).

2.6 Characterization of Mycosynthesized Silver Nanoparticles

After 48 h of incubation at a shaking incubator at 37°C and 150 rpm, the color change in the reaction mixture was monitored visually to detect the formation of AgNPs. In the next stage, AgNPs were characterized by UV-Vis spectrophotometer (UV-1650 PC SHIMADZU, Chrom Tech, USA) in the Department of Applied Sciences at the University of Technology in Iraq. Before the UV-Vis spectrophotometer examination, the AgNPs were sonicated for 20 min; moreover, the existence of the greatest absorbance (λmax) and the production of AgNPs were determined using UV-Vis spectra of AgNPs in the region of 200 to 800 nanometers, and deionized water was used as a baseline. X-Ray Diffraction (XRD- Panalytical X Pert Pro) analysis was used to determine the structural nature of AgNPs. A thin layer of AgNPs has been prepared on glass slides,and the sample was then examined at 40 kV and scanned at 2θ ranging from10° to 70°. The grain size of AgNPs was calculated using the Scherrer equation as below:

$D=\mathrm{K\; \lambda }/\beta \mathrm{Cos\theta }$

Where D represents crystallite size, K is the shape factor, λ signifies the X-ray wavelength, β indicates the line broadening at half the maximum intensity, and θ means the Bragg angle. Furthermore, mycosynthesized AgNPs were characterized by field emission scanning electron microscope (FESEM) and energy dispersive analysis of X-ray (EDX) analysis to identify the size, shape, and all the elements that exist in the sample of AgNPs. The sample was spread on a smooth surface of carbon ground and then was coated with a thin layer of gold and examined. Zeta potential analysis was done using Zeta plus analyzer to determine the stability of AgNPs, and the analysis depends on dynamic light scattering measurements assay. The analysis was performed at 25°C, and the dispersion was diluted to an appropriate volume with deionized water ( 13 - 15 ).

2.7 Cytotoxicity Effect of Silver Nanoparticles against HT-29 Cells

To determine the cytotoxic effect of AgNPs, the MTT assay was done using 96-well plates. HT-29 cells, a human colon cancer cell line (HT-29), were seeded at 1×104cells/well. The cells were treated with AgNPs at various concentrations after 24 h or when a confluent monolayer was achieved. After 72 h of treatment, the cell viability was determined by removing the medium adding 28 µL of a 2 mg/mL MTT solution and incubating the cells for 2.5 h at 37 °C. Following the removal of the MTT solution, the crystals in the wells were solubilized by adding 130 µL of DMSO (Dimethyl Sulphoxide) and incubating at 37 °C for 15 min with shaking. The absorbency was measured at 492 nm using a microplate reader, and the test was conducted in triplicate. The following equation was used to compute the rate of cell growth inhibition (the percentage of cytotoxicity) ( 16 ):

Inhibition rate = A- B/A*100

Where A is the optical density of control, and B signifies the optical density of the samples ( 17 ). The cells were seeded into 24-well micro-titration plates at a density of 1×10⁵ cells ml⁻¹ and cultured for 24 h at 37°C to examine their form under an inverted microscope. The cells were then treated for 24 h to AgNPs at IC₅₀. The plates were stained with crystal violet stain and incubated at 37°C for 10-15 min after the exposure period. The stain was carefully wiped away with tap water until all of the dye was gone. The cells were seen under a 40×magnification inverted microscope with photographs recorded using a digital camera mounted to the microscope ( 18 ).

2.8 Antioxidant Properties of Silver Nanoparticles

Antioxidant activity of NPs was measured using stable DPPH radicals with minor adjustments according to ( 19 ). The samples were combined with 450 µL of DPPH solution, and 100% ethanol was added to bring the total volume of the combination to one ml. At a concentration of 10 µg/ml, ascorbic acid served as a positive control. For 30 min, both samples and the control were kept in the dark at room temperature. 517 nm was used to determine absorbance. Scavenging activity was calculated using the following equation formula ( 19 ):

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

2.9. Effect of Silver Nanoparticles on the Chromosomal Pattern

2.9.1 Blood Specimen

Totally, five milliliters of venous blood were taken from a 25-year-old male who was not exposed to any type of chemical and physical pollution (vocational exposure) and had no genetic diseases (cancers or congenital distortion), no smoking history, and no consumption of any kind of medications for the treatment of chronic and acute diseases during the sampling period to be ensured that his blood sample was a dependable source of chromosomes in our current study.

2.9.2 Chromosomes Preparation

The preparation technique was followed as described by AbbasHaleem, Abbas ( 20 ) by adding 0.5 ml of peripheral blood to 4.5 ml of whole culture medium (RPMI-1640) equipped with 10 µg/ml of phytohemagglutinin that acted as a stimulator, two different concentrations of AgNPs that prepared biologically by C. albicans supernatant (0.0, 25, 50) µg/ml, and a test tube containing 0.65 µg/ml of methotrexate as a positive control. The tubes were placed in an incubator in the slanted form at 37°C for 70 h, 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 h, after that, the implant was placed in the centrifuge at 2000 rpm speed for 10 min. The precipitate was suspended in 5 mL of hypotonic calcium chloride solution after the supernatant was discarded, which was gradually added with continuous shaking. Subsequently, the tubes were returned to the incubator for another 45 min to remove the red blood cells by exploding them in the potassium chloride solution and swelling of lymphocytes to be ready for the chromosomes preparation.

The tubes were then placed in the centrifuge at a speed of 2000 rpm for 10 min to remove the supernatant, and the sediment was suspended in a cold fixative solution prepared immediately by mixing three parts of absolute methanol with one part of glacial acetic acid. This solution was added as a drop on the wall of the tube with continuous shaking till a volume of approximately 5 ml was obtained. Afterward, the centrifugation process was carried out at a speed of 2000 rpm. This process was reiterated several times till the solution became colorless. The precipitated cells were suspended with 1.5-1 ml of the fixative solution, mixed by a clean and dry Pasteur pipette and got ready for installation on cool and wet slides. A certain size of the prepared cells was taken from above, and at the height of about 30 cm, seven spaced drops were dropped on the glass slide to obtain a good spreading of the chromosomes for the easy observation of chromosomal changes.

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

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

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

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

2.10. Statistical Analysis

The data were analyzed in SPSS software (version 24) through the two-way analysis of variance.

3. Results

3.1 Identification of C. albicans Isolates

The colonies of C. albicans isolates seemed large, round, raised, smooth, glabrous, and white to cream-colored on Sabouraud dextrose agar medium (Figure1). The identification of the isolates was made according to ( 21 ). Under microscopic examination with an oil immersion lens and crystal violet stain, the yeast cells exhibited a blue to a purple color indicating that they could retain crystal violet stain as observed in (Figure 2). These microscopic morphological features were recognized according to ( 22 ). Furthermore, the isolates of C. albicans were diagnosed by the VITIC system.

Figure 1.C. albicans colonies on Sabouraud Dextrose Agarmedium

Figure 2.C. albicans with Gram stains under a microscope

Extracellular Mycosynthesis of Silver Nanoparticles

After 48 h of incubation in a shaking incubator in dark conditions at 37°C and 150 rpm, the color change in the reaction mixture was changed from pale yellow to brownish color as shown in figure 3 (A and B).

Figure 3. Extracellular mycosynthesis of silver nanoparticles using the supernatant of C. albicans(A): before mycosynthesis, (B): after 48 h at 37°C in dark conditions

3.3. Characterization of Silver Nanoparticles

3.3.1. UV-Vis Spectroscopy

The formed AgNPs by C. albicans were further specified by UV-vis spectroscopy. The appearance of the highest absorbance (λmax) at the wavelength of 429 nanometers indicated the creation of AgNPs.

3.3.2 X-Ray Diffraction

XRD showed two peaks at 2θ related to Ag nanoparticles, (38.47 o and 44.47o) corresponding to (111) and (200) planes, respectively, when comparing with JCPDS File no. 04-0783 as shown in figure 4.

Figure 4. Characterization of silver nanoparticles using X-Ray Diffraction

3.3.3 Energy Dispersive Analysis of X-ray

EDX analysis for AgNPs showed the weight percentage of silver in the sample (82.4%). The presence of a 3eV optical absorbance band implies the existence of AgNPs. At the same time, other signals of Au, C, O, and Cl atoms appear in the graph of EDX as shown in figure 5.

Figure 5. Characterization of silver nanoparticles using the energy dispersive analysis of X-ray analysis

3.3.4 Field Emission Scanning Electron Microscope

The shape and the size of mycosynthesized AgNPs were characterized through FESEM micrographs, and as can be observed in figure 6 (A, B, and C), the morphology of AgNPs was spherical, and its size was 40.19 nm.

Figure 6. Characterization of silver nanoparticles using the field emission scanning electron microscope analysis at different magnificationsA: at 1µm, B: at 200 nm, and C: at 200 nm with a different nanoscale bar

3.3.5 Zeta Potential

The Zeta potential of mycosynthesized AgNPs was 23.02 mV with a single peak indicating repulsion among the mycosynthesized AgNPs as can be observed in figure 7.

Figure 7. Zeta potential analysis of silver nanoparticles

3.4 Cytotoxicity Effect of Mycosynthesized Silver Nanoparticles against HT-29 Cells

HT-29 cells were used to test the cytotoxicity of mycosynthesized AgNPs. The ability of AgNPs to decrease the proliferation of test cells was used to determine their anti-cancer efficacy. As shown in figures 8 and 9 (A and B), mycosynthesized AgNPs had a highly substantial cytotoxic action against cancer cell lines but not against normal cell lines.

Figure 8. Cytotoxic effect of nanoparticles against HT-29 cells; IC₅₀= 24.10 µg/ml

Figure 9.(A) Control untreated HT-29 cells; (B) Morphological changes in HT-29 cell after treated with silver nanoparticles

3.5. Antioxidant Properties of Silver Nanoparticles

The DPPH method is an agreeable, fast, and easy method for counting the free radical scavenging activity. Figure10 shows the mycosynthesized AgNPs that have antioxidant properties. This property is increased by increasing the concentrations of silver nanoparticles. The maximum antioxidant activity was 83.6% at the concentration of 100 μg/ml, while the lowest antioxidant activity was 17.0% at the concentration of 6.25 μg/ml (Table 1).

Figure 10. Antioxidant activity of nanoparticles using DPPH assay and the ascorbic acid act as the positive control

3.6 Chromosomal Analysis

Table 2 shows that the total chromosomal abnormality (TCA) is directly proportional to an increase in the concentration of AgNPs. The decrease in the BI and MI indicators was the result of cell death or cessation at some stages of interphase and the effect of AgNPs on their cleavage.

4. Discussion

The color alteration in the mycosynthesis of AgNPs by C. albicans was the first indicator of the formation of AgNPs because of the reduction of Ag ions and Plasmon resonance. These findings are consistent with those of a prior study conducted by Mourad Magdi, Mourad ( 23 ). In the characterization of AgNPs using a UV-visspectrophotometer, the appearance of λ max at the wavelength of 429 nanometers indicated the formation of AgNPs, which was because of the phenomenon of the surface Plasmon resonance that occurred due to the excitation of the surface Plasmon present on the outer surface of the AgNPs. This got excited due to the applied electromagnetic field. These findings are quite similar to the results of a study conducted by Bhat and NayakBhat, Nayak ( 10 ) who discovered that AgNPs biosynthesized by C. albicans had an absorption peak at 430 nm. The appearance of two peaks at 2θ (38.47o and 44.47o) in the XRD analysis are related to AgNPs and the crystalline nature of mycosynthesized AgNPs. The crystallite size was58.5 nm using the Scherrer equation. These findings are consistent with the results of a study performed by Abdehgah, Khodavandi ( 24 ) who discovered that C. albicans biosynthesizing AgNPs had a crystalline form. The benefit of determining the size of nanoparticles depends on their effectiveness and behavior as antibacterial. As it is known, nanoparticles increase their effectiveness at smaller sizes.

In the spectrum of EDX analysis, signals of Au, C, O, and Cl atoms may have been originated from the biomolecules found on the surface of the AgNPs and the appearance of Au in the EDX analysis of AgNPs due to the presence of substrate over which the NPs sample was held during FESEM microscopy. These results are in line with the findings of the previous studies ( 25 , 26 ). This weight percentage is close to that ina study carried out by Dhabalia, Ukkund ( 27 ) who reported the weight percentage of AgNPs as85.7%. The spherical form of mycosynthesized AgNPs was revealed by FESEM, and their size was 40.19 nm.

This is less than the average size of AgNPs biosynthesized by C. albicans as reported by Bhat, Nayak ( 10 ). They discovered that the average size of AgNPs biosynthesized by C. albicans was between 60.88 and 65.57 nm in their study. The shape and the size of mycosynthesized AgNPs that were gained from this study were in agreement with those in the study carried out by Dhabalia and Ukkun Dhabalia, Ukkund ( 27 ). They reported the average size of AgNPs that were biosynthesized by C. albicansas 30-70 nm with a spherical shape. Moreover, these results are consistent with the findings of the prior studies that used other fungi ( 28 , 29 ). The negative Zeta potential of mycosynthesized AgNPs was discovered indicating dissonance among green produced AgNPs and raising the stability of the compound. Because of the moderate negative Zeta potential value of AgNPs, it is obvious that they are intermediate poly distributed in the sample, and the electrostatic repulsive force between them leads to a poor inhibition of nanoparticle clotting. In addition, AgNPs exhibit a state of primary instability in the solution. These results are consistent with the findings in the earlier studies conducted byNiknejad, Nabili ( 30 ), Kim, Kuk ( 31 ), as well as Elamawi, Al-Harbi ( 32 ).

Bio-manufactured AgNPs have been used on various types of cancerous cells, such as skin cancer cells. It has been observed that tumor progression is inhibited by AgNPs and controls the progression of the disease without causing toxicity to normal cells. This study finds that AgNPs have the ability to suppress the growth of HT-29 cell lines, and this effect is concentration-dependent manner. The IC₅₀was 24.10 µg/ml. These results are consistent with the findings of the earlier studies conducted by Ismail, Ahmed ( 33 ), as well as Yehia and Al-Sheikh ( 34 ).

Many cells produce reactive oxygen species (ROS), including single oxygen, irritating carbonyl (C=O), and hydrogen peroxide, which can damage cell components, such as proteins, lipids, and DNA resulting in cell death. The use of AgNPs to combat ROS yielded outstanding results. Antioxidants are well-known for their ability to scavenge free radicals and protect the body from a variety of ailments, including heart disease and cancer. The use of AgNPs to combat ROS yielded novel outcomes ( 8 ). The mycosynthesized AgNPs by C. albicans show maximum antioxidant activity (83.6%) at the concentration of 100 μg/ml. This is in line with the findings of other studies conducted by Menon, Agarwal ( 35 ), as well as Vorobyova, Vasyliev ( 36 ).

The study finds that AgNPs affect chromosomal patterns through an increase in the TCA index, and the reason for this may be the effect of the toxin that binds to cell receptors located in the plasma membrane and leads to the process of sensing and contributing to the response process to these substances and stimulating the systems responsible for the process of toxicity machine. The effect of any cleaved material is by activating the genetic material of the cell since the genes responsible for growth are activated to give orders to enter the mitotic cycle within a specific period that depends on the cell type and the efficiency of the stimulating material in the presence of AgNPs. The cell will not be able to go through its four phases during the normal period of division. It may have exceeded the first mitotic cycle; however, it will not be able to pass in the second and third ( 37 , 38 ).

5. Conclusion

The findings of this study reveal that mycosynthesized AgNPs can be made in a simple, safe, cost-effective, and environmentally friendly way by employing yeast C. albicans, compared to other methods of preparing AgNPs, such as physical and chemical methods. AgNPs showed a cytotoxic effect on the HT-29 using the MTT assay, and no cytotoxic effect appeared on normal cells. Furthermore, AgNPs showed antioxidant activity using the DPPH assay. Because of their special characteristics of silver and nano size, mycosynthesized AgNPs look promising in pharmaceutical, biological, and other fields, providing that safety data is generated to verify their safety while excluding the possibility of toxicity.

Authors' Contribution

Study concept and design: R. A. H.

Acquisition of data: N. N. H.

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

Drafting of the manuscript: R. A. H.

Critical revision of the manuscript for important intellectual content: N. N. H.

Statistical analysis: N. N. H.

Administrative, technical, and material support: R. A. H.

Ethics

All procedures performed in this study involving human participants were in accordance with the ethical standards of the University of Technology, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

Grant Support

No funding support by our institution was provided. The authors are responsible for their funding support.

Acknowledgement

The authors are grateful to the Department of Applied Sciences Laboratories, University of Technology, Baghdad, Iraq, for providing experimental support.

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