Abstract
Increasing resistance of microbes to available antimicrobial agents has necessitated the production of new compounds with wider activity and lower toxicity. Candida is one of the pathogenic fungal strains that are highly prevalent in oral diseases. The goal of this study was to optimize the biosynthesis of selenium nanoparticles (NPs) with the suitable antifungal activity on Candida albicans oral pathogen. For this purpose, selenium NPs were synthesized by the green method using the Halomonas elongata bacterium. To optimally synthesize NPs with the maximal antifungal properties, 9 experiments were planned applying the Taguchi method. In the planned experiments, the efficacy of 3 factors, sodium selenite concentration, glucose concentration, and incubation time at 3 various levels was studied, and the most desirable conditions with the suitable performance were determined. Then, optimizing the synthesis of the studied nanoparticles, their properties were evaluated using Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), electron transmission microscopy (TEM), and thermogravimetric analysis (TGA) to confirm the synthesis of nanoparticles with suitable conditions. The results demonstrated that the synthesized NPs in the experiment 9 condition showed the best performance and prevented more than 70% of fungal growth. Since selenium NPs were effective as an antifungal agent, they can be used in the structure of mouthwashes as an antimicrobial agent in the prevention and treatment of many oral diseases.
1. Introduction
Antimicrobial resistance has become the biggest global health threat and it threatens people of all ages. The increasing resistance of various infections to common drugs has led to an increase in the number of victims [1–4].
Tooth decay is also an irreversible microbial disease. Approximately 30% of the world’s population has decays in their permanent teeth, which has made tooth decays one of the most common diseases in the world [5, 6]. The increase of microorganisms such as bacteria and fungi in the mouth and teeth is one of the most important reasons for the increase in the rate of tooth decay. Fungal infections have recently increased, and Candida albicans is the most important pathogenic fungus that causes disease in the mouth and teeth. An excessive increase in this pathogen penetrates the dentin and causes an infection in the tooth pulp, which eventually results in severe pain, tooth nerve death, tooth loss, and systemic infections [7, 8].
One of the most promising approaches of the current century to meet human needs is the use of nanotechnology [9, 10]. Nanoscale materials have unique properties, such as magnetic, optical, mechanical, and catalytic, and have special applications such as biomedical applications. In nanotechnology, a compound with a size between 1 and 100 nanometers is defined as a nanoparticle, which acts as a separate unit in terms of properties and motion [11, 12].
Recent developments in nanotechnology and nanoscience have focused mainly on the synthesis and development of metal NPs. The synthetic methods available for nanoparticles still face problems such as targeted optimization. Physical and chemical methods of nanoparticle synthesis use toxic and expensive chemicals that allow the absorption of toxic chemicals on the surface of nanoparticles [13]. The use of environmentally friendly biosynthesis methods does not have the problems of chemical and physical methods [14].
Microorganisms (bacteria, algae, fungi, and yeasts) and plants are the most important biological agents used to synthesize green nanoparticles [15–17]. The reason for using this type of synthesis is its ease and simplicity. In addition, this method does not produce any toxic and hazardous waste materials in the environment. The mechanism of biosynthesis of nanoparticles includes two cases, intracellular and extracellular, which will have different mechanisms of nanoparticle synthesis for different biological factors [18].
Selenium is one of the essential and effective elements in the functioning of the human body, which is very important. Selenium is used to synthesize stable selenium-containing organic compounds that have antioxidant, antitumor, immune system stabilizing, enzyme inhibitory, and antimicrobial applications [19]. The high bioactivity and low toxicity observed in selenium NPs have led many researchers to be interested in the synthesis of these nanoparticles in different ways. Recent research has shown that biosynthesized selenium NPs are less chemically toxic than bulk selenium particles. In the method of green synthesis, biomolecules play the role of reducing and stabilizing agents of selenium metal NPs [20].
Problems with candida biofilm infections are on the rise today. Therefore, finding new ways to combat the causes of such fungal infections seems necessary. The utilization of nanotechnology as novel human knowledge to make antifungal compounds with higher quality and greater productivity for prevention and treatment can be important. This study aimed to optimize the biosynthesis of selenium NPs and evaluate their antifungal activity on the Candida albicans oral pathogen.
2. Experimental
2.1. Synthesis of Selenium Nanoparticles
For the green synthesis of selenium NPs, Halomonas elongata (IBRC-M 10433) was prepared from the bacterial archive of the Iranian biological resource center. To prepare the bacterial culture medium, 0.2 g of glucose, 3 g of NaCl, 0.028 g of K2HPO4, and 0.0001 g of FeSO4 were combined to a container containing 100 ml of distilled water and stirring was applied on a magnetic stirrer to obtain a clear solution. The culture medium was sterilized in an autoclave and then an isolated bacterial colony was added to the Erlenmeyer flasks. Erlenmeyer flasks containing bacteria and culture medium were incubated at 37°C for 48 h. To isolate the bacteria from the culture medium, centrifugation was performed at 5000 rpm for 5 min and the supernatant from the precipitate was detached and used for the produce of nanoparticles [21]. Nine experiments were planned to optimize the synthesis conditions of selenium NPs by Taguchi method [22]. In the planned experiments, the efficacy of 3 factors of sodium selenite concentration (0.2, 0.4, and 0.8 mg/ml), glucose concentration (2.5, 5, and 7.5 mg/ml), and incubation time (24, 48, and 72 h) at 3 different levels were studied to determine the best situation that has the most suitable performance. According to the designed experimental conditions, the culture medium was prepared with different nutrient compositions by changing the amount of glucose. The prepared supernatant was then mixed in equal proportions with solutions comprising 0.2, 0.4, and 0.8 mg/ml sodium selenite. The obtained solutions were incubated at 30°C for 24, 48, and 72 h in an incubator shaker at 140 rpm. The nanoparticles produced by centrifugation were separated and purified at 5000 rpm for 15 min. The obtained solutions were then placed in an oven at 80°C for 24 h to provide nanoparticles for structural analysis [21].
2.2. Characterization
The FTIR spectrum of the selenium nanoparticles was provided in the range of 400–4000 cm−1 at room temperature. X-ray test was accomplished by PHILIPS’s PW1730 device in the range of 20 to 80°. SEM images of selenium NPs were provided to study their morphology and size applying TESCAN MIRA III field emission scanning electron microscope (FESEM). X-ray energy diffraction spectroscopy was used to investigate and determine the composition of the synthesized nanoparticles. For this purpose, after preparing the samples, using an EDX detector on field emission scanning electron microscope, the spectra of the constituent elements of the samples were prepared. Ultraviolet-visible spectroscopy of selenium NPs synthesized using Halomonas elongata was achieved by the device made by Thermo in the range of 200–800 nm. The thermal behavior of selenium NPs was investigated by thermogravimetric analysis (TGA) using device model Q600 made by TA company.
2.3. Antifungal Activity
The antifungal properties of selenium NPs on Candida albicans was examined by the colony forming unit (CFU) method. For achieving this goal, Candida albicans was incubated on a Sabouraud dextrose agar (SDA) medium at 28°C for 48 h to provide a fresh colony. Then, slightly of the colony was dissolved in deionized water to gain an almost concentration of 106 CFU/ml. Then, solutions containing Sabouraud dextrose agar and 9 synthesized nanoparticles (1 mg/ml) were provided. The obtained solutions were added into the plates and after freezing the culture medium, 100 μL of the fungal suspension prepared on the culture medium was completely cultured applying a swap. In the control group, 100 μL of fungal suspension was cultured on a pure Sabouraud dextrose agar medium. The number of colonies was counted for control and nine experimental groups after incubation at 28°C for 96 h, and all groups had three replications. The rate of fungal growth inhibition for each of the produced nanoparticles was determined by the presented equation:where is the mean growth of colonies in the control group and is the mean growth of colonies in the experimental group.
3. Results and Discussion
3.1. Antifungal Analysis
Some selenium compounds, such as selenium sulfide (SeS2), have long been used for antifungal purposes. However, few studies have been performed on the antifungal activity of selenium NPs against resistant fungi such as Candida albicans. For this purpose, in this study, the optimal conditions for the production of selenium NPs with the suitable antifungal properties based on 9 experiments planned by the Taguchi method and the efficacy of synthesized nanoparticles under various conditions on the growth inhibition of Candida albicans were investigated (Table 1). The results demonstrated that the nanoparticles fabricated under experimental conditions 9 (sodium selenite 0.8 mg/ml, glucose 7.5 mg/ml, and 48 h incubation time) had the best antifungal properties against Candida albicans, and under those conditions, the greatest inhibition of fungal growth (more than 70%) was obtained.
The effect of studied factors on the growth inhibition of Candida albicans is presented in Table 2. The results illustrated that glucose and sodium selenite at level 3 and incubation time at level 2 had the greatest efficacy on preventing the growth of Candida albicans.
Table 3 displays the interaction of studied factors on the growth inhibition of Candida albicans. Glucose at the third level and incubation time at the second level had the highest interaction with each other and on the growth inhibition of Candida albicans at 50.79. Sodium selenite and glucose in the third level exhibited a remarkable interaction on the growth inhibition of Candida albicans (42.65). The lowest interaction intensity index was observed between the third level of sodium selenite and the second level of incubation time (10.75).
Table 4 shows the analysis of variance of the factors affecting the growth inhibition of Candida albicans. The greatest efficacy on inhibiting the growth of Candida albicans was in sodium selenite with an efficacy of 29.95%, incubation time of 20.25%, and glucose of 10.07%, respectively.
After reviewing the data and analyzing the effect of each of the factors and their interaction, the optimal conditions for the produce of selenium NPs with the maximal antifungal properties were estimated (Table 5). According to the results, sodium selenite had the greatest role, glucose had the slightest role in the growth inhibition of Candida albicans, and the incubation time illustrated an efficacy between these two factors and near to sodium selenite. The third level was found to be the most suitable level for sodium selenite and glucose and the second level for incubation time.
Based on the findings, it was estimated that the produced nanoparticles in optimal conditions prevented about 72% of fungal growth.
The results of previous studies showed that the selenium element at the nanoscale has antimicrobial activity. Metal NPs have been developed as antimicrobial agents due to their unique properties such as high surface to volume ratio, specific surface area, quantum effects, increased surface reactivity, and their specific chemical and physical properties. Nanoparticles can kill resistant pathogens by reacting with the thiol (-SH) protein group, affecting membrane permeability, DNA damage, oxidative stress, mitochondrial membrane dysfunction, altered gene expression, and altered cell morphology [23, 24].
3.2. FTIR Analysis
The Fourier transform infrared spectroscopy of selenium NPs shows the different functional groups involved in the reduction process (Figure 1). The Fourier transform infrared spectroscopy of selenium n NPs has two main peaks, located at 3423 cm−1 and 1653 cm−1, respectively, related to the tensions of the hydroxyl groups. The peak observed in the range of 1290 cm−1 corresponds to the functional groups of C = O, −NH, and −NH2. The stability of selenium NPs is owing to the presence of these functional groups. These functional groups are also known as reducing agents by converting sodium selenite to elemental selenium [25].
3.3. XRD Analysis
The XRD spectrum prepared from selenium NPs is presented in Figure 2. The sharp and narrow peaks suggested the formation of well crystallized selenium NPs. The peaks centered at 2θ values of 23.5o, 29.2o, 41.4o, 43.3o, 45.4o, 52.5o, 55.7o, and 62.7o were related to the crystal planes of (100), (101), (110), (102), (111), (201), (112), and (202) of (JCPDS card No. 06–362) standard. In addition, the prepared spectrum indicates the polymorphism of synthesized selenium NPs [26].
3.4. SEM Analysis
Scanning electron microscopy images of selenium NPs confirmed the formation of these nanoparticles by bacteria. From the image shown in Figure 3(a), it can be seen that many of the nanoparticles formed are spherical. The results obtained from the histogram diagram for calculating the nanoparticle size distribution are given in Figure 3(b). The size of the synthesized nanoparticles was in the size range of less than 50 nm and NPs with a size of 11 nm having the highest frequency.
(a)
(b)
3.5. EDX Analysis
The presence of selenium was determined as nanoscale particles obtained from bacteria from the EDX spectrum. The EDX spectrum prepared from the synthesized selenium NPs is presented in Figure 4. In the spectrum of selenium oxide NPs, in addition to oxygen and selenium, carbon and silicon elements were also observed, which indicates the presence of some impurities due to the biological synthesis of nanoparticles. In previous studies, selenium NPs were produced using other types of bacteria and the presence of some impurities with nanoparticles was reported [27–29].
3.6. TEM Analysis
Figure 5(a) shows the TEM image made of synthesized selenium NPs. The image showed that the synthesized NPs have a spherical shape and size suitable for use in various fields. Evaluation of particle size by TEM showed that the dispersion of synthesized NPs was mostly in the range of 5 to 25 nm and the highest frequency was observed in the range of 5 to 10 nm (Figure 5(b)). Nanoparticles with small sizes have better penetration into the cell structure and can cause dysfunction and the death of pathogens.
(a)
(b)
3.7. UV-Vis Analysis
The optical properties of selenium NPs were determined using ultraviolet absorption spectroscopy (Figure 6). The prepared spectrum provided an absorption peak at 267 nm, possibly due to the presence of aromatic amino acids. Therefore, this absorption peak indicates the possible adhesion of protein materials on the surface of selenium NPs [30, 31].
3.8. Thermal Analysis
Thermal analysis of the synthesized selenium NPs was performed using TGA analysis and is shown in Figure 7. The first stage of weight loss of selenium NPs was observed in the range of 100°C, which can be due to the removal of volatiles and moisture on the surface of the nanoparticles. The second stage, between 100–480°C, showed a significant weight loss due to the widespread degradation of selenium NPs. In addition, thermal degradation up to 800°C was observed for selenium NPs [32].
4. Conclusions
The results showed that biologically synthesized selenium NPs using Halomonas elongata bacterium according to 9 experiments designed by Taguchi method had desirable antifungal properties against Candida albicans. The favorable structural characterizations of the synthesized nanoparticles were proved by diverse analysis methods. In addition, according to the results, it was predicted that the synthesized NPs in optimal situation stopped the growth of Candida albicans up to 72%. Selenium NPs have suitable antifungal properties against Candida albicans and can be effective in combating fungal diseases.
Data Availability
The data supporting the findings of this study are all included within the article.
Conflicts of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors gratefully acknowledge the Research Council of Kermanshah University of Medical Sciences (grant number: 980674) for the financial support.