World J Microbiol Biotechnol DOI 10.1007/s11274-014-1703-3
ORIGINAL PAPER
Biosynthesis and characterization of silver nanoparticles produced by Pleurotus ostreatus and their anticandidal and anticancer activities Ramy S. Yehia • Hashem Al-Sheikh
Received: 23 January 2014 / Accepted: 14 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The biosynthesis of nanoparticles has received increasing interest because of the growing need to develop safe, cost-effective and environmentally friendly technologies for the synthesis of nano-materials. In this study, silver nanoparticles (AgNPs) were synthesized using a reduction of aqueous Ag? ions with culture supernatant from Pleurotus ostreatus. The bioreduction of AgNPs was monitored by ultra violet-visible spectroscopy and the obtained AgNPs were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy techniques. TEM studies showed the size of the AgNPs to be in the range of 4–15 nm. The formation of AgNPs might be an enzyme-mediated extracellular reaction process. Furthermore, the antifungal effect of AgNPs against Candida albicans as compared with commercially antifungal drugs was examined. The effect of AgNPs on dimorphic transition of C. albicans was tested. The anticancer properties of AgNPs against cells (MCF-7) were also evaluated. AgNPs caused a significant decrease in cell viability of an MCF-7 cell line (breast carcinoma). Exposure of MCF-7 cells with AgNPs resulted in a dosedependent increase in cell growth inhibition varying from 5 to 78 % at concentrations in the range of 10–640 lg ml-1. The present study demonstrated that AgNPs have potent antifungal, antidimorphic, and anticancer activities. The current research opens a new avenue for the green synthesis of nano-materials. R. S. Yehia (&) H. Al-Sheikh Department of Biological Sciences, College of Science, King Faisal University, Al-Hasa 31982, Saudi Arabia e-mail: [email protected]; [email protected] R. S. Yehia Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt
Keywords Pleurotus ostreatus Biosynthesis Silver nanoparticles Antifungal Antidimorphic Anticancer
Introduction Nanoparticles are particles that have one dimension 100 nm or less in size. They tend to react differently than larger particles of the same composition because of their large surface area, thus allowing them to be used in novel applications (Abou El-Nour et al. 2010). Nanoparticles serve as the fundamental building blocks of nanotechnology (Vahabi et al. 2011). Because of their wide application in numerous areas such as electronics, catalysis, chemistry, energy, and medicine, the commercial requirement of nanoparticles is increasing. New applications for nanoparticles and nanomaterials are emerging rapidly (Otsuka et al. 2003; Nalwa 2000). The use of environmentally benign materials such as plant leaf extracts, bacteria and fungi for the synthesis of silver nanoparticles (AgNPs) offers numerous benefits of eco-friendliness, low cost production and minimum period and compatibility for pharmaceutical and biomedical applications, as they do not require toxic chemicals for their synthesis (Parashar et al. 2009). During the synthesis procedure, the use of chemical methods including toxic chemicals on the surface of nanoparticles and non-polar solvents limits their applications in biomedical and clinical fields. Therefore, there is a need for the development of a clean, safe, biocompatible, cost-effective, non-toxic, sustainable, and environmental friendly method for synthesizing nanoparticles. Compared with traditional synthetic methods, biological systems are a novel proposal for the production of nanomaterials (Bansal et al. 2011). Fungi are useful biological agents for the synthesis of metal nanoparticles. The use of fungi is potentially exciting because they secrete large
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amounts of enzymes and their biomass is easy to manage (Bhainsa and D’Souza 2006). For example, Duran et al. (2005) found that the pathogenic fungus Fusarium oxysporum secrets enzyme hydrogenase extracellularly in the broth when cultured in synthetic medium. In addition, this enzyme demonstrates excellent redox properties and acts as an electron shuttle in metal reduction. Another novel biological method consists of a two-step mechanism for the synthesis of AgNPs using Verticillum (Mukherjee et al. 2001a, b). It is evident that electron shuttles and/or other reducing agents such as hydroquinones released by bacteria and fungi are capable of reducing metal ions to nanoparticles. Also, some species of fungi such as Aspergillus sp. and Trichoderma sp. were reported to synthesize nanoparticles extracellularly (Bhainsa and D’Souza 2006; Binupriya et al. 2010; Jaidev and Narasimha 2010; Vahabi et al. 2011). Mushrooms are known to have anti-inflammatory, cardiovascular, antitumor, antiviral, antibacterial, hepatoprotective and hypotensive activities in biological systems (Wasser and Weis 1999; Bernardshaw et al. 2005). Many varieties of naturally occurring mushrooms are found to have promising antioxidant and anticancer properties and prolong longevity (Mizuno 2000). Edible mushrooms are well known for their antioxidant, antimicrobial, antiinflammatory, antitumor and anticancer activities (Ajith and Janardhanan 2007; Turkoglu et al. 2007). Mushrooms characteristically contain many different bioactive compounds with diverse biological activity, and the content and bioactivity of these compounds depend on how the mushroom is prepared and consumed (Chang 1996). Sanghi and Verma (2009a) reported that efficiently synthesize AgNPs by white rot fungi Trametes versicolor and Phaenerochaete chrysosporium (Sleytr et al. 1999). This study therefore explores an in vitro approach for the biosynthesis of AgNPs using edible mushroom Pleurotus ostreatus.
Materials and methods Test microorganism Pleurotus ostreatus belonging to class Basidiomycetes was kindly provided by Moubarak City for Science and Technology, Alexandria, Egypt and maintained in potato dextrose agar slant at 4 °C. All chemicals and media used were of analytical grade. Biological synthesis and characterization of silver nanoparticle
(Sanghi and Verma 2009b). Flasks were inoculated with culture and incubated in an orbital shaker (100 rpm) for 72 h at 25 °C. After 3 days of incubation, the fungal biomass was harvested using plastic sieves and then washed extensively with distilled water to remove any medium components. Fresh clean biomasses were weighed, transferred to 200 ml of MilliQ water, and incubated in a shaker (100 rpm) for 72 h at 25 °C. Then, the biomass was filtered through Whatman filter paper No. 1 and the cell free filtrate was used for experiments. To the 50 ml of cell filtrate in a 250 ml conical flask, 8.4 mg of silver nitrate 1 mM was mixed and agitated in a shaker (150 rpm) at 25 °C in the dark. Simultaneously, controls without AgNO3 (cell free filtrate) were produced. Another negative control containing AgNO3 alone was maintained under the same conditions as above. The formation of nanoparticles was confirmed by an ultraviolet–visible (UV–Vis) spectrophotometer (Optizen 2120 UV, Mecasys, Korea). Samples were withdrawn at predetermined time intervals (0, 24, 48, 72 h) and the spectra were recorded from 350 to 700 nm. After primary detection, the cell-free medium was supplemented with AgNO3, lyophilized, and subjected to Fourier transform infrared (FTIR) spectroscopy in the region of 4,000–400 cm-1 (Perkin Elmer, USA) to identify the chemical constituents of the AgNPs in this region. Samples for transmission electron microscopy (TEM) analysis were prepared as follows. The sample was sonicated (Vibronics VS 80) for 5 min. AgNPs were loaded on carbon-coated copper grids, and solvent was allowed to evaporate under Infrared light for 30 min. TEM measurements were performed on a Phillips model CM 20 instrument, operated at an accelerating voltage of 200 kV, and the shape and size of the AgNPs were characterized. Nitrate reductase assay The enzyme-nitrate reductase in fungal filtrate was assayed according to the procedure followed by Harley (1993). Five milliliter aliquots of 5-day fungal filtrate were mixed with 5 ml of assay medium (30 mM KNO3 and 5 % propanol in 0.1 M phosphate buffer of pH 7.5) and incubated in the dark for 60 min. After incubation, nitrites formed in the assay mixture were estimated by adding 2.5 ml of sulfanilamide and NEED [N-(1-naphthyl) ethylene diamine dihydrochloride] solutions. The pink color that developed was measured by an UV–Vis spectrophotometer. Enzyme activity was expressed in terms of n moles of nitrite ml-1 h-1. Antifungal efficiency of silver nanoparticles Test organism and growth conditions
Fungal biomass used for biosynthetic experiments was grown aerobically in liquid growth medium which composed of 5.0 g l-1 malt extract and 10.0 g l-1 glucose
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A total of 35 strains of one fungal species were used in this study. Candida albicans, C. glabrata, C. parapsilosis, and
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C. krusei were obtained from the Microanalytical Centre, Faculty of Science, Cairo University. Candida spp. was cultures in Sabouraud Dextrose Agar at 35 °C.
were cultured as a monolayer in RPMI-1640 medium with 5 % FBS and 1 % penicillin–streptomycin cocktail at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % O2.
Antifungal susceptibility testing
Measurement of cell viability by MTT assay
Antibiotics, fluconazole, and amphotericin B were obtained from Sigma-Aldrich. AgNPs were obtained from P. ostreatus by the method described above. Fluconazole and AgNPs were dissolved in distilled water, while amphotericin B was dissolved in 100 % dimethyl sulfoxide (SigmaAldrich). The minimum inhibitory concentration (MIC) for Candida spp. was evaluated using a broth microdilution method based on the Clinical and Laboratory Standards Institute (CLSI 2002) method. RPMI-1640 medium buffered to pH 7.0 with 3-(N-morpholino) propanesulfonic acid was used as the culture medium, and the inoculum size of Candida spp. was 1.5 9 103 cells ml-1. The microdilution plates inoculated with fungi were then incubated at 35 °C, and the turbidity of the growth control wells was observed every 24 h. The 80 % inhibitory concentration (IC80) was expressed as the lowest concentration that inhibited 80 % of the growth compared with the growth of controls. Growth was assessed using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) by monitoring absorption at 405 nm. In the current study, amphotericin B and fluconazole were used as positive controls toward fungi. Amphotericin B is a fungicidal agent widely used in treating serious systemic infections (Hartsel and Bolard 1996), and fluconazole is used in the treatment of superficial skin infections caused by dermatophytes and Candida species (Amichai and Grunwald 1998).
The effect of AgNPs of (10–600 lg ml-1) on the viability of cells was determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazoliumbromide (MTT) assay. Cells were plated at 1 9 105 cells per well in 200 ll of complete culture medium containing 10–600 lg ml-1 concentrations of AgNPs in 96-well micro-titer plates. Each concentration of Ag? nanoparticles was repeated in five wells. After incubation for the desired times at 37 °C in a humidified incubator, cell viability was determined. Fifty microliters of MTT was added to each well and incubated for 2 h after which the plate was centrifuged at 6009g for 5 min at 4 °C. The MTT solution was removed from the wells by aspiration. Then, 0.1 ml of buffered DMSO was added to each well, and plates were shaken. The absorbance was measured on a microtiter plate reader (Tecan, Switzerland) at a wavelength of 540 nm.
Effects on the dimorphic transition of C. albicans Candida albicans was maintained by subculturing in a liquid yeast extract/peptone/dextrose (YPD) medium. Cultures of yeast cells (blastoconidia) were maintained in a liquid YPD medium at 37 °C. To induce mycelial formation, cultures were directly supplemented with 20 % of fetal bovine serum (FBS). The dimorphic transition in C. albicans was investigated from cultures containing 2 mg ml-1 of Nano-Ag (at the IC80), which were incubated for 48 h at 37 °C (Jung et al. 2007; Sung et al. 2007). The dimorphic transition to mycelial forms was detected by phase contrast light microscopy. Anticancer activity of silver nanoparticles Cell lines and culture A human breast carcinoma (MCF-7) cell line was obtained from the National Cancer Institute (Cairo, Egypt). Cells
Results and discussion Fungi are extremely good candidates for the synthesis of metal nanoparticles. In our study, AgNPs were synthesized using a reduction of aqueous Ag? with the culture supernatants of P. ostreatus at 25 °C. When AgNO3 was incubated with the fungal filtrate, it turned a dark brown color, while the negative control flask remain unchanged during a 72 h incubation period. The generation of the dark brown color was due to the surface plasmon resonance (SPR) exhibited by the nanoparticles (Wiley et al. 2006). SPR shifts to a longer wavelength as the particle size increases (Zhao et al. 2006). The UV–Vis spectra were recorded from P. ostreatus at different reaction times (Fig. 1). The time at which the aliquots were removed for measurement is indicated next to the respective curves in Fig. 1. The strong SPR centered at about 450 nm is characteristic of colloidal silver. These results were compatible with the reports of Huang et al. (2007) and Verma et al. (2010). Ahmad et al. (2003) demonstrated that silver nitrate solution incubated with spent mushroom substrate synthesis of AgNPs purified solution yielded a maximum absorbance at 436 nm. The spectra show an increase in intensity of silver solution with time, indicating the formation of an increased number of AgNPs in the solution. The solution was extremely stable even after a month of reaction, with no evidence of particle aggregation. FTIR spectroscopy is a useful tool for quantifying secondary structures in metal nanoparticle protein interactions
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Zero time 24h 48h 72h
Absorbance
1 0.8 0.6 0.4 0.2 0 350
400
450
500
550
600
650
700
Wave length
Fig. 1 UV–Vis spectra recorded for fungal extracellular filtrate amended with AgNO3 at various time intervals
Fig. 3 TEM micrograph showing silver nanoparticles at different size
Fig. 2 FTIR spectrum of AgNPs formed after 72 h of incubation of the culture supernatant of P. ostreatus with silver nitrate
by the absorption of infrared radiation through the resonance of non-centro symmetric (IR active) modes of vibration. Figure 2 shows the spectrum bands at 1,640 and 1,460 cm-1 are due to –C=O and N–H stretch vibrations present in the amide linkages of the proteins, respectively (Balaji et al. 2009). The peak at 3417.6 cm-1 refers to the stretching vibrations of primary amines (Vigneshwaran et al. 2007). The molecular vibrational positions of these bands are in agreement with earlier studies on native proteins by Labrenz et al. (2000). Thus, the FTIR measurement indicates that the secondary structures of proteins in P. ostreatus fungi are not affected by their interaction with Ag? ions or nanoparticles, as well as the number of aggregates. The carbonyl groups of amino acid residues and peptides have a strong ability to bind to silver ions (Balaji et al. 2009). It was accounted that proteins could bind to nanoparticles either through free amine or cysteine groups in proteins and thereby stabilizing the AgNPs (Mandal et al. 2005). The proteins present over the AgNPs surface acts as capping agents. TEM analysis provided further insight into the size and shape of AgNPs. TEM images recorded from the AgNPs placed on a carbon-
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coated copper TEM grid is shown in Fig. 3. AgNPs were polydispersed with a spherical shape. Most of the nanoparticles shown in the micrograph are in the range of 4–15 nm. The extracellular proteins secreted by the fungus are responsible for the bioreduction of silver ions. Hence, the role of reductases in the fungal filtrate was investigated by nitrate reductase assay. In the current study, it was clearly observed that culture filtrates of P. ostreatus exhibited nitrate reductase activity 200 nmol ml-1 h-1. Nitrate reductase activity of the fungus indicated a possible mechanism of the reduction of silver nitrate to AgNPs. Various studies have indicated that nitrate reductase enzyme is an important factor for the biosynthesis of metal nanoparticles (Klittich and Leslie 1988; Ottow and Von Klopotek 1969; Lioyd 2003) in Fusarium oxysporum Ahmad et al. (2003) and Bacillus licheniformis (Kalimuthu et al. 2008). Furthermore, the nitrate reductase activity of the culture supernatant of Penicillium spp. was 270 nmol ml-1 h-1 (Bhaskara Rao et al. 2010) and 200 and 250 nmol ml-1 h-1 of Trichoderma harzianum and Trichoderma asperellum, respectively (Devi et al. 2013). The antifungal activities of amphotericin B and fluconazole that are widely used against many fungal infections were used as positive controls for comparison with the antifungal activity of AgNPs. The results of MIC were determined by means of the broth microdilution method after 48 h of incubation of Candida spp. in culture media containing amphotericin B or fluconazole and AgNPs (Table 1). AgNPs in an IC80 range of 5–28 lg ml-1,
World J Microbiol Biotechnol Table 1 Antifungal activity of silver nanoparticle against Candida spp. as compared with some antifungal drugs Fungal strains (no. of strains)
IC80 (lg ml-1)
Table 2 Effect of AgNPs on cell growth inhibition of breast carcinoma cell MCF-7 Dose (lg ml-1)
AgNP
Amphotericine B
Flucanazole
C. albicans (3)
5–7
8
13–19
C. tropicalis (2)
7–28
5
16
C. glabrata (4) C. parapsilosis (1)
16 10
7 5–7
23–33 16
C. krusei (2)
4–11
5–7
13–19
showed significant antifungal activity against Candida species. AgNPs exhibited a similar activity with amphotericin B toward all strains tested with IC80 values of 5–8 lg ml-1. However, it had a more powerful activity than fluconazole, with IC80 values of 13–33 lg ml-1. However, this compound exhibited less potent activity than amphotericin B, with IC80 values of 5–7 lg ml-1 for C. tropicalis and C. glabrata (Table 1). It is well known that Ag ions and Ag-based compounds have potent antimicrobial activities (Furno et al. 2004). These inorganic nanoparticles have a distinctive advantage over traditional chemical antimicrobial agents. The greatest problem caused by chemical antimicrobial agents is multidrug resistance. Therefore, an unconventional way to overcome the drug resistance of various microorganisms is desperately required for medical devices. Ag ions and Ag salts have been used as antimicrobial agents for decades in various fields because of their growth-inhibitory capacity against microorganisms (Hamouda et al. 2000). Our results are compatible with those of Noorbakhsh (2011) who found that growth of T. rubrum was inhibited when using 10 lg ml-1 AgNPs alone. Similarly, Kim et al. (2008) demonstrated significant antifungal activity of Nano-Ag, at an IC80 range of 1–7 lg ml-1 against T. mentagrophytes and Candida spp. Data from the present study are also consistent with those of Panacek et al. (2009) who determined MIC of AgNPs for C. albicans and C. tropicalis were 0.21, 0.84 mg l-1, respectively. In other reports, Keuk-Jun et al. (2009) demonstrated the inhibition of C. albicans using 2 lg ml-1 of AgNPs. Many studies have measured the antifungal susceptibility in C. albicans, but most deal with yeast-phase cells only and do not consider the ability of these agents to prevent transition to the hyphal-form, which is an important factor in disease pathogenesis. Furthermore, reliable data on the antimycotic and antidimorphic properties of AgNPs are not available; thus, the current study determined the medicinal status of AgNPs, especially related to growth and dimorphic transition of C. albicans. To explicate the antifungal activity of AgNPs and the dimorphic transition of C. albicans induced
0
Percentage inhibition of cell proliferation 0 ± 0.0
10
5 ± 0.01
20 40
12 ± 0.02 17 ± 0.1
80
35 ± 0.4
160
49 ± 0.2
320
65 ± 0.5
640
78 ± 0.9
Values represent mean ± SE of 3 different assay at P B 0.01
by Nano-Ag was investigated. The dimorphic transition of C. albicans from the yeast form to mycelial form is responsible for pathogenicity, with mycelial shapes being principally found during the invasion of host tissue. The mycelial form can be induced by temperature, pH, and serum (McLain et al. 2000). Serum-induced mycelia were significantly inhibited from extending and forming in the presence of AgNPs (Fig. 4c), but the mycelia formed were normal in the absence of AgNPs (Fig. 4b). Similarly, Juneyoung et al. (2010) demonstrated that Nano-Ag inhibited the dimorphism of C. albicans. The MTT assay is an uncomplicated and consistent technique to determine cell viability and is used for the screening of anti-proliferative agents. Table 2 summarizes the percentages of cell growth inhibition (CGI) at different concentrations of AgNPs. AgNPs displayed a dosedependent anti-proliferative effect against MCF-7 cells. Increasing concentrations of AgNPs induced greater inhibition of cell proliferation 78 %. Overall, AgNPs were found to inhibit the proliferation of MCF-7 cells. Exposure of MCF-7 cells to AgNPs for 24 h resulted in a celldependent increase in CGI varying from 5 to 78 % at concentrations in the range of 10–640 lg ml-1. The biosynthesized AgNPs also had effective cytotoxic activity against tumor cells. Despite the widespread use of synthetic AgNPs, very few studies have been conducted to determine the cytotoxic effects of biosynthesized AgNPs. Here, we measured apoptosis to measure cytotoxic effects. Our results are in accord with those of Devi et al. (2012) who observed in vitro cytotoxic activity against the human laryngeal cancer Hep-2, MCF-7, and human colon cancer HT29 cell lines at different AgNPs concentrations. Lower cytotoxicity of synthesized AgNPs against a normal Vero cell line was observed with an IC50 of 95 lg ml-1. Similarly, Bhat et al. (2013) found that AuNPs biosynthesized using edible mushroom Pleurotus florida exhibited significant dose-dependent anti-proliferative activity against different cancer cell lines at dilutions in the range of
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Fig. 4 The effect of AgNPs on the dimorphic transition of C. albicans. a Control without 20 % FBS and Nano-Ag (yeast form), b cells treated with 20 % FBS only, c cells treated with 2 lg ml-1 of AgNPs
10–30 lg ml-1. Our data are consistent with those of Bhimba et al. (2012) who reported in vitro cytotoxicity of AgNPs against Hep-2 cell lines at different concentrations. Cytotoxicity analysis of samples show a direct dose– response relationship where cytotoxicity increased at higher AgNPs concentrations. The anticancer properties of AgNPs may be attributed to their functionalization with organic moieties and irregular shape. It was hypothesized that cell apoptosis was the mechanism induced by biosynthesized AgNPs (Sukirtha et al. 2012). Currently, cancer claims the lives of approximately seven million people worldwide on an annual basis. Hence, in recent years, the search for novel cancer therapeutics from natural products has been reported against various cancer cell lines. Cytotoxic effects are inversely proportional to the size of the bioactive compound AgNPs. In conclusion, it is predicted that nanotechnology will have an impact on the global economy by 2015. The use of AgNPs should emerge as a novel approach for anticandidal and anticancer therapies, and when the molecular mechanism of targeting is better understood, the application of AgNPs is likely to expand further. The present study demonstrated AgNPs might be an effective alternative to antifungal agents for future therapies in Candida infection. Therefore, it can be expected that AgNPs might be potential anti-infective agents for human fungal diseases. The present work also explored the potential anticancer activity of biologically synthesized AgNPs in an in vitro tumor cell line (MCF-7), and indicated they might be an effective alternative for the treatment of tumors.
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Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar SR, Khan MI, Parischa R, Ajayakumar PV, Alam M, Kumar R, Sastry M (2001b) Extracellular biosynthesis of biometallic Au–Ag alloy nanoparticles. Nano Lett 1:515–519 Nalwa HS (2000) Hand book of Nanostructured Materials and Nanotechnology: Electrical properties. Academic Press, Waltham Noorbakhsh F (2011) Antifungal effects of silver nanoparticle alone and with combination of antifungal drug on dermatophyte pathogen Trichophyton rubrum. IPCBEE 5:364–367 Otsuka H, Nagasaki Y, Kataoka K (2003) PEGylated nanoparticles for biological and pharmaceutical applications. Adv Drug Deliv Rev 55:403–419 Ottow JCG, Von Klopotek A (1969) Enzymatic reduction of iron oxide by fungi. Appl Microbiol 18:41–43 Panacek A, Kola´r M, Vecerova´ R, Prucek R, Soukupova´ J, Krystof V, Hamal P, Zboril R, Kvı´tek L (2009) Antifungal activity of silver nanoparticles against Candida spp. Biomaterials 30(31):6333–6340 Parashar V, Parashar R, Sharma B, Pandey AC (2009) Parthenium leaf extract mediated synthesis of silver nanoparticles: a novel approach towards weed utilization. Dig J Nanomater Bios 4(1):45–50 Sanghi R, Verma P (2009a) Bio-synthesis of silver nanoparticles using white rot fungus. In: Singh J (ed) Information technology and environmental management. MD Publications Pvt. Ltd., New Delhi Sanghi R, Verma P (2009b) Biomimetic synthesis and characterization of protein capped silver nanoparticles. Bioresour Technol 100(1):501–504 Sleytr UB, Messner P, Pum D, Sa´ra M (1999) Crystalline bacterial cell surface layers (S-layers): from supramolecular cell structure to biomimetics and nanotechnology. Angew Chem Int Ed Engl 38:1034–1054 Sukirtha R, Priyanka KM, Antony JJ, Kamalakkannan S, Thangam R, Gunasekaran P, Krishnan M, Achiraman S (2012) Cytotoxic effect of green synthesized silver nanoparticles using Melia azedarach against in vitro HeLa cell lines and lymphoma mice model. Proc Biochem 47:273–279 Sung WS, Lee IS, Lee DG (2007) Damage to the cytoplasmic membrane and cell death caused by lycopene in Candida albicans. J Microbiol Biotechnol 17(11):1797–1804 Turkoglu A, Duru ME, Mercan N (2007) Antioxidant and antimicrobial activity of Russula delica Fr: an edible wild mushroom. Eur J Anal Chem 2(1):54–67 Vahabi K, Mansoori G, Karimi V (2011) Biosynthesis of silver nanoparticles by fungus Trichoderma reesei. Insciences J 1(1):65–79 Verma VC, Kharwar RN, Gange AC (2010) Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus. Nanomedicine (Lond) 5:33–40 Vigneshwaran N, Ashtaputre NM, Varadarajan PV, Nachane RP, Paralikar KM, Balasubramanya RH (2007) Biological synthesis of silver nanoparticles using the fungus Aspergillus flavus. Mater Lett 61:1413–1418 Wasser SP, Weis AL (1999) Medicinal properties of substances occurring in higher basidiomycetes mushrooms: current perspectives. Int J Med Mushrooms 1:31–62 Wiley BJ, Im SH, Li ZY, McLellan J, Siekkinen A, Xia Y (2006) Maneuvering the surface plasmon resonance of silver nanostructures through shape-controlled synthesis. J Phys Chem B 110:15666–15675 Zhao Y, Jiang Y, Fang Y (2006) Spectroscopy property of Ag nanoparticles. Acta A 65:1003–1006
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ORIGINAL PAPER
Biosynthesis and characterization of silver nanoparticles produced by Pleurotus ostreatus and their anticandidal and anticancer activities Ramy S. Yehia • Hashem Al-Sheikh
Received: 23 January 2014 / Accepted: 14 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The biosynthesis of nanoparticles has received increasing interest because of the growing need to develop safe, cost-effective and environmentally friendly technologies for the synthesis of nano-materials. In this study, silver nanoparticles (AgNPs) were synthesized using a reduction of aqueous Ag? ions with culture supernatant from Pleurotus ostreatus. The bioreduction of AgNPs was monitored by ultra violet-visible spectroscopy and the obtained AgNPs were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy techniques. TEM studies showed the size of the AgNPs to be in the range of 4–15 nm. The formation of AgNPs might be an enzyme-mediated extracellular reaction process. Furthermore, the antifungal effect of AgNPs against Candida albicans as compared with commercially antifungal drugs was examined. The effect of AgNPs on dimorphic transition of C. albicans was tested. The anticancer properties of AgNPs against cells (MCF-7) were also evaluated. AgNPs caused a significant decrease in cell viability of an MCF-7 cell line (breast carcinoma). Exposure of MCF-7 cells with AgNPs resulted in a dosedependent increase in cell growth inhibition varying from 5 to 78 % at concentrations in the range of 10–640 lg ml-1. The present study demonstrated that AgNPs have potent antifungal, antidimorphic, and anticancer activities. The current research opens a new avenue for the green synthesis of nano-materials. R. S. Yehia (&) H. Al-Sheikh Department of Biological Sciences, College of Science, King Faisal University, Al-Hasa 31982, Saudi Arabia e-mail: [email protected]; [email protected] R. S. Yehia Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt
Keywords Pleurotus ostreatus Biosynthesis Silver nanoparticles Antifungal Antidimorphic Anticancer
Introduction Nanoparticles are particles that have one dimension 100 nm or less in size. They tend to react differently than larger particles of the same composition because of their large surface area, thus allowing them to be used in novel applications (Abou El-Nour et al. 2010). Nanoparticles serve as the fundamental building blocks of nanotechnology (Vahabi et al. 2011). Because of their wide application in numerous areas such as electronics, catalysis, chemistry, energy, and medicine, the commercial requirement of nanoparticles is increasing. New applications for nanoparticles and nanomaterials are emerging rapidly (Otsuka et al. 2003; Nalwa 2000). The use of environmentally benign materials such as plant leaf extracts, bacteria and fungi for the synthesis of silver nanoparticles (AgNPs) offers numerous benefits of eco-friendliness, low cost production and minimum period and compatibility for pharmaceutical and biomedical applications, as they do not require toxic chemicals for their synthesis (Parashar et al. 2009). During the synthesis procedure, the use of chemical methods including toxic chemicals on the surface of nanoparticles and non-polar solvents limits their applications in biomedical and clinical fields. Therefore, there is a need for the development of a clean, safe, biocompatible, cost-effective, non-toxic, sustainable, and environmental friendly method for synthesizing nanoparticles. Compared with traditional synthetic methods, biological systems are a novel proposal for the production of nanomaterials (Bansal et al. 2011). Fungi are useful biological agents for the synthesis of metal nanoparticles. The use of fungi is potentially exciting because they secrete large
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amounts of enzymes and their biomass is easy to manage (Bhainsa and D’Souza 2006). For example, Duran et al. (2005) found that the pathogenic fungus Fusarium oxysporum secrets enzyme hydrogenase extracellularly in the broth when cultured in synthetic medium. In addition, this enzyme demonstrates excellent redox properties and acts as an electron shuttle in metal reduction. Another novel biological method consists of a two-step mechanism for the synthesis of AgNPs using Verticillum (Mukherjee et al. 2001a, b). It is evident that electron shuttles and/or other reducing agents such as hydroquinones released by bacteria and fungi are capable of reducing metal ions to nanoparticles. Also, some species of fungi such as Aspergillus sp. and Trichoderma sp. were reported to synthesize nanoparticles extracellularly (Bhainsa and D’Souza 2006; Binupriya et al. 2010; Jaidev and Narasimha 2010; Vahabi et al. 2011). Mushrooms are known to have anti-inflammatory, cardiovascular, antitumor, antiviral, antibacterial, hepatoprotective and hypotensive activities in biological systems (Wasser and Weis 1999; Bernardshaw et al. 2005). Many varieties of naturally occurring mushrooms are found to have promising antioxidant and anticancer properties and prolong longevity (Mizuno 2000). Edible mushrooms are well known for their antioxidant, antimicrobial, antiinflammatory, antitumor and anticancer activities (Ajith and Janardhanan 2007; Turkoglu et al. 2007). Mushrooms characteristically contain many different bioactive compounds with diverse biological activity, and the content and bioactivity of these compounds depend on how the mushroom is prepared and consumed (Chang 1996). Sanghi and Verma (2009a) reported that efficiently synthesize AgNPs by white rot fungi Trametes versicolor and Phaenerochaete chrysosporium (Sleytr et al. 1999). This study therefore explores an in vitro approach for the biosynthesis of AgNPs using edible mushroom Pleurotus ostreatus.
Materials and methods Test microorganism Pleurotus ostreatus belonging to class Basidiomycetes was kindly provided by Moubarak City for Science and Technology, Alexandria, Egypt and maintained in potato dextrose agar slant at 4 °C. All chemicals and media used were of analytical grade. Biological synthesis and characterization of silver nanoparticle
(Sanghi and Verma 2009b). Flasks were inoculated with culture and incubated in an orbital shaker (100 rpm) for 72 h at 25 °C. After 3 days of incubation, the fungal biomass was harvested using plastic sieves and then washed extensively with distilled water to remove any medium components. Fresh clean biomasses were weighed, transferred to 200 ml of MilliQ water, and incubated in a shaker (100 rpm) for 72 h at 25 °C. Then, the biomass was filtered through Whatman filter paper No. 1 and the cell free filtrate was used for experiments. To the 50 ml of cell filtrate in a 250 ml conical flask, 8.4 mg of silver nitrate 1 mM was mixed and agitated in a shaker (150 rpm) at 25 °C in the dark. Simultaneously, controls without AgNO3 (cell free filtrate) were produced. Another negative control containing AgNO3 alone was maintained under the same conditions as above. The formation of nanoparticles was confirmed by an ultraviolet–visible (UV–Vis) spectrophotometer (Optizen 2120 UV, Mecasys, Korea). Samples were withdrawn at predetermined time intervals (0, 24, 48, 72 h) and the spectra were recorded from 350 to 700 nm. After primary detection, the cell-free medium was supplemented with AgNO3, lyophilized, and subjected to Fourier transform infrared (FTIR) spectroscopy in the region of 4,000–400 cm-1 (Perkin Elmer, USA) to identify the chemical constituents of the AgNPs in this region. Samples for transmission electron microscopy (TEM) analysis were prepared as follows. The sample was sonicated (Vibronics VS 80) for 5 min. AgNPs were loaded on carbon-coated copper grids, and solvent was allowed to evaporate under Infrared light for 30 min. TEM measurements were performed on a Phillips model CM 20 instrument, operated at an accelerating voltage of 200 kV, and the shape and size of the AgNPs were characterized. Nitrate reductase assay The enzyme-nitrate reductase in fungal filtrate was assayed according to the procedure followed by Harley (1993). Five milliliter aliquots of 5-day fungal filtrate were mixed with 5 ml of assay medium (30 mM KNO3 and 5 % propanol in 0.1 M phosphate buffer of pH 7.5) and incubated in the dark for 60 min. After incubation, nitrites formed in the assay mixture were estimated by adding 2.5 ml of sulfanilamide and NEED [N-(1-naphthyl) ethylene diamine dihydrochloride] solutions. The pink color that developed was measured by an UV–Vis spectrophotometer. Enzyme activity was expressed in terms of n moles of nitrite ml-1 h-1. Antifungal efficiency of silver nanoparticles Test organism and growth conditions
Fungal biomass used for biosynthetic experiments was grown aerobically in liquid growth medium which composed of 5.0 g l-1 malt extract and 10.0 g l-1 glucose
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A total of 35 strains of one fungal species were used in this study. Candida albicans, C. glabrata, C. parapsilosis, and
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C. krusei were obtained from the Microanalytical Centre, Faculty of Science, Cairo University. Candida spp. was cultures in Sabouraud Dextrose Agar at 35 °C.
were cultured as a monolayer in RPMI-1640 medium with 5 % FBS and 1 % penicillin–streptomycin cocktail at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % O2.
Antifungal susceptibility testing
Measurement of cell viability by MTT assay
Antibiotics, fluconazole, and amphotericin B were obtained from Sigma-Aldrich. AgNPs were obtained from P. ostreatus by the method described above. Fluconazole and AgNPs were dissolved in distilled water, while amphotericin B was dissolved in 100 % dimethyl sulfoxide (SigmaAldrich). The minimum inhibitory concentration (MIC) for Candida spp. was evaluated using a broth microdilution method based on the Clinical and Laboratory Standards Institute (CLSI 2002) method. RPMI-1640 medium buffered to pH 7.0 with 3-(N-morpholino) propanesulfonic acid was used as the culture medium, and the inoculum size of Candida spp. was 1.5 9 103 cells ml-1. The microdilution plates inoculated with fungi were then incubated at 35 °C, and the turbidity of the growth control wells was observed every 24 h. The 80 % inhibitory concentration (IC80) was expressed as the lowest concentration that inhibited 80 % of the growth compared with the growth of controls. Growth was assessed using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) by monitoring absorption at 405 nm. In the current study, amphotericin B and fluconazole were used as positive controls toward fungi. Amphotericin B is a fungicidal agent widely used in treating serious systemic infections (Hartsel and Bolard 1996), and fluconazole is used in the treatment of superficial skin infections caused by dermatophytes and Candida species (Amichai and Grunwald 1998).
The effect of AgNPs of (10–600 lg ml-1) on the viability of cells was determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazoliumbromide (MTT) assay. Cells were plated at 1 9 105 cells per well in 200 ll of complete culture medium containing 10–600 lg ml-1 concentrations of AgNPs in 96-well micro-titer plates. Each concentration of Ag? nanoparticles was repeated in five wells. After incubation for the desired times at 37 °C in a humidified incubator, cell viability was determined. Fifty microliters of MTT was added to each well and incubated for 2 h after which the plate was centrifuged at 6009g for 5 min at 4 °C. The MTT solution was removed from the wells by aspiration. Then, 0.1 ml of buffered DMSO was added to each well, and plates were shaken. The absorbance was measured on a microtiter plate reader (Tecan, Switzerland) at a wavelength of 540 nm.
Effects on the dimorphic transition of C. albicans Candida albicans was maintained by subculturing in a liquid yeast extract/peptone/dextrose (YPD) medium. Cultures of yeast cells (blastoconidia) were maintained in a liquid YPD medium at 37 °C. To induce mycelial formation, cultures were directly supplemented with 20 % of fetal bovine serum (FBS). The dimorphic transition in C. albicans was investigated from cultures containing 2 mg ml-1 of Nano-Ag (at the IC80), which were incubated for 48 h at 37 °C (Jung et al. 2007; Sung et al. 2007). The dimorphic transition to mycelial forms was detected by phase contrast light microscopy. Anticancer activity of silver nanoparticles Cell lines and culture A human breast carcinoma (MCF-7) cell line was obtained from the National Cancer Institute (Cairo, Egypt). Cells
Results and discussion Fungi are extremely good candidates for the synthesis of metal nanoparticles. In our study, AgNPs were synthesized using a reduction of aqueous Ag? with the culture supernatants of P. ostreatus at 25 °C. When AgNO3 was incubated with the fungal filtrate, it turned a dark brown color, while the negative control flask remain unchanged during a 72 h incubation period. The generation of the dark brown color was due to the surface plasmon resonance (SPR) exhibited by the nanoparticles (Wiley et al. 2006). SPR shifts to a longer wavelength as the particle size increases (Zhao et al. 2006). The UV–Vis spectra were recorded from P. ostreatus at different reaction times (Fig. 1). The time at which the aliquots were removed for measurement is indicated next to the respective curves in Fig. 1. The strong SPR centered at about 450 nm is characteristic of colloidal silver. These results were compatible with the reports of Huang et al. (2007) and Verma et al. (2010). Ahmad et al. (2003) demonstrated that silver nitrate solution incubated with spent mushroom substrate synthesis of AgNPs purified solution yielded a maximum absorbance at 436 nm. The spectra show an increase in intensity of silver solution with time, indicating the formation of an increased number of AgNPs in the solution. The solution was extremely stable even after a month of reaction, with no evidence of particle aggregation. FTIR spectroscopy is a useful tool for quantifying secondary structures in metal nanoparticle protein interactions
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Zero time 24h 48h 72h
Absorbance
1 0.8 0.6 0.4 0.2 0 350
400
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500
550
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Wave length
Fig. 1 UV–Vis spectra recorded for fungal extracellular filtrate amended with AgNO3 at various time intervals
Fig. 3 TEM micrograph showing silver nanoparticles at different size
Fig. 2 FTIR spectrum of AgNPs formed after 72 h of incubation of the culture supernatant of P. ostreatus with silver nitrate
by the absorption of infrared radiation through the resonance of non-centro symmetric (IR active) modes of vibration. Figure 2 shows the spectrum bands at 1,640 and 1,460 cm-1 are due to –C=O and N–H stretch vibrations present in the amide linkages of the proteins, respectively (Balaji et al. 2009). The peak at 3417.6 cm-1 refers to the stretching vibrations of primary amines (Vigneshwaran et al. 2007). The molecular vibrational positions of these bands are in agreement with earlier studies on native proteins by Labrenz et al. (2000). Thus, the FTIR measurement indicates that the secondary structures of proteins in P. ostreatus fungi are not affected by their interaction with Ag? ions or nanoparticles, as well as the number of aggregates. The carbonyl groups of amino acid residues and peptides have a strong ability to bind to silver ions (Balaji et al. 2009). It was accounted that proteins could bind to nanoparticles either through free amine or cysteine groups in proteins and thereby stabilizing the AgNPs (Mandal et al. 2005). The proteins present over the AgNPs surface acts as capping agents. TEM analysis provided further insight into the size and shape of AgNPs. TEM images recorded from the AgNPs placed on a carbon-
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coated copper TEM grid is shown in Fig. 3. AgNPs were polydispersed with a spherical shape. Most of the nanoparticles shown in the micrograph are in the range of 4–15 nm. The extracellular proteins secreted by the fungus are responsible for the bioreduction of silver ions. Hence, the role of reductases in the fungal filtrate was investigated by nitrate reductase assay. In the current study, it was clearly observed that culture filtrates of P. ostreatus exhibited nitrate reductase activity 200 nmol ml-1 h-1. Nitrate reductase activity of the fungus indicated a possible mechanism of the reduction of silver nitrate to AgNPs. Various studies have indicated that nitrate reductase enzyme is an important factor for the biosynthesis of metal nanoparticles (Klittich and Leslie 1988; Ottow and Von Klopotek 1969; Lioyd 2003) in Fusarium oxysporum Ahmad et al. (2003) and Bacillus licheniformis (Kalimuthu et al. 2008). Furthermore, the nitrate reductase activity of the culture supernatant of Penicillium spp. was 270 nmol ml-1 h-1 (Bhaskara Rao et al. 2010) and 200 and 250 nmol ml-1 h-1 of Trichoderma harzianum and Trichoderma asperellum, respectively (Devi et al. 2013). The antifungal activities of amphotericin B and fluconazole that are widely used against many fungal infections were used as positive controls for comparison with the antifungal activity of AgNPs. The results of MIC were determined by means of the broth microdilution method after 48 h of incubation of Candida spp. in culture media containing amphotericin B or fluconazole and AgNPs (Table 1). AgNPs in an IC80 range of 5–28 lg ml-1,
World J Microbiol Biotechnol Table 1 Antifungal activity of silver nanoparticle against Candida spp. as compared with some antifungal drugs Fungal strains (no. of strains)
IC80 (lg ml-1)
Table 2 Effect of AgNPs on cell growth inhibition of breast carcinoma cell MCF-7 Dose (lg ml-1)
AgNP
Amphotericine B
Flucanazole
C. albicans (3)
5–7
8
13–19
C. tropicalis (2)
7–28
5
16
C. glabrata (4) C. parapsilosis (1)
16 10
7 5–7
23–33 16
C. krusei (2)
4–11
5–7
13–19
showed significant antifungal activity against Candida species. AgNPs exhibited a similar activity with amphotericin B toward all strains tested with IC80 values of 5–8 lg ml-1. However, it had a more powerful activity than fluconazole, with IC80 values of 13–33 lg ml-1. However, this compound exhibited less potent activity than amphotericin B, with IC80 values of 5–7 lg ml-1 for C. tropicalis and C. glabrata (Table 1). It is well known that Ag ions and Ag-based compounds have potent antimicrobial activities (Furno et al. 2004). These inorganic nanoparticles have a distinctive advantage over traditional chemical antimicrobial agents. The greatest problem caused by chemical antimicrobial agents is multidrug resistance. Therefore, an unconventional way to overcome the drug resistance of various microorganisms is desperately required for medical devices. Ag ions and Ag salts have been used as antimicrobial agents for decades in various fields because of their growth-inhibitory capacity against microorganisms (Hamouda et al. 2000). Our results are compatible with those of Noorbakhsh (2011) who found that growth of T. rubrum was inhibited when using 10 lg ml-1 AgNPs alone. Similarly, Kim et al. (2008) demonstrated significant antifungal activity of Nano-Ag, at an IC80 range of 1–7 lg ml-1 against T. mentagrophytes and Candida spp. Data from the present study are also consistent with those of Panacek et al. (2009) who determined MIC of AgNPs for C. albicans and C. tropicalis were 0.21, 0.84 mg l-1, respectively. In other reports, Keuk-Jun et al. (2009) demonstrated the inhibition of C. albicans using 2 lg ml-1 of AgNPs. Many studies have measured the antifungal susceptibility in C. albicans, but most deal with yeast-phase cells only and do not consider the ability of these agents to prevent transition to the hyphal-form, which is an important factor in disease pathogenesis. Furthermore, reliable data on the antimycotic and antidimorphic properties of AgNPs are not available; thus, the current study determined the medicinal status of AgNPs, especially related to growth and dimorphic transition of C. albicans. To explicate the antifungal activity of AgNPs and the dimorphic transition of C. albicans induced
0
Percentage inhibition of cell proliferation 0 ± 0.0
10
5 ± 0.01
20 40
12 ± 0.02 17 ± 0.1
80
35 ± 0.4
160
49 ± 0.2
320
65 ± 0.5
640
78 ± 0.9
Values represent mean ± SE of 3 different assay at P B 0.01
by Nano-Ag was investigated. The dimorphic transition of C. albicans from the yeast form to mycelial form is responsible for pathogenicity, with mycelial shapes being principally found during the invasion of host tissue. The mycelial form can be induced by temperature, pH, and serum (McLain et al. 2000). Serum-induced mycelia were significantly inhibited from extending and forming in the presence of AgNPs (Fig. 4c), but the mycelia formed were normal in the absence of AgNPs (Fig. 4b). Similarly, Juneyoung et al. (2010) demonstrated that Nano-Ag inhibited the dimorphism of C. albicans. The MTT assay is an uncomplicated and consistent technique to determine cell viability and is used for the screening of anti-proliferative agents. Table 2 summarizes the percentages of cell growth inhibition (CGI) at different concentrations of AgNPs. AgNPs displayed a dosedependent anti-proliferative effect against MCF-7 cells. Increasing concentrations of AgNPs induced greater inhibition of cell proliferation 78 %. Overall, AgNPs were found to inhibit the proliferation of MCF-7 cells. Exposure of MCF-7 cells to AgNPs for 24 h resulted in a celldependent increase in CGI varying from 5 to 78 % at concentrations in the range of 10–640 lg ml-1. The biosynthesized AgNPs also had effective cytotoxic activity against tumor cells. Despite the widespread use of synthetic AgNPs, very few studies have been conducted to determine the cytotoxic effects of biosynthesized AgNPs. Here, we measured apoptosis to measure cytotoxic effects. Our results are in accord with those of Devi et al. (2012) who observed in vitro cytotoxic activity against the human laryngeal cancer Hep-2, MCF-7, and human colon cancer HT29 cell lines at different AgNPs concentrations. Lower cytotoxicity of synthesized AgNPs against a normal Vero cell line was observed with an IC50 of 95 lg ml-1. Similarly, Bhat et al. (2013) found that AuNPs biosynthesized using edible mushroom Pleurotus florida exhibited significant dose-dependent anti-proliferative activity against different cancer cell lines at dilutions in the range of
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Fig. 4 The effect of AgNPs on the dimorphic transition of C. albicans. a Control without 20 % FBS and Nano-Ag (yeast form), b cells treated with 20 % FBS only, c cells treated with 2 lg ml-1 of AgNPs
10–30 lg ml-1. Our data are consistent with those of Bhimba et al. (2012) who reported in vitro cytotoxicity of AgNPs against Hep-2 cell lines at different concentrations. Cytotoxicity analysis of samples show a direct dose– response relationship where cytotoxicity increased at higher AgNPs concentrations. The anticancer properties of AgNPs may be attributed to their functionalization with organic moieties and irregular shape. It was hypothesized that cell apoptosis was the mechanism induced by biosynthesized AgNPs (Sukirtha et al. 2012). Currently, cancer claims the lives of approximately seven million people worldwide on an annual basis. Hence, in recent years, the search for novel cancer therapeutics from natural products has been reported against various cancer cell lines. Cytotoxic effects are inversely proportional to the size of the bioactive compound AgNPs. In conclusion, it is predicted that nanotechnology will have an impact on the global economy by 2015. The use of AgNPs should emerge as a novel approach for anticandidal and anticancer therapies, and when the molecular mechanism of targeting is better understood, the application of AgNPs is likely to expand further. The present study demonstrated AgNPs might be an effective alternative to antifungal agents for future therapies in Candida infection. Therefore, it can be expected that AgNPs might be potential anti-infective agents for human fungal diseases. The present work also explored the potential anticancer activity of biologically synthesized AgNPs in an in vitro tumor cell line (MCF-7), and indicated they might be an effective alternative for the treatment of tumors.
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