Assessment of antibacterial and anticancer capability of silver nanoparticles extracellularly biosynthesized using Aspergillus terreus

The present study explores biosynthesis of silver nanoparticles (AgNPs) employing extracellular extract of Aspergillus terreus ITCC 9932.15. Modulation of various variables that dictate the biosynthesis of AgNPs, suggested of optimal AgNPs synthesis using AgNO3, 1 mM at pH 8 and temperature, 35 °C. The biosynthesis of AgNPs was observed to be time dependent with incremental particle synthesis till 24 h. Various studies were undertaken to authenticate formation and characterization of AgNPs for size, crystallinity and biomolecules involved. A sharp SPR peak observed at 420 nm in the UV–vis absorption spectra validated synthesis of nanoparticles. These particles exhibited spherical morphology with size ∼25 nm and −16 mV of zeta potential. Further, the existence of proteins and other biomolecules onto the surface of AgNPs was confirmed with FTIR studies. The SAED pattern investigated by employing TEM authenticated the crystallinity of AgNPs. The AgNPs also exhibited potential antibacterial activity against Gram-negative and Gram-positive bacteria (E. coli and P. aeruginosa). In addition, remarkable anticancer activity was obtained in breast cancer cell line (MCF-7).


Introduction
Nanoparticles have found plethora of applications in different domains including diagnosis, energy, cosmetics, enzyme immobilization, biosensors and biodegradation. Amongst all nanoparticles, silver nanoparticles (AgNPs) have been thoroughly harnessed owing to its excellent antimicrobial properties. The antimicrobial effects could be attributed to interaction with the thiol groups of pivotal proteins and enzymes, introduction of oxidative stress and blockade of DNA replication [1]. In addition to antimicrobial efficacy, AgNPs have been shown to exhibit excellent anticancer effects that includes, lung cancer, breast cancer, skin cancer and hepatocellular carcinoma. Uptake of AgNPs by cancer cells could lead to ROS generation, diminished cell migration, enhanced oxidative stress that ultimately results in programmed cell death [2].
Chemical synthesis approach which involves reduction of silver salt using a reducing agent is the most customary approach of AgNPs synthesis. The green synthesis approach that utilizes extracts from plants or microbes is rapidly emerging as method of choice [3][4][5][6][7]. Interestingly, besides plant extracts researchers have also synthesized AgNPs using silk fibroin (from Bombyx mori) and UV radiation [8][9][10][11][12][13] Application of fungal biomass for biosynthesis of AgNPs is preferred to be of great interest due to the inherent metabolic diversity involved. There are two approaches for biosynthesis of AgNPs using fungi i.e. extracellular and intracellular biosynthesis. Intracellular biosynthesis consist of addition of AgNO 3 to the fungal culture whereby it gets incorporated and reduced to AgNPs, accompanied by tedious extraction steps involving chemical exposure, filtration and centrifugation to dislodge biomass and free AgNPs [14,15]. In contrast extracellular biosynthesis involves addition of filtrate obtained after cultivation of fungus in water to AgNO 3 [16][17][18]. The second approach is broadly implicated as it offers ease of recovery of AgNPs. Further, the fungi shows considerable tolerance to metals that facilitates enhanced secretion of extracellular proteins that in turn imparts high stability to the synthesized AgNPs [19,20]. Furthermore, the fungal biomass appears to be more stable to high pressure and agitation in comparison to plant extracts, a positive requisite for large-scale synthesis. In addition, nanoparticles with desired characteristics could be synthesized by modulation of synthesis variables including pH, time, temperature and amount of fungal biomass [21]. In a recent study, AgNPs were synthesized from Penicillium oxalicum and tested against several pathogenic organisms [22]. A repertoire of fungi have been explored for synthesis of metallic nanoparticles that includes Ag, Au, Cu, Pd, Ti and Zn etc [23].
Amongst multifarious fungi, Aspergillus strains such as A. fumigatus and A. flavus have been reported for extracellular synthesis of AgNPs, while A. niger synthesized AgNPs has been investigated to decipher the antibacterial mechanism in E. coli [23][24][25][26]. Amidst numerous fungi, A. fumigatus has been reported to facilitate rapid synthesis i.e. within 10 min, where nanoparticles displayed high stability and monodispersity. The present study explores the biosynthesis of AgNPs employing extracellular extract of A. terreus ITCC 9932.15, accompanied by optimization of synthesis variables that consists of AgNO 3 concentration, pH, temperature and incubation time. The antibacterial potential of AgNPs was explored against Pseudomonas aeruginosa and Escherichia coli, followed by in vitro anticancer potential in human breast cancer cells (MCF 7).

Materials
For the biosynthesis of AgNPs silver nitrate (AgNO 3 ) was procured from Merck Limited, India. Nutrient agar and Nutrient broth were purchased from Hi-Media Laboratories, India. Dulbecco's Modified Eagle Medium high glucose (DMEM HG), Fetal Bovine Serum (FBS), 0.25% Trypsin-EDTA were purchased from Life Technologies, USA. Dulbecco's phosphate buffered saline (PBS) without calcium and magnesium chloride, Sodium bicarbonate, Resazurin sodium salt was purchased from Sigma Aldrich, India. Kanamycin was purchased from Himedia, India. Filtration was done using Whatman No.1 filter paper and double distilled water (in-house prepared) was used for the preparation of extract and other solutions.

Fungus
A. terreus ITCC 9932.15 was evaluated for its ability to produce silver nanoparticles. The strain was acquired from the Microbial Catalysis and Process Engineering Laboratory, Department of Microbiology, Central University of Rajasthan, Rajasthan, India. Serial dilution technique was employed to isolate and purify the strain from soil samples collected from Ajmer (26°27′ 0″ N, 74°38′ 0″ E), Rajasthan, India. Identification of the purified culture was undertaken by Indian Type Culture Collection (ITCC), IARI, New Delhi, India. The strain was identified as A. terreus and sustained on PDA (potato dextrose agar) slants with periodical subculturing for further experiments.

Cultivation and preparation of A. terreus filtrate
Cultivation of A. terreus was done using MGYP (Malt extract, Glucose, Yeast extract, Peptone) media under shaking conditions (96 h, 30°C, 150 rpm). Following incubation, the filtrate was centrifuged (10 000 rpm, 4°C, 10 min; Hanil Combi 514R centrifuge) to obtain the mycelia. The supernatant was discarded and the pellet was weighed. The wet weight of the mycelial pellet was ∼25 gm. Further, the pellet was suspended in pre-sterilized distilled water (50 ml) and incubated (120 rpm, 24 h, 30°C). Following incubation, mycelium was obtained through filtration using Whatman filter paper. Further, the obtained filtrate was scanned using UV-vis spectrophotometer and stored (4°C) for future studies.

Extracellular biosynthesis of AgNPs
A. terreus filtrate was employed for extracellular biological production of AgNPs. Briefly, 1 mM solution of silver nitrate (AgNO 3 ) was added to the fungal filtrate and incubated (35°C, 150 rpm) under dark conditions. Additionally, AgNO 3 (1 mM) solution incubated under parallel settings was used as control.

UV-visible spectroscopic analysis of biosynthesized AgNPs
The extracellular biosynthesis of AgNPs was monitored by the observable variations occurred in the solution with time. Aliquots (1 ml) were withdrawn at regular intervals and scanned (300-600 nm wavelength) by UVvisible spectrophotometer (Evolution 201, Thermo Scientific, USA) for validation of biological synthesis of AgNPs.

Structural and functional characterization of AgNPs
Surface architecture and size of synthesized AgNPs was studied using TEM. Furthermore, the nanoparticles were subjected to zeta potential analysis employing Zetasizer and occurrence of silver was confirmed using EDX analysis. Macromolecular functional groups involved in the synthesis of AgNPs were analyzed using FTIR. The sample preparations for characterization studies include separation of AgNPs from the reaction by centrifugation (10 000 rpm, 10 min; Fresco 17, Thermo Fisher Scientific, USA). The pellet containing AgNPs was washed twice with double distilled water and resuspended in distilled water. TEM analysis was performed by placing solution on the copper grid and further drying. Whereas, Zeta potential and FTIR analysis was done using whole suspension of AgNPs.

Derivation of various factors for AgNPs synthesis
Four key parameters affecting AgNPs synthesis viz concentration of AgNO 3 , pH, temperature and time were optimized by varying one variable at a time. The range of parameters used for the optimization was: AgNO 3 concentration, 0.25-2 mM; pH, 4-12; temperature, 25°C-55°C and time, 0-24 h. Effect of the parameters was analyzed in terms of AgNPs synthesis by scanning the solution at regular intervals (300-600 nm).

Evaluation of antibacterial potential
Biosynthesized AgNPs were evaluated for their antimicrobial activity against P. aeruginosa (Gram positive) and E. coli (Gram negative) by employing disk diffusion method. The disk diffusion was performed by inoculating the pure bacterial cultures in Luria-Bertini (LB) broth followed by incubation under shaking conditions (overnight, 37°C) till the optical density attains a value of 0.5 Mcfarland turbidity (1-2×10 8 CFU ml −1 ). Culture thus obtained was then evenly spreaded on agar plates. Subsequently, sterile disks impregnated with 4-5 μl of volume with varying amounts of AgNPs (50, 100, 150, 200 μg ml −1 ); were placed over the solidified agar plates using micro-pipette, incubated overnight at 37°C proceeded by measuring the zone of inhibition. Kanamycin was used as a positive control for disk diffusion assay. The experiments were performed with replicates.
2.9. Analysis of anticancer activity 2.9.1. Cell culture Human breast cancer cell line (MCF 7), obtained from NCCS, Pune, India was used for the study. Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum (FBS) was used to maintain the cells. The cells were placed in incubator (37°C, 5%CO 2 atmosphere, 24-48 h), trypsinized and centrifuged (1500 rpm, 5 min). Afterwards, at a density of 8000 cells/well, seeding of the cells was done in 96 well plate and incubated (37°C, 5%CO 2 atmosphere, 24 h) for adherence to the plate surface.

Anticancer assay
A range of AgNPs dilutions (2,4,8,16,32,64, 128 μg ml −1 ) were prepared using double distilled water and added to each well of 96 well plate and incubated (24 h) with subsequent Resazurin sodium salt (Sigma-Aldrich, USA) addition, followed by measuring either absorbance or fluorescence intensities. The deviation in the absorbance or fluorescence on the basis of cellular metabolic activity could be measured. Resazurin sodium salt (10 μl) was added at 0.1 mg ml −1 concentration, following 24 h of AgNPs exposure and incubated (37°C, 5% CO 2 atmosphere) for 3 h. The absorbance was measured at both 570 and 600 nm using ELISA plate reader (Multiskan GO, Thermo Fisher Scientific).

Biosynthesis of AgNPs
The fungal strain employed in the study was identified as A. terreus ITCC 9932.15 by ITCC, IARI, New Delhi, India. The spores of A. terreus appear brown in colour with yellow pigmentation and irregular shape ( figure 1(A)). The fungal filtrate displayed maximum absorbance at 290 nm, indicating the presence of proteins in the filtrate (1D). Further, it was confirmed by performing Bradford assay. The concentration of the protein was found to be 41.6 μg ml −1 . Following this, Incubation of 1 mM AgNO 3 with the fungal extracellular filtrate resulted in biosynthesis of AgNPs. The synthesis of AgNPs was evident from the change in colour to brown from initial colourless, while, the control where only filtrate was taken remained colourless (figures 1(B) and (C)). This colour transformation could be due to the surface plasmon resonance (SPR) of the AgNPs. Further, the biosynthesis of AgNPs was confirmed by UV-vis spectrophotometer scans within range of 200-600 nm wavelengths ( figure 1(D)). The AgNPs synthesis was observed to enhance with time i.e. 1 h to 24 h, with a prominent peak at 420 nm.
The series of biosynthesis studies were done in order to optimize the conditions for optimal AgNPs synthesis. The initial optimization studies were performed at room temperature and involved varied concentration if AgNO 3 solution (0.25 mM-2 mM). As evident from the data, AgNPs synthesis occurred at two AgNO 3 concentrations i.e. 0.5 mM and 1 mM ( figure 2(A)). Further, increase in the strength of AgNO 3 solution resulted in widening of the absorption spectra. This observation could be speculated to be due to either aggregation of AgNPs or simply increase in the size. Variation in the pH i.e. pH 4 to12, of the AgNO 3 solution revealed of maximal AgNPs synthesis at pH 8, while lower nanoparticles synthesis took place at acidic and high alkaline pH ( figure 2(B)). Extreme pH be it acidic or alkaline affects the structure of proteins present in the fungal extract or it may even lead to denaturation rendering unstable AgNPs [27,28]. Our results were in concurrence to the studies where pH 9 was observed to be optimal for AgNPs synthesis [29]. It could be speculated that the pH may induce ionization by transfer of electron at alkaline pH of 8, with different metabolites present in the solution along side AgNO 3 salt; which resulted in formation of AgNPs. Furthermore, the studies done to investigate the influence of temperature suggested of optimal AgNPs synthesis at 35°C (figure 2(C)). Remarkable inhibition in synthesis above 45°C may probably be due to loss of protein activity owing to the denaturation [28]. Time duration of the biosynthesis reaction was also investigated that revealed enhancement of AgNPs formation up to 24 h ( figure 2(D)). Post optimization studies, AgNPs were synthesized at 35°C with 1 mM AgNO 3 at pH 8 accompanied with 24 h incubation. Further, no aggregation was seen upon incubation of AgNPs upto 15 days (figure 3), indicating stability of the suspension which is in agreement with the observation of Xue et al [30].
The size of the nanoparticles can be determined by the equation: where A is corresponds to scattering process (is 3/4 for silver) and go is the velocity of bulk scattering, and v F is the Fermi velocity as given by [31]. The range of size was found to be around 25 nm.

Characterization of AgNPs
High Resolution-Transmission Electron Microscope (HR-TEM) was employed to investigate the size and shape of the AgNPs; the particles thus synthesized were observed to be nearly spherical in shape along with even distribution throughout the sample with minor agglomerates. The average size of the AgNPs was found to be  4(C)). Further, the elemental composition of AgNPs was investigated by EDX analysis ( figure 5(A)). The EDX spectrum showed a strong peak between 3 and 3.2 keV, which could assigned to the binding energy of silver thus, inferring to the synthesis of  AgNPs of crystalline nature. The spectrum revealed the presence of higher amounts of Ag (83.17%) and lower amounts of Cl (16.83%) weight % while the atomic % was of Ag and Cl was 61.9 and 38.1, respectively. Zeta potential of AgNPs as evaluated on Zetasizer was observed as −16 mV, which suggests of remarkable stoutness ( figure 5(B)). The stability of the AgNPs could be corroborated to the negative values as it prevents aggregation [32]. Another contributing factor could be the presence of capping molecules onto the surface of nanoparticles. FTIR studies of filtrate exhibited prominent peaks situated at 3414 cm Thus, the presence of these peaks establishes the participation of amide and imine bonds in capping of AgNPs. This could further be ascribed to the availability of proteins and enzymes in extracellularly secreted fungal extract. High molecular weight proteins have been speculated to be involved in the nanoparticle synthesis wherein the pivotal role is played by NADH-dependent reductase [33]. The presence of this enzyme onto the surface of nanoparticles as well as into the extract was confirmed by fluorescence spectroscopy [33]. Further, an absorption signature peak for the halo compounds was also observed at 671 cm −1 . Earlier, A. terreus has been shown to produce metabolites with chlorine substitution including geodin and erdin [34]. Looking at the FTIR data it can concluded that several biomolecules present in the extracellular fungal extract have potential role in biosynthesis and capping of nanoparticles.

Antibacterial potential of AgNPs
In the present study, disk diffusion assay was utilized to study the antibacterial potential of AgNPs towards Gram positive (P. aeruginosa) and Gram negative bacteria (E. coli) (figure 7). Effective antibacterial activity was observed when bacteria were exposed to high concentration (200 μg ml −1 ) of AgNPs. At AgNPs concentration of 200 μg ml −1 the zone of inhibition was 12 mm while at 50 μg ml −1 it was reduced to only 8 mm in case of P. aeruginosa. This trend was followed in case of E. coli i.e. the zone of inhibition was 12 and 8 mm at 200 and 50 μg ml −1 , respectively. The zone of inhibition of the positive control (Kanamycin) was found to be around 15 mm for both the bacteria. The antimicrobial potential of AgNPs could be attributed to plethora of mechanisms. It could be due to the degradation of bacterial DNA as suggested by Tamboli et al [1]. Cell membrane blebbing and escape of essential biomolecules due to alteration of membrane permeability followed by blockade of essential enzymes has also been reported to contribute to antimicrobial effects [32,35]. Further, it was proposed that the silver nanoparticles impart antibacterial activity differently in gram-negative and grampositive bacteria. The antibacterial effect was stronger in gram-negative bacteria than gram-positive. This was attributed to the cell wall thickness of gram positive bacteria (30 nm) to gram-negative bacteria (3-4 nm) [36]. One of the most common factor that is responsible for antibacterial efficacy is the size of AgNPs. The smaller the size of the nanoparticles, the larger is its surface area. This enhances the contact area of small-sized AgNPs to the cell in relative to the large sized nanoparticles [37]. Also, it is evident from several results that the antibacterial activity depends on the concentration of AgNPs [38,39].

Anticancer potential of AgNPs
Human breast cancer cell line MCF-7 was engaged to evaluate the anticancer potential of AgNPs (figure 8). The cell viability decreased from 80%-35% with increase in the concentration of AgNPs from 2-128 μg ml −1 . The IC 50 value of the nanoparticles was calculated to be 25.24±0.990 μg ml −1 . The size of AgNPs dictates the toxic potential, as it determines the uptake along with the potential interactions with the thiol enzymes [40]. Similarly, in another study it was demonstrated that the hemolysis, protein leakage and other cellular functions are affected by the AgNPs shape and size [41]. Thus, AgNPs display selective participation by disrupting the mitochondrial respiratory chain and formation of reactive oxygen species resulting in damage to the nucleic acids [42]. The disruption of vital cellular processes could also be cited as one of the reason behind toxicity of nanoparticles [40]. In another study, dose dependent anticancer efficacy has been reported by AgNPs synthesized by Aspergillus fumigatus in vivid cancer cell lines [43,44]. The AgNPs showed varying IC 50 values from 31.1-45.4 μg ml −1 depending on the cell line studied [43]. The anticancer potential of AgNPs varies from one to another species, used for biosynthesis. The IC 50 value of 1.47, 2.46 and 3.12 μg ml −1 has been reported for AgNPs biosynthesized using A. japonicus, A. niger and A. michelle respectively in MCF-7 cells [45].

Conclusion
This study evidenced the potential of fungal strain A. terreus ITCC 9932.15 for synthesis of AgNPs. Spherical shape particles with uniform size distribution of ∼25 nm and −16 mV of zeta potential were synthesized by the extracellular fungal extract. In vitro assays suggested of potent antibacterial and anticancer efficacy of AgNPs. Thus, the AgNPs synthesized holds a great promise in effective anti-tumour drug delivery. This overcomes the disadvantages of conventional therapies by crossing the biological barriers and targeted delivery of drugs. The plasmonic properties can be implemented in theranostic approaches, where the AgNPs can be utilised in diagnosis as well as treatment of cancer. However, clinical trials are one of the most important steps that is required to be followed for future direction of its applications. Despite of its valuable properties, AgNPs have toxicity issues that is necessary to be mitigated by meticulous pre-clinical study and elaborate datasets of its toxicities and pharmacological issues.