Biosynthesis and Biological Properties of Nano-Silver from Aspergillus terreus Towards Antibacterial and Antitumor Applications

Abstract

Background: Nanomaterials have emerged as a transformative approach in modern pharmaceutical applications, offering advanced benefits compared to conventional therapies. Among available pharmaceutical nanomaterials, silver nanoparticles (AgNPs) have been reported with broad-spectrum antimicrobial potential and drug delivery potency. Nevertheless, some studies suggested that chemical synthesis of AgNPs might result in redundant chemicals, posing environmental and health risks. To minimize undesired products, a promising approach is to biologically synthesize this potent nanomaterial. Methods: This study ultilized an eco-friendly system for AgNPs synthesis using Aspergillus terreus isolated from the air. Physical properties of biosynthesized AgNPs were evaluated by UV–visible spectroscopy, dynamic light scattering, and scanning electron microscopy analysis. Antibacterial activity of biosynthesized AgNPs was examined by well diffusion and minimum inhibitory concentration, while in vitro cytotoxicity was used to determine the antitumor activity of AgNPs. Results: The biosynthesized AgNPs had a size of around 60 nm, a PDI inferior to 0.2, and a zeta potential of −30 mV. They exhibited potent antibacterial activity against both Gram-positive and Gram-negative pathogens. Additionally, these nanoparticles also exerted a selective antiproliferative effect on MCF-7, A549, and MDA-MB-231 cell lines. Conclusions: Our research presented the potential of biosynthesized AgNPs using Aspergillus terreus for antimicrobial and anticancer applications, offering an eco-friendly and sustainable alternative to traditional chemical methods.

1. Introduction

Nanotechnology has revolutionized different aspects of modern medicine, offering opportunities for advancements in diagnostics, therapeutics, and antimicrobial applications [1,2,3,4,5]. Indeed, nanoparticles (NPs) have emerged as essential tools based on their potent biological properties, unique physicochemical properties, such as a high surface-to-volume ratio, quantum confinement effects, and tunable morphology [6,7]. These characteristics have favoured their applications in broad-range approaches, including targeted drug delivery, pollutant remediation, and biomedical engineering [8,9].

Silver nanoparticles (AgNPs) are currently drawing significant attention for their antimicrobial and anticancer activities, especially their biocompatibility and effectiveness against the drug-resistant phenomenon. Their versatility also extends to applications in imaging and therapeutic interventions [10]. Nevertheless, several studies have suggested that conventional chemical and physical syntheses of AgNPs are normally associated with toxic reagents and energy-intensive processes, posing significant environmental and safety risks [11].

To address these limitations, bio/green synthesis has emerged as a sustainable and eco-friendly alternative. This approach leverages biological agents such as plants, bacteria, and fungi to facilitate the reduction of silver ions into nanoparticles [12,13,14]. Among those, fungi have been demonstrated with promising potential as a biofactory for AgNP production based on their abundant ability to secrete reducing agents like enzymes, proteins, and metabolites. These natural compounds not only enable the synthesis of biocompatible nanoparticles but also stabilize them, resulting in well-defined structures for biomedical applications and a higher yield of synthesis [15].

Fungal-mediated biosynthesis of AgNPs offers many advantages, including scalability, chemical solvent reduction, simplicity, and sustainability [16,17,18]. Fungal species such as Schizophyllum commune and Geopora sumneriana were reported for their ability to produce AgNPs with potent antimicrobial and anticancer properties [19]. Indeed, biosynthesized AgNPs can disrupt the microbial membrane, induce oxidative stress, which interferes with DNA replication and cause cell death. This mechanism of action makes AgNPs highly effective against biofilm-forming and multidrug-resistant pathogens. Furthermore, the anticancer activity of these nanoparticles has been documented with selective cytotoxicity through apoptosis induction, mitochondrial disruption, and reactive oxygen species generation [20]. Among different fungi, Aspergillus terreus (A. terreus), a filamentous species, has emerged as a promising source for AgNP biosynthesis. In this research, we investigate the potential of A. terreus for biosynthesizing AgNPs with bioactivity against bacterial and cancer cell proliferation. Our results thus indicate a promising bio-approach for AgNP production, which may serve as a sustainable solution for the development of nanotherapy.

2. Materials and Methods

2.1. Isolation and Culture of A. terreus

A. terreus was isolated from the air and cultured on Potato dextrose agar (PDA) medium (MH096, HiMedia, Maharashtra, Mumbai, India) under aseptic conditions [21]. The pure fungal colonies were maintained at room temperature and routinely sub-cultured to ensure their viability and growth. To prepare biomass for AgNP synthesis, A. terreus spores were inoculated into 100 mL of Potato Dextrose Broth (PDB) medium (GM403, HiMedia, Maharashtra, Mumbai, India) and incubated on an orbital shaker at 150 rpm at room temperature for 72 h.

2.2. Identification of A. terreus

The isolated A. terreus was identified by two methods: morphological identification and molecular identification based on the Internal Transcribed Spacer (ITS) sequence analysis by PCR.

2.2.1. Morphological Identification

The morphological identification of A. terreus was performed by observing macroscopic and microscopic characteristics of the fungal strain grown on PDA medium [22]. Specific macroscopic features, including colony colour, diffusible pigments, growth rate, and distinctive characteristics such as exudates, colour particles, and odor, were documented. Microscopic analysis involved observing aerial and substrate mycelia, spore mass colour, sporophore morphology, and the arrangement of spore chains [23]. These observations were performed using the Lactophenol Cotton Blue (S016, HiMedia, Maharashtra, Mumbai, India) tease mount technique under a light microscope to document both asexual and sexual structures.

2.2.2. Molecular Identification

For molecular identification, seven-day-old fungal colonies were used. Genomic DNA was extracted following a modified protocol and subsequently subjected to PCR amplification based on the ITS region. The ITS-1 forward primer (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 reverse primer (5′-GCTGCGTTCTTCATCGATGC-3′) were used for amplification [24]. The PCR cycling was set as follows: 95 °C for 1 min, followed by 35 cycles of 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 7 min. Amplified PCR products were analyzed by electrophoresis and then sequenced. The ITS sequences obtained were analyzed using BioEdit software version 7.2 and compared with entries in the National Center for Biotechnology Information (NCBI) database to confirm the identity of A. terreus.

2.3. Biosynthesis of AgNPs

The fungal biomass was harvested by filtering the culture through Whatman filter paper and washing three times with sterile water to remove residual culture medium. A total of 5 g of washed fungal biomass was transferred to a 250 mL Erlenmeyer flask containing 50 mL of sterile distilled water. The flask was incubated on an orbital shaker at 150 rpm at 25–30 °C for another 72 h to stimulate the release of bioactive compounds, including enzymes and metabolites. The mixture was filtered through a Whatman filter to obtain the crude cell-free filtrate, which was used for the biosynthesis of AgNPs [25].

To synthesize AgNPs, 50 mL of the crude cell-free filtrate was mixed with 10 mL of a 10 mM AgNO₃ solution in a 250 mL Erlenmeyer flask and agitated manually for 30 s to ensure homogeneous mixing. The reaction mixture with a pH of 7.0 was incubated in the dark at 25–30 °C for 72 h to prevent photoactivation. A control experiment was conducted using distilled water and crude cell-free filtrate without the addition of AgNO₃ for non-specific reactions. The formation of AgNPs was visually monitored by a colour change from light yellow to brownish yellow, which is specific for nanoparticle synthesis [26]. The supernatant was removed by centrifugation at 21,420× g for 10 min, and the AgNP pellet was washed 3 times with sterile demineralized water. The AgNP pellet was dried, weighed, and re-dispersed in sterile demineralized water to prepare a stock suspension at 10 mg/mL.

2.4. Characterization of Silver Nanoparticles

2.4.1. UV-Visible Spectroscopy

The biosynthesized AgNPs were characterized using a UV-Vis spectrophotometer (Ultrospec 3100 pro, Biochrom, Cambridge, UK) [27]. The absorbance spectrum was recorded within the wavelength range of 200–700 nm to detect the characteristic surface plasmon resonance (SPR) peak, which confirms the formation of AgNPs. Sterilized deionized water was used as the blank for all UV-Vis spectrophotometric analyses to ensure baseline correction and eliminate background absorbance.

2.4.2. Scanning Electron Microscopy (SEM)

The morphology of the biosynthesized nanoparticles was assessed using scanning electron microscopy. For SEM analysis, the reaction mixture was centrifuged at 21,420× g for 10 min, and the resulting pellet was washed three times with sterile water to remove any impurities. The purified AgNPs were dried at room temperature in a desiccator for 24 h, then coated with a thin layer of gold using a rotary pump coater (Q150R ES Plus, Quorum Technologies, East Sussex, UK). Thereafter, SEM analysis was conducted using a field-emission scanning electron microscope (Phenom ProX, Thermo Fisher Scientific, MA, USA).

2.4.3. Powder X-Ray Diffraction (PXRD)

Powder X-ray diffraction (PXRD) patterns were obtained using a Bruker D8 Advance diffractometer, operating at 40 kV and 40 mA, with a Cu Kα radiation source (λ = 1.54178 Å). The PXRD data were collected in the 5° to 50° range with a step time of 0.25 s per 0.020°.

2.4.4. Size and Zeta Potential Analysis

The particle size, polydispersity index (PDI), and zeta potential of the AgNPs suspension were determined using Zetasizer Nano ZS (Malvern Panalytical, Worcestershire, UK) at ambient temperature (approximately 25 °C). Dynamic light scattering (DLS) was employed to determine the particle size and PDI, while electrophoretic light scattering was used to measure the zeta potential. Before analysis, the AgNP suspensions (10 mg/mL) were diluted with deionized water to obtain a concentration of 0.1 mg/mL, and the particle size was based on intensity using hydrodynamic diameter.

2.5. Antibacterial Activity of Biosynthesized AgNPs

2.5.1. Preparation of Bacterial Strains

The antibacterial activity of AgNPs was assessed for both Gram-positive and Gram-negative bacterial strains, including Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300), Methicillin-sensitive Staphylococcus aureus (MSSA, ATCC 25923), Klebsiella quasipneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Shigella flexneri (ATCC 12022), Salmonella enterica (ATCC 13076), Proteus vulgaris (ATCC 49132), and Streptococcus faecalis (ATCC 19433). The bacteria were maintained on nutrient agar slants at 4 °C. To initiate bacterial growth, frozen stocks stored at −80 °C were streaked onto Mueller-Hinton agar (MHA) medium (M173, HiMedia, Maharashtra, Mumbai, India) and incubated overnight at 37 °C for 12 h. Following incubation, three to five colonies from the plates were transferred to test tubes containing 4 mL of Tryptone soya Broth (TSB) medium (LQ009A, HiMedia, Maharashtra, Mumbai, India). The cultures were incubated at 37 °C overnight until the stationary growth phase was reached. The bacterial cell density was measured using a UV-Vis spectrophotometer (Ultrospec 3100 pro, Biochrom, Cambridge, UK) at 600 nm (OD₆₀₀). The optical density (OD) of the bacterial suspensions was adjusted to a range of 0.08–0.12 to standardize the inoculum for subsequent antibacterial assays. This preparation ensured consistent bacterial growth conditions and reliable evaluation of the antibacterial efficacy of biosynthesized AgNPs.

2.5.2. Well Diffusion Assay

The antibacterial activity of biosynthesized AgNPs was assessed using the well diffusion method [28]. Bacterial suspensions were prepared in sterile saline and adjusted to a turbidity equivalent to the 0.5 McFarland standard, corresponding to approximately 1 × 10⁸ CFU/mL. MHA plates containing 15 mL of medium were inoculated by evenly spreading the bacterial suspension across the agar surface using a sterile cotton swab to ensure uniform distribution. Wells were created in the agar using a sterile stainless-steel cylinder. Each well was filled with 50 μL of the AgNP solution (equivalent to 1024 µg/mL) using a micropipette. Negative controls, consisting of wells filled with sterile distilled water, and positive controls, containing 50 μL of ciprofloxacin (equal to 5 ug ciprofloxacin for each well), a standard antibiotic solution, were included on each plate. After the incubation at 37 °C for 24 h, the antibacterial activity was assessed by measuring the diameter of the inhibition zones (ZOI) surrounding each well in millimeters. The results were recorded for comparison with control samples, providing insights into the efficacy of the biosynthesized AgNPs against the tested bacterial strains.

2.5.3. Minimum Inhibitory Concentration (MIC)

The MIC of the biosynthesized AgNPs was determined using the broth microdilution method in sterile 96-well plates. From the stock suspension at 10 mg/mL, the AgNPs solution was diluted to a concentration of 2048 µg/mL. To create a concentration gradient, 100 µL of serial two-fold dilutions of biosynthesized AgNP solution were performed across the wells of the plate using MHB (M391, HiMedia, Maharashtra, Mumbai, India), with concentrations ranging from 2048 µg/mL in the first column to the lowest concentration in the final column. Each well received 100 µL of a standardized bacterial suspension, prepared at a density of 1 × 10⁶ CFU/mL, to achieve a total volume of 200 µL per well, 200 µL of MHB medium to confirm sterility, while the positive control contained 100 µL of MHB and 100 µL of the bacterial suspension without AgNPs to verify bacterial growth. The plates were incubated at 37 °C for 24 h. Following incubation, 50 µL of a 0.15 mg/mL resazurin solution (62758-13-8, Thermo Fisher Scientific, Waltham, MA, USA) was added to each well to assess bacterial viability. The plates were further incubated in the dark for an additional 4 h. The resazurin solution served as a colourimetric indicator of bacterial metabolic activity, with a blue colour indicating no bacterial growth (negative sample) and a pink colour indicating active bacterial growth (positive sample). The MIC was defined as the lowest concentration of AgNPs that completely inhibited bacterial growth, as indicated by the absence of turbidity and retention of the blue colour in the resazurin assay.

2.6. Anticancer Activity of Biosynthesized AgNPs

2.6.1. Cell Culture

The anticancer potential of the biosynthesized AgNPs was assessed using human cancer cell lines, including MCF-7 (ab257303, Abcam, Cambridge, UK), A549 (CCL-185, ATCC, Manassas, VA, USA), and MDA-MB-231 (HTB-26, ATCC, Manassas, VA, USA). HEK293T cells (CRL-1573, ATCC, Manassas, VA, USA) were used as a non-cancerous control to evaluate the cytotoxicity of the AgNPs. All cell lines were cultured in DMEM (11965092, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (11570506, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (15140122, Thermo Fisher Scientific, Waltham, MA, USA). The cultures were maintained at 37 °C in a humidified incubator with 5% CO₂ to ensure optimal growth conditions for the cells.

2.6.2. Cytotoxicity Assay

The cytotoxicity of biosynthesized AgNPs was evaluated using the MTT assay (E-CK-A341, Elabscience, Houston, TX, USA) [29]. Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight under standard culture conditions. The cells were cultured for 24–48 h until they reached 70–80% confluence. Once confluence was achieved, the old medium was aspirated, and the wells were washed twice with 1X PBS (10010023, Thermo Fisher Scientific, Waltham, MA, USA) to remove any residual medium or debris. Following the washing step, 100 µL of two-fold serial dilutions of the stock AgNPs (prepared in culture medium) at concentrations ranging from 0.625 mg/mL to 10 mg/mL were added to the respective wells. Culture medium was used as a negative control. The plates were incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO₂. After the incubation period, the medium containing AgNPs was removed, and the wells were washed twice with 1X PBS to eliminate unbound nanoparticles. Then, 100 µL of fresh culture medium and 10 µL of MTT solution (5 mg/mL) (PB180519, Elabscience, Houston, TX, USA) were added to each well, followed by a 4 h incubation to allow the formation of formazan crystals. The samples were dissolved in 50 µL of DMSO (AAJ66650AD, Thermo Fisher Scientific, Waltham, MA, USA), and the absorbance was measured at 570 nm using a microplate reader. Cell viability was determined by normalizing the OD values of treated cells to those of untreated control cells, according to the following equation [30]:

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)={\displaystyle \frac{ODtreatedcells}{ODuntreatedcells}}\times 100$$

2.7. Statistical Analysis

All experiments were performed in triplicate, and data were expressed as mean ± standard deviation. Statistical significance was determined using one-way ANOVA, with a p-value < 0.05 considered significant. All analyses were performed using GraphPad Prism version 8 (GraphPad Software, Boston, MA, USA).

3. Results

3.1. Morphological Characterization and Molecular Identification of A. terreus

The isolation of A. terreus from air samples was confirmed through a combination of macroscopic, microscopic, and molecular analyses. Colonies of A. terreus grown on PDA displayed rapid growth, forming dense, velvety, and suede-like mycelial mats with a distinctive cinnamon-brown colouration (Figure 1A). The reverse side of the colonies exhibited pigmentation ranging from pale yellow to deep, dirty brown (Figure 1B). These macroscopic characteristics are consistent with descriptions in fungal taxonomic literature and underscore the rapid-growing nature of A. terreus.

Subsequent microscopic examination confirmed the identity of A. terreus. The conidial heads were observed to be compact, columnar, and biseriate, which are hallmark features of this species. Conidiophores were smooth-walled, short, and hyaline, terminating in biseriate phialides located on the upper half of hemispherical vesicles. The conidia were globose to subglobose or ellipsoidal in shape, smooth-walled, and ranged in colour from hyaline to slightly yellow, with diameters of 2–2.5 µm (Figure 1C). Additionally, septate hyphae occasionally bore solitary conidia or aleurioconidia, further supporting the identification of the isolate as A. terreus (Figure 1D).

Molecular identification of A. terreus was conducted through sequencing the ITS region. The obtained ITS sequence was subjected to BLAST v2.16.0 analysis of the NCBI database (Figure 1E). That revealed 100% query coverage and 100% sequence identity with A. terreus, with an E-value of 0, confirming the identification of our fungal isolate.

3.2. Biosynthesis and Properties of AgNPs

The isolated Aspergillus terreus was subsequently used for the biosynthesis of AgNPs, as outlined in Figure 2A. The formation of AgNPs using the cell filtrate of A. terreus was confirmed through visual observation and spectral analysis. Upon the addition of AgNO₃ to the cell filtrate, a distinct colour change was observed, transitioning from a clear yellow to a brownish-yellow hue, indicative of the reduction of silver ions and the formation of AgNPs (Figure 2B). In contrast, no colour change was observed in the cell filtrate without AgNO₃ (Figure 2C). The brownish-yellow colour intensity of the cell filtrate containing AgNO₃ remained stable after 72 h of incubation, suggesting that the biosynthesized AgNPs were well-dispersed in the solution and no significant aggregation occurred. Further confirmation of AgNP formation was obtained through UV-Vis spectroscopic analysis. The spectral data revealed a characteristic SPR absorption peak at 405 nm, a representative feature of AgNPs (Figure 2D), whereas similar peaks were not obtained in the cell filtrate sample without AgNO₃. These results demonstrated the formation of biosynthesized AgNPs by the cell filtrate of A. terreus, with stable dispersion and characteristic optical properties.

3.3. Characterization of Biosynthesized AgNPs

The morphology and size distribution of the biosynthesized AgNPs were characterized by SEM, DLS, and PXRD analyses. SEM images showed that the AgNPs were predominantly spherical with smooth surfaces (Figure 3A). Their appearance was well-dispersed, with no significant aggregation observed, indicating a uniform and controlled synthesis process. The biosynthesized AgNPs also presented a broad size distribution, with the majority of particles falling within the 90–130 nm range and peaking at 100–110 nm (Figure 3B). From DLS analysis, our AgNPs presented a monodisperse size distribution, with an average hydrodynamic diameter of 69.46 ± 3.7 nm and a PDI of 0.182 ± 0.031. Furthermore, a zeta potential of −30 ± 5 mV was also determined for the obtained AgNPs.

The crystalline structure of the biosynthesized AgNPs was determined by PXRD (Figure 3C). The PXRD patterns of the AgNP-containing samples were two sharp diffraction signals appear at 2θ ≈ 31.5° and 45°, while the characteristic reflections of face-centered cubic (fcc) silver at 2θ ≈ 38.1°, 44.3°, 64.4°, and 77.5°, corresponding to the (111), (200), (220), and (311) planes, were undetectable. The lack of defined Ag diffraction peaks suggested that the formed silver species did not possess long-range crystalline order. In addition, strong interactions between silver species and surrounding bioorganic components or capping agents could effectively inhibit crystal growth, resulting in reduced crystallinity and broadened or suppressed diffraction features. Thus, the absence of Ag reflections did not exclude the presence of metallic silver in the material, as has been widely reported for bio-stabilized or ligand-protected Ag nanoparticles [31,32].

Furthermore, diffraction peaks observed at approximately 2θ ≈ 31.5° and 45° are not characteristic of metallic silver and are therefore unlikely to originate from crystalline Ag phases. Similar unassigned diffraction signals in this angular range have previously been attributed to crystalline or amorphous organic phases associated with biomolecules or organic stabilizing agents present in biosynthesized nanoparticle systems. In particular, unassigned peaks appearing at 2θ ≈ 27.96°, 32.28°, and 46.18° have been reported to arise from organic components rather than metallic phases, supporting the interpretation that the observed diffraction signals in the present study are related to organic matrices involved in the stabilization of AgNPs rather than bulk silver crystallites.

3.4. Antibacterial Activity of AgNPs

The antibacterial activity of the biosynthesized AgNPs was evaluated using the well diffusion assay and MIC determination. The well diffusion assay demonstrated that AgNPs effectively inhibited the growth of both Gram-negative and Gram-positive bacterial strains, as evidenced by the formation of clear zones of inhibition around the wells containing AgNPs (Figure 4A). At a concentration of 1024 µg/mL of biosynthesized AgNPs, the largest zones of inhibition were observed for MSSA, Shigella flexneri, and Escherichia coli, with diameters of 12.95 mm, 12.5 mm, and 12.05 mm, respectively. Other bacterial strains, including MRSA, Streptococcus faecalis, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella quasipneumoniae, exhibited moderate sensitivity to AgNPs, with inhibition zone diameters ranging from 10.4 to 11.55 mm. In contrast, Proteus vulgaris displayed higher resistance, with the smallest zone of inhibition measuring 8.55 mm (Table S1).

MIC determination further supported the antibacterial efficacy of AgNPs, with Gram-negative bacteria showing greater sensitivity compared to Gram-positive strains. Among Gram-positive bacteria, MRSA, MSSA, and S. faecalis exhibited MIC values of 256 µg/mL (1/8 of the initial concentration) (Figure 4B,E). Similarly, S. enterica and P. vulgaris displayed an MIC of 256 µg/mL (Figure 4C,F). In contrast, Gram-negative bacteria, including E. coli and K. quasipneumoniae, demonstrated lower MIC values of 128 µg/mL (1/16 of the initial concentration) (Figure 4E,F). Notably, S. flexneri and P. aeruginosa exhibited the highest sensitivity to AgNPs, with MIC values of 64 µg/mL and 32 µg/mL, respectively (Figure 4C,D). The superior antibacterial effect of AgNPs against P. aeruginosa was consistent with findings from previous studies (Figure 4B–G). These results highlight a broad-spectrum antibacterial potential of AgNPs, with varying levels of sensitivity across different bacterial strains (Figure 4G). The enhanced efficacy against Gram-negative bacteria, particularly P. aeruginosa and S. flexneri, suggests promising applications of AgNPs in addressing antimicrobial resistance and treating infections caused by multidrug-resistant pathogens.

3.5. Anticancer Activity of AgNPs

AgNPs derived from other fungi were demonstrated to exert anti-proliferative effects against some cancer cells, such as MCF7, HeLa, or HepG2 [33,34]. Thus, we also evaluated our biosynthesized AgNPs for their anti-proliferative capacity towards cancer cell lines from different tumour sources or hormone response. The biosynthesized AgNPs also exhibited potent anticancer activity against the tested cell lines, including MCF7, A549, and MDA-MB-231, with comparatively lower cytotoxic effects on the non-cancerous HEK293T cells. A clear dose-dependent reduction in cell viability was observed across all cancerous cell lines, with increasing concentrations of AgNPs leading to significant cytotoxicity. Particularly, the non-cancerous HEK293T cells showed minimal cytotoxicity at lower concentrations of AgNPs, with a more gradual decline in viability at higher doses (Figure 5A). Whereas the MCF7 cells showed the highest sensitivity to AgNPs, with a marked decline in viability at lower concentrations, followed by A549 cells, which also exhibited substantial dose-dependent cytotoxic effects (Figure 5B,C). MDA-MB-231 cells, while affected, displayed comparatively greater resistance, indicating a less pronounced reduction in viability at equivalent AgNP concentrations (Figure 5D). This data suggested selective targeting of cancer cells by our AgNPs, with significant efficacy against breast and lung cancer cell lines while sparing non-cancerous cells.

4. Discussion

This study represents an environmentally sustainable approach in nanotechnology by utilizing the biosynthesis of AgNPs using A. terreus isolated from the air. By exploiting fungal-mediated synthesis, our research demonstrates an eco-friendly alternative in addition to conventional chemical and physical methods for producing AgNPs with potent biological activities.

The use of A. terreus for AgNP biosynthesis highlights the potential of fungi as biocatalysts based on their ability to secrete bioactive metabolites, including enzymes and proteins, which act as reducing and stabilizing agents. While conventional methods often require some toxic chemicals and energy-intensive processes, this approach is environmentally benign and cost-effective. In addition, fungal systems offer many advantages, such as higher biomass production, ease of cultivation, and the secretion of significant quantities of extracellular metabolites for nanoparticle stabilization [35,36,37]. In this study, A. terreus exhibited promising potential as a bio-source for producing AgNPs, which were validated by different analytical methods. Our approach aligns with global initiatives to adopt sustainable practices in nanotechnology as well as establish A. terreus as a potential candidate for large-scale nanoparticle synthesis.

The obtained biosynthesized AgNPs presented a spherical shape, uniform distribution, and minimal aggregation, as confirmed by SEM and DLS analyses. The monodisperse nature of the nanoparticles might enhance their bioactivity and applicability in biomedical contexts. In comparison to chemically synthesized AgNPs, our biosynthesized AgNPs represent a slight increase in size compared to chemical synthetic ones (60 nm vs. 5–20 nm) but still in the range of 10–100 nm [38,39]. However, the zeta potential was similar between our AgNPs and the chemically synthetic ones (around −30 mV), reflecting a good stability in the colloidal system [38,39]. Other bio-related methods for AgNP synthesis, including the use of herbal extracts as reducing agent that were documented to produce AgNPs with sizes ranging from 10–40 nm and zeta potential around −30 mV [40,41,42]. In a study of Sharma et al., the authors used Talaromyces purpureogenus for producing AgNPs with a size of 240.2 nm, a PDI of 0.720, and a zeta potential around −20 mV [43]. In contrast, the AgNPs obtained from a study of Wang et al., using Aspergillus sydowii, had a size of around 24 nm [34]. Despite the differences in size, those AgNPs were recorded with various biological activities, including antibacterial and anticancer properties. These findings suggest that the size of AgNPs may vary according to the source of reducing agent. Though the specific mechanisms of biogenic synthesis of AgNPs have not been fully elucidated, some studies indicated that reduced nicotinamide adenine dinucleotide (NADH) produced by A. terreus might account for the formation of AgNPs [44,45]. Further studies on the impact of reducing the source on the biological activities of the obtained AgNPs are needed.

Our AgNPs exerted broad-spectrum antibacterial activity for both Gram-positive and Gram-negative bacterial strains. We observed a good antibacterial efficacy against Pseudomonas aeruginosa and Shigella flexneri as evidenced by low MIC values (32 µg/mL and 64 µg/mL, respectively), although a higher resistance of Gram-negative bacteria to chemically synthesized AgNPs was noted in some studies [46,47,48]. The enhanced activity can be attributed to the uniform size and stability of the nanoparticles, which improve interaction with bacterial membranes and oxidative stress induction. In a study of Urnukhsaikhan et al., the synthesized AgNPs from Carduus crispus extract showed an antibacterial activity on E. coli and M. luteus [49]. The AgNPs from Rhus coriaria L. extract inhibited the growth of different bacterial strains such as S. Mutans, E. faecalis, S. sobrinus, S. salivarius, and L. acidophilus [50]. Fungi-derived AgNPs were frequently reported to have better antibacterial properties on more bacterial strains and lower MIC values. For instance, Sharma et al. used Talaromyces purpureogenus for the synthesis of AgNPs with a potent antibacterial activity on Listeria monocytogenes, Escherichia coli, Shigella dysenteriae, and Salmonella typhi [43]. A better antibacterial property of AgNPs synthesized using fungi compared to the chemical method was also reported in a study of Qiao et al., using the endophytic fungus Letendraea sp. WZ07 [51]. Our study obtained a larger antibacterial zone of E. coli and M. luteus compared to the AgNPs from Carduus crispus extract [49].

A selective cytotoxicity was detected for our biosynthesized AgNPs towards different cancer cell lines (MCF7, A549, and MDA-MB-231) while sparing non-cancerous HEK293T cells. The anticancer properties of AgNPs synthesised from bio-related methods have been reported, but slightly differ from the sources. In particular, AgNPs synthesised from the fruit extract of Azadirachta indica demonstrated a high cytotoxicity against A549 lung cancer cells, while AgNPs from Carduus crispus showed a low cytotoxicity against HepG2 cells [49,52]. In this work, a selective anticancer activity was noticed, indicating the potency of A. terreus as a source of AgNP synthesis. This selective targeting may be attributed to mechanisms reported in previous studies, involving apoptosis induction through ROS generation and mitochondrial disruption [53,54,55]. However, the resistance of MDA-MB-231 cells requires a deeper understanding of the AgNP molecular mechanism of action. These also suggest that the anticancer activity of biosynthesized AgNPs may be dependent on the source of reducing agents. Future studies on comparing and selecting optimal sources for AgNPs should be performed.

Collectively, our data presented a convenient, eco-friendly, and cost-effective method for biosynthesis of AgNPs using A. terreus with potent antibacterial and anticancer activities. Future direction is the utilization of biosynthesized AgNPs alone or in combination with drugs/bioactives for enhancing and selectively targeting specific cancer cells or pathogenic microorganisms.

Our study has several limitations. First, while the biosynthesis of AgNPs by A. terreus was confirmed, the specific biochemical pathways and metabolites responsible for reduction and stabilization were not identified. Investigating these pathways could improve reproducibility and enable the optimization of nanoparticle synthesis. Second, we did not examine the underlying molecular mechanisms of the antibacterial and anticancer activities in detail. Advanced molecular techniques, such as transcriptomic and proteomic analyses, could provide insights into these mechanisms. Third, the dependence on environmental conditions during biosynthesis may pose challenges for scalability and reproducibility, particularly for industrial applications. Future research should focus on optimizing the biosynthesis process to enhance yield and consistency, identifying molecular pathways involved in the bioactivity of AgNPs, and conducting in vivo evaluations to validate their therapeutic potential. Fourth, incorporating advanced characterization techniques, such as Fourier-transform infrared spectroscopy (FTIR) and Single-Crystal X-ray diffraction (SCXRD), could also provide a deeper understanding of the nanoparticle surface chemistry and crystalline structure. By addressing these limitations, the application of fungal-mediated AgNP synthesis could be expanded to industrial, biomedical, and environmental domains, paving the way for a more sustainable and innovative approach to nanotechnology.

5. Conclusions

This study represents A. terreus as a promising source for the biosynthesis of AgNPs, highlighting a sustainable and environmentally friendly approach to nanotechnology. The biosynthesized AgNPs demonstrated remarkable uniformity, stability, and potent antibacterial and selective anticancer properties, outperforming many findings reported in recent years. Despite certain limitations, this research establishes a robust foundation for the utilization of fungal-mediated AgNP synthesis in biomedical and environmental applications, offering significant potential for advancing sustainable nanotechnology innovations.