Biosynthesis of multifunctional silver nanoparticles using a Penicillium verrucosum RPF011 extract for antibacterial and anticancer applications

Abstract

This study reports the green synthesis of silver nanoparticles (PV-Ag NPs) using a pigment-producing environmental fungus, Penicillium verrucosum (PV) RPF011, as a biological reducing and stabilizing agent. The formation of nanoparticles was initially evidenced by a distinct color change from light to dark brown, corresponding to the reduction of Ag⁺ ions, and was further confirmed by UV–visible spectroscopy showing a characteristic surface plasmon resonance peak at 433 nm. Fourier transform infrared (FTIR) analysis indicated the involvement of proteinaceous biomolecules in the reduction and capping processes, ensuring nanoparticle stability. X-ray diffraction (XRD) patterns revealed the crystalline nature of the synthesized nanoparticles, while high-resolution transmission electron microscopy (HR-TEM) analysis demonstrated that the PV-Ag NPs were predominantly spherical and polydisperse, with an average particle size of 78 ± 2 nm. The biologically synthesized nanoparticles exhibited potent antibacterial activity against clinically relevant pathogens, including Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Furthermore, in vitro cytotoxicity assays revealed significant anticancer activity against the human lung carcinoma A549 cell line, with an IC₅₀ value of 53.22 ± 1.5 µg/mL. The enhanced bioactivity of PV-Ag NPs can be attributed to their nanoscale dimensions and the presence of bioactive capping agents derived from the fungal extract. Overall, the findings highlight the potential of P. verrucosum-mediated silver nanoparticles as an eco-friendly and cost-effective nanoplatform with dual therapeutic applications in antimicrobial and anticancer treatments. This study underscores the promise of fungal-mediated nanotechnology in developing novel nano-drug formulations for managing infectious diseases and lung cancer.

Graphical abstract

Introduction

Biofunctionalized nanoparticles (NP) are an innovative material known for their remarkable functionalities across nearly all biomedical science and engineering fields [1–3]. Various promising metals, such as silver, gold, copper, and palladium, have garnered significant interest from researchers [4]. Among these, silver is commonly employed for nanoparticle production due to its unique chemical stability, catalytic characteristics, and conductivity [5, 6]. Silver’s antibacterial properties have led to its widespread use in medicine [7]. However, current (chemical and physical) methods for fabricating silver nanoparticles (Ag NPs) often rely on harmful chemicals, are costly, require advanced experimental setups, and can lead to environmental contamination [8, 9]. Therefore, there is a pressing need to develop eco-friendly approaches to synthesize biofunctionalized Ag NPs that minimize environmental pollution.

One promising technique is the biosynthesis of nanoparticles, which uses plants and microorganisms (bacteria, actinomycetes, algae, and fungi) to produce metal nanoparticles [10–13]. This method is more sustainable and biosafe compared to traditional chemical and physical approaches. In addition, plants and other microbes can synthesize NPs both extracellularly and intracellularly, and these extracts operate as reducing agents, changing the chemical composition of metals through a process of protection against hazardous substances [14, 15]. Plant extracts and microbial extracts used in NP synthesis contain molecules such as carbonyl groups, amines, amides, terpenoids, phenolics, proteins, and pigments. Among natural sources, fungi have been widely exploited as biological agents due to their low cost, high biomass output, low toxicity, and low energy consumption [16, 17]. Fungi produce a wide range of metabolites, enzymes, and extracellular proteins [18]. These biomolecules operate as reducing agents while simultaneously capping NPs, so limiting their size and stability [19, 20]. Additionally, fungal secondary metabolites such as phytochelatin, alterosolol, periconicin, chitin, and galactomannans contribute to the reduction of Ag+ ions in the synthesis of nanoparticles [21–23]. These compounds possess antibacterial, antioxidant, antiviral, and anti-inflammatory properties [10–20]. For example, Taxol (paclitaxel) is used in chemotherapy to fight breast, lung, and cervical cancers [10]. In addition, a broad spectrum of filamentous fungi, including Alternaria, Aspergillus, Fusarium, and Penicillium, are employed to generate Ag NPs [14, 20, 24–33]. Silver nanoparticles have excellent antimicrobial characteristics, are durable in extreme stress settings, and can be used to treat a variety of ailments [26, 34].

In this research, we emphasize the fabrication of bioactive Ag NPs using the extract of the endophytic fungus Penicillium verrucosum (PV) for antimicrobial and anticancer applications. Recently, silver nanoparticles coated with fungal extracts have been shown to significantly enhance antimicrobial and anticancer treatments [10, 14, 24, 28, 35–37]. The eco-friendly fabrication of Ag NPs enhances their biocompatibility, sustainability, and biomedical effectiveness in anticancer treatments [13, 15, 16]. In a one-pot synthesis method, bioactive components from fungal extract were bonded to the surface of the Ag NPs [19]. Furthermore, Ag NPs have recently garnered significant interest as potential anticancer agents due to their ability to induce apoptosis in various human cancer cells. Nanotechnology-based cancer treatment strategies have gained popularity in the medical industry over the past decade [2–4, 7, 16]. In the current study, the antibacterial effectiveness of the synthesized PV-Ag NPs was tested against human pathogenic bacterial strains, including E. coli, K. pneumoniae, and S. aureus. The anticancer potential of PV-Ag NPs was evaluated using A549 lung cancer cell lines. Intracellular apoptosis and mitochondrial damage were identified through fluorescence assays. It is expected that these bioactive PV-Ag NPs would serve as an effective and biocompatible material for both antibacterial and anticancer treatments.

Materials and methods

Materials

Silver nitrate, potato dextrose agar (PDA), potato dextrose broth (PDB), Nutrient broth, Mueller Hinton agar (MHA), Milli-Q water, gentamicin, DMEM medium, cisplatin, streptomycin, and methanol were purchased from Himedia Chemicals Company, India. JC-1 stain, Acridine Orange, Ethidium Bromide, and MTT were obtained from Sigma Aldrich, USA. All chemicals and reagents were used immediately, without additional purification. Human pathogenic bacteria, Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 3384), and Staphylococcus aureus (MTCC 7443), were obtained from the Microbial Type Culture Collection at the Institute of Microbial Technology (IMTECH), Chandigarh. Human lung cancer cells, A549 cells, were purchased from the National Centre for Cell Science (NCCS, Pune, India).

Collection of the fungal strain

The fungus Penicillium verrucosum (PV), referenced in GenBank as PP732489, was previously identified as a contaminant in MS medium collected from the Plant Tissue Culture Lab within the Department of Biotechnology, Microbiology, and Bioinformatics at National College (Autonomous) in Tiruchirappalli, Tamil Nadu. This specific strain of PV was subsequently chosen for its potential to synthesize silver nanoparticles, a process that harnesses the organism’s unique properties. To ensure viable and active cultures for experimentation, PV is routinely maintained on potato dextrose agar (PDA) in a controlled environment at a consistent temperature of 28 ± 2 °C, which is conducive to optimal growth and development (Fig. 1A).

Fig. 1.

A Morphology of Penicillium verrucosum (PV) (28 °C, 7days), B Mass culture of Penicillium verrucosum in PDB media and Biosynthesis of PV-Ag NPs

Biomass extract preparation

The fungus was carefully inoculated into 200 mL of PDB and incubated at 28 ± 2 °C for 96 h, in a rotary shaker, set at 120 rpm, ensuring optimal aeration and mixing of the culture. After the incubation, the fungal biomass was collected and filtered using Whatman filter paper No. 1 to separate the mycelium from the liquid medium. To eliminate any remaining undesirable components from the excess culture, the biomass was thoroughly rinsed with sterile distilled water. Subsequently, 10 g of the fungal biomass was suspended in a 250 mL flask containing 100 mL of sterile distilled water, and the mixture was agitated again at 120 rpm for an additional 48 h, maintaining the temperature at 28 ± 2 °C. After this second incubation, the resulting fungal filtrate was carefully separated using Whatman filter paper and used to synthesize silver nanoparticles (PV-Ag NPs), paving the way for potential applications in various fields.

Biosynthesis of Ag NPs

The bioactive PV-Ag NPs were synthesized according to a previous method [19]. To begin, approximately 90 mL of a 1 mM silver nitrate solution was blended with 10 mL of fungal extract and incubated overnight in a dark environment. This process led to a color change of the precursor solution from a vibrant pink to a dark brown, indicating silver nanoparticle formation. The colloidal PV-Ag NPs were then separated through ultracentrifugation at 10,000 rpm for 30 min. The product was rinsed three times with deionized water to eliminate any unreacted precursor contaminants. Afterwards, the hydrated PV-Ag NPs were dried in a hot air oven at 60 °C for 24 h, resulting in a finely powdered form. The PV-Ag NPs powder was then stored at 4 °C for experimentation (Fig. 1B).

Characterization of PV-Ag NPs

To confirm the successful synthesis of silver nanoparticles (Ag NPs), UV-visible spectroscopy was conducted using a UV-3600 spectrophotometer (Shimadzu, Japan). The crystalline structure was examined using a powder X-ray diffraction (XRD) instrument (Shimadzu, XRD6000). The XRD analysis was conducted over a 2θ range of 10–80 degrees. The identification of functional groups within the Ag NPs was performed using a Shimadzu 8400 FTIR Spectrophotometer. The IR range was from 4000 to 400 cm⁻¹. Scanning electron microscopy (SEM) and High-resolution transmission electron microscopy (HR-TEM) were used to assess the surface morphology of the PV-Ag NPs. To evaluate the stability and size distribution of the synthesized PV-Ag NPs, both a Zeta analyzer and dynamic light scattering (DLS) were used (Malvern Instruments Ltd., UK).

Antibacterial assay

The modified agar well diffusion method [15] was employed to assess the antibacterial activity of the synthesized PV-Ag NPs against human pathogens, including Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Bacterial isolates were sub-cultured in nutrient broth, and an overnight culture of each strain (1 × 10⁸ CFU/mL) was uniformly swabbed (200 µL) onto Muller–Hinton agar (MHA) plates using sterile swabs. Different concentrations of PV-Ag NPs (10, 20, 30, and 40 µg/mL) were added to wells, with gentamicin (1 mg/mL) as a positive control. After incubating at 28 ± 2 °C for 24 h, we measured the zones of inhibition in millimeters (mm). To ensure reliability, these experiments were independently repeated three times.

Minimum bactericidal concentration of PV-Ag NPs

The minimum bactericidal concentration of PV-Ag NPs was evaluated against the human pathogenic bacterial strains E. coli, K. pneumoniae, and S. aureus. The study was carried out in a 24-well plate, where 100 µL of bacterial culture was added to each well, along with PV-Ag NPs at different doses ranging from 10 to 100 µg/mL. The plates were incubated overnight at 37 °C under dark conditions. The absorbance of the PV-Ag NPs-treated culture medium was quantified by an ELISA plate reader. The total percentage of bacterial inhibition was quantified by comparing the treated and control groups.

MTT assay

The cytotoxic effects of PV-Ag NPs on lung cancer cells were assessed in vitro using the MTT cytotoxicity assay [36]. Initially, different concentrations of PV-Ag NPs were dispersed in Dulbecco’s Modified Eagle Medium (DMEM). Human lung cancer cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate and incubated at 37 °C for 4 h to allow for cell attachment. After washing the cells with phosphate-buffered saline (PBS) to remove unattached cells, varying doses of PV-Ag NPs (20–100 µg/mL) were added to each well, maintaining a total volume of 100 µL. The cells were then incubated for 24 h at 37 °C, allowing for interaction with the nanoparticles. After this, 10 µL of MTT solution was added, and the cells were incubated for an additional 2 h to enable the reduction of MTT by viable cells. The absorbance was measured at 570–630 nm using a microplate reader.

JC-1 staining for mitochondrial membrane potential loss

The mitochondrial transmembrane potential (MMP) in A549 cells was assessed using the JC-1 staining method [38]. The cells were treated with PV-Ag nanoparticles (NPs), silver nitrate, and the positive control cisplatin, and incubated at 37 °C with JC-1 stain (1 µg/mL) for 30 min. The excess stain was removed with PBS solution. Fluorescent absorbance intensity was measured using a microplate reader (Spectra Max) at excitation and emission wavelengths of 488/525 nm, allowing for quantification of MMP loss. Stunning cellular images were captured with a Nikon ECLIPSE fluorescence microscope at 40x magnification, showcasing the details of cellular dynamics.

Hoechst staining for assessment of nuclear damage

The cellular nuclear changes and apoptotic mechanisms of PV-Ag nanoparticles (NPs) were assessed through Hoechst staining [17]. A549 Cells (1 × 10³) were treated for 48 h with the anticancer drug cisplatin and PV-Ag NPs at various concentrations. Untreated cells served as a negative control (0.1% DMSO), while those treated with cisplatin acted as the positive control. After treatment, the cells were washed with PBS and exposed to a 10 µg/mL Hoechst stain, incubated in the dark for 30 min. Following three washes with PBS to remove excess stain, changes in cell morphology and nuclear damage were examined under a fluorescent microscope with excitation at 350/460 nm. The percentages of apoptotic cells and nuclear membrane blebbing were analyzed across three individual fields of view, highlighting the intriguing effects of PV-Ag NPs in cancer treatment.

Assessment of ROS generation

PV-Ag NPs-generated reactive oxygen species (ROS) were evaluated by 2′,7′- dichlorofluorescein diacetate (DCFH-DA) fluorescence assay [38]. The fully grown A549 cells were incubated with 10 µM of DCFH-DA fluorescent probe at 37 °C for 30 min under CO₂ flow. Cells were exposed to different concentrations of PV-Ag NPs, silver nitrate, and cisplatin ( 20–50 nM). Before flow cytometry, cells were washed with serum-free media and resuspended in 1000 µL of PBS solution. ROS levels were quantified by a microplate reader (Molecular Device Spectramax M3) at 485 and 535 nm excitation and emission wavelengths.

Biocompatibility of PV-Ag NPs nanoparticles

Fibroblast cells (L929) were used to assess the biocompatibility of the PV-Ag NPs. The MTT calorimetric technique was utilized to examine the cytotoxic effect of PV-Ag NPs. The L929 cells were cultured in 96-well plates with DMEM containing 10% of FBS. The cells were treated with PV-Ag NPs and the commercial anticancer drug cisplatin at different concentrations (10 to 100 µg/mL). The wells with PBS solution alone were considered as a negative control. The PV-Ag NPs post-exposed cells were maintained for 24 h at 37 °C, under 5% CO₂ flow. Further, 10 µL of MTT solution was added to each well and again incubated for 4 h for the dye fixation process. After the excess of MTT stain was removed by washing with 100 µL DMSO solution. The percentage of cell viability was quantified from the optical density (OD) of the culture medium under a microplate reader at 490 nm wavelength. This formula quantified the percentage of cell viability.

The morphological change of PV-Ag NPs-treated L929 cells was observed under a fluorescence microscope. Cells were treated with the IC₅₀ concentration of PV-Ag NPs, incubated for 24 h at 37 °C under continuous CO₂ flow. Then, 50 µL of AO/EB live/dead stain solution was added to each well and maintained under optimal conditions. The intracellular changes and dead cells were visualized under fluorescence microscopy.

AO/EtBr dual staining technique

The AO/EB staining assay was performed according to Govindan et al. [37]. The A549 lung cancer cells were pre-seeded in a 96-well culture plate and treated with an IC₅₀ concentration of PV-Ag NPs. Further, nanoparticle-treated A549 cells were incubated for 48 h at 37 °C under a CO₂ incubator. After the incubation period, the cells were trypsinized for microscopic observation. About 20 µl of AO/EB live/dead stains were added to the cell medium. The malformations of treated cells were evaluated under a fluorescent microscope (Carl-Zeiss/Axio). For quantitative studies, approximately 100 cells were counted, and mean values were calculated from triplicate experiments using ImageJ software.

Statistical analysis

All experimental results were presented as means (n = 3) with standard deviation. A significance level of p < 0.05 was set for all tests. Spectral graphs were created using Prism 8.0 software. IC₅₀ and IC₉₀ concentrations were expressed as the mean standard deviation. The results were analyzed with a one-way ANOVA using Origin 8.5 software. **p < 0.05 is statistically significant.

Results and discussion

Mycosynthesis of silver nanoparticles (Ag NPs)

Metal nanoparticles are produced using green chemistry or biological processes. Microorganisms such as bacteria and fungi employ cellular enzymes and secondary metabolites to convert metal ions into NPs like Ag, Au, Zn, and Cu [2, 3, 39]. When exposed to high levels of metallic ion stress, fungi develop a defensive mechanism that decreases metal ion toxicity by precipitating metal ions at the nanoscale to create NPs [9, 10]. Thus, fungi have emerged as a promising source of NPs due to their ability to withstand such metallic ion stress [13]. In this study, we produced Ag NPs from an extract of the previously characterized environmental fungus Penicillium verrucosum. The interaction of the fungal extract and silver nitrate solution resulted in a dark brown precipitate, indicating the formation of Ag NPs (Fig. 1A–B). The excitation of Ag NPs generates a color shift in the reaction mixture [15]. The bioactive components of the fungal extract reduce Ag ions, creating stable and ecologically benign Ag nanoparticles [19, 24].

UV-Vis and powder XRD analysis

The biosynthesis of silver nanoparticles (Ag NPs) through Penicillium verrucosum (PV) fungal filtrates was characterized by a remarkable transformation in color, which transitioned from a vibrant pink to a rich, deep black (Fig. 2A). This striking change occurred after 24 h of incubation with silver nitrate, indicating a successful Ag NP formation [10–12]. Extracellular enzymes from PV fungal cells are essential for reducing silver ions into Ag NPs [13]. To further substantiate this process, UV-vis spectroscopy was employed, revealing pronounced plasmon absorption bands at 433 nm (Fig. 2A). This wavelength underscores the distinctive optical properties of silver nanoparticles, i.e., between 400 and 450 nm [19, 20]. This is due to the phenomenon of surface plasmon resonance (SPR), which occurs when electrons oscillate collectively in the conductive band [14]. In contrast, no absorbance occurred in the fungal extract. A peak at 433 nm indicates the release of active components from PV mycelia, which reduce and cap silver nanoparticles. The enzymatic activity of the aqueous solution triggered the bioreduction of Ag + to Ag0 ions in the colloidal solution, with the appearance of a deep black colour. In addition, light scattering intensity varies with scattering angle, particle refractive index, and distance. However, this approach only considers particle size and the wavelength of incoming light [13]. Biomolecules of fungal extract which bind to the surface of these nanoparticles in a process called capping, which prevents agglomeration and enhances the stability of the formed PV-Ag NPs. This mechanism underscores the dual role both reducing silver ions and stabilizing the nanoparticles through capping [27, 28]. PV-Ag NPs displayed a brown color when suspended in water. The formation of brown color due to the oscillation modes generated by the electromagnetic field in the visible spectrum, along with the collective oscillations of conductive electrons. The extract showed a color change from yellow to dark brown while preparing Ag NPs [13, 19].

Fig. 2.

A UV–visible absorption spectrum of PV-Ag NPs (red), Fungal extract (black), B Powder XRD spectrum of PV-Ag NPs

In this investigation, X-ray diffraction (XRD) analysis was used to explore the crystalline structure of the synthesized PV-AgNPs. The XRD patterns demonstrated a series of sharp diffraction peaks corresponding to the (100), (111), (200), (220), and (311) lattice planes, appearing at 2θ values of 12.98, 32.20, 33.78, 38.17, 46.21, 48.70, 66.62, and 74.98°. The resultant crystal structure aligned closely with the JCPDS File No. 04-0783, confirming the cubic structure typical of silver nanoparticles (Fig. 2B). This comprehensive analysis highlights the successful synthesis and unique characteristics of Ag NPs derived from Penicillium verrucosum, showcasing their potential applications in various fields. This result is consistent with the findings of Gupta et al. [24] and Taha et al. [14], who employed XRD analysis to determine the crystalline and metallic character of Ag NPs produced from P. oxalicum and P. citrinum.

FTIR and surface morphological analysis

The functional groups of synthesized PV-Ag NPs were effectively analyzed using FTIR spectroscopy across a range of 450–4000 cm^− 1. Notably, the fungal extract-mediated PV-Ag NPs displayed distinct IR bands at approximately 3436, 2075, 1630, 1384, and 668 cm^− 1. The broad absorption band observed at 3436 cm^− 1 indicates the O–H stretching vibration of phenolic compounds in the fungal extract, highlighting the extract’s rich phenolic content. Previous research [24, 40] has shown that the hydroxyl group acts as a bio-reduction agent during silver nanoparticle formation. Moreover, the sharp and intense peaks around 2075 and 1384 cm^− 1 correspond to the C–C, C–H, and C–O stretching vibrations associated with alcohols, esters, and ethers present in the extract, suggesting diverse functional groups that contribute to the nanoparticles’ synthesis. The band observed at 1630 cm^− 1 is linked to the bending vibrations of amide I and II proteins, further emphasizing the extract’s biological significance [24]. Interestingly, the FTIR readings of the P. verruscum extract revealed quite consistent results, with only a slight reduction in intensity (Fig. 3A). Overall, the similarities in wavenumbers and positions of the IR spectrum between the PV-Ag NPs and the fungal extract underscore a promising correlation that may enhance our understanding of the synthesis process and functionalization of these nanoparticles. Furthermore, FTIR data confirmed the presence of bioactive substances such as amino acids, carboxylates, alkenes, and carbohydrates, which have been identified as possible reducing agents for metal and metal oxide NP production [22, 28, 40–43] and confirmed the chemical purity of Ag NPs [43].

Fig. 3.

A FTIR spectrum of Penicillium verrucosum extract (black) and synthesized PV-Ag NPs (red), B–C SEM and TEM images of PV-Ag NPs

SEM and TEM analysis

The investigation into the mean particle size and morphology of biosynthesized PV-Ag NPs was conducted using scanning electron microscopy (SEM), yielding insightful results. The SEM images, which illustrate PV-Ag NPs generated from Penicillium verrucosum (PV), reveal a predominantly irregular and hexagonal morphology [20]. The particle sizes ranged from 20.8 to 31.3 nanometers, showcasing the effectiveness of the biosynthesis process. Additionally, the SEM images highlight slightly aggregated, hexagonal nanoparticles (Fig. 3B). While the materials exhibit some irregular size distribution, it is noteworthy that the average particle size remained below 200 nanometers, suggesting a controlled synthesis process [19]. Further analyses using high-resolution transmission electron microscopy (HR-TEM) provided detailed insights into the morphological characteristics and particle sizes (Fig. 3C). The SEM images also depict spongy crystals with agglomerated particles, which can be attributed to the presence of a chitosan outer coating. These findings contribute valuable knowledge to the field and suggest avenues for further research in nanoparticle synthesis and applications.

DLS and zeta potential analysis

The Dynamic Light Scattering (DLS) analysis of the PV-Ag NPs indicates a Z-average size of 166.4 nm, along with a polydispersity index (PdI) of 0.332 (Fig. 4A). The presence of this peak reflects the satisfactory quality of the produced Ag NP. In another study, Cladosporium and Purpureocillium have similar DLS values of 165.6 and 166.7 nm, respectively [40]. PdI measures the average homogeneity of particles in a solution, ranging from 0 to 1. The polydispersity of Ag NPs from Aspergillus sydowii and Penicillium brasilianum NP5 was similar [28, 43, 44]. Furthermore, the Zeta potential measurement for the bioactive PV-Ag NPs in aqueous solution (Fig. 4B) was recorded at -75.2 mV, suggesting excellent stability in suspension. Negative zeta potential values indicate Ag NPs are covered by negatively charged biomolecules, which improves stability by reducing repulsion between particles and charged nanoparticles interacting with other molecules and ions [10–13]. This investigation used no stabilizers, indicating that the released fungal metabolites served as both reducing agents and nanoparticle stabilizers [19, 22, 25]. In dynamic light scattering, nanoparticles are dispersed in a liquid, meaning they carry a hydration shell of bound solvent molecules. The diameter of the particle is the moving layer, known as the hydrodynamic diameter. Due to the electrostatic interaction of water molecules with nanoparticles, the size of the particles is increased. TEM requires samples to be dried and placed under a high vacuum. The solvent evaporates, and any organic stabilizing layers collapse, leaving only the electron-dense, desiccated core. Due to the pure and ultra-fine imaging techniques, the particle size in TEM analysis shows small sizes. Overall, these results highlight the promising characteristics of the synthesized PV-Ag NPs [24, 26].

Fig. 4.

A DLS particle size distribution, B Zeta potential analysis of PV-Ag NPs, C Minimum inhibitory concentration of PV-Ag NPs, fungal extract, and silver nitrate against human pathogens

Antibacterial activity of PV-Ag NPs

Most pathogenic bacteria have developed resistance to all antibiotics presently used [45], necessitating the search for more potent alternatives. The antibacterial activity of biosynthesized Ag NPs was reported previously [32, 43]. The antibacterial properties of PV-Ag NPs were investigated using the agar diffusion method, showing their effectiveness in reducing bacterial growth, particularly against S. aureus, K. pneumoniae, and E. coli. Results indicated significant inhibition of these pathogens with a clear dose-dependent effect. Our findings align with those of Rudrappa et al. [28] and Raut et al. [40], who found that Cladosporium cladosporoides, Penicillium brasilianum NPs, Penicillium chrysogenum, and Purpureocillium lilacinum-derived Ag NPs inhibited Gram-negative bacteria more than Gram-positive ones [27]. Gram-negative bacteria contain a thin coating of peptidoglycan that allows Ag+ ions to enter the cell and a lipopolysaccharide layer with a negative charge that attracts Ag+ ions, leading to increased absorption and the breakdown of the cell wall [28, 32]. The inclusion of fungal extract enhanced the release of bioactive molecules, further improving antibacterial activity. Specifically, the treated plates exhibited inhibition (Table 1) zones of 18 mm for S. aureus, 20 mm for K. pneumoniae, and 19 mm for E. coli, demonstrating the ability of PV-Ag NPs to penetrate bacterial cell walls and induce damage via reactive oxygen species. ROS have antibacterial properties by inhibiting DNA synthesis, causing membrane damage and leakage, suppressing protein creation, causing mitochondrial malfunction, and inhibiting the electron transport chain, leading to cell lysis [26, 28, 34, 46]. These findings suggest that the combination of fungal extract and PV-Ag NPs presents a promising approach for wound healing and nursing care, particularly with maximum effectiveness observed at a concentration of 40 µg/mL. The results indicated a significant reduction in bacterial growth even at the lowest concentrations tested, demonstrating that these nanofibers effectively inhibited the growth of all tested bacterial strains at lower doses. The minimum inhibitory concentration (IC₅₀) values of test pathogens were found to be 43.12 ± 1.02 µg/mL for S. aureus and 52.30 ± 1.03 µg/mL for E. coli, and 45.21 ± 1.03 µg/mL respectively (Fig. 5). Interestingly, the agar well diffusion assay results revealed a broad range of bacterial inhibition starting from the lowest doses (Fig. 4C).

Table 1.

Antibacterial activity of PV-Ag NPs against human pathogens

Bacterial strains	Zone of inhibition (mm) /Sample concentrations (µg/mL)
	10	20	30	40	PC
Escherichia coli	15 ± 0.84	16 ± 0.32	18 ± 0.58	19 ± 0.78	31 ± 0.15
Klebsiella pneumoniae	17 ± 0.52	18 ± 0.76	18 ± 0.70	20 ± 1.12	32 ± 0.90
Staphylococcus aureus	16 ± 0.90	17 ± 0.74	18 ± 0.54	18 ± 0.94	30 ± 0.56

Values are presented in average (n = 3 ± SD), Positive control (PC)-Gentamycin

Fig. 5.

Assessment of antibacterial potential of PV-Ag NPs against human infectious pathogens (E. coli, K. pneumoniae, and S. aureus) using well diffusion assay with various concentrations (10–40 µg/mL) of PV-Ag NPs. Positive control: standard antibiotic Gentamicin (10 µg/well)

Cytotoxicity of PV-Ag NPs against A549 cells

The unique biological characteristics of PV-Ag NPs open up promising possibilities in the biomedical field. These nanocomposites can be used effectively in bioimaging, cancer treatment, and infection prevention. While their cytotoxicity and chemical composition require careful consideration for human use, understanding these factors can enhance safety and efficacy [35]. Our study assessed the cytotoxic effects of PV-Ag NPs on A549 cells using the MTT assay. The results showed significant toxicity against human lung cancer cells, with an IC₅₀ value of 53.22 ± 1.5 µg/mL, suggesting the potential of PV-Ag NPs for lung cancer treatment (Fig. 6). This highlights the effectiveness of our PV-Ag NPs at lower concentrations, indicating their promising role in targeting A549 lung cancer cells. Higher doses of PV-Ag NPs significantly lowered cell viability, caused morphological abnormalities, and led to more apoptotic cells, whereas lower doses resulted in a larger percentage of healthy cells. Figure 8 illustrates the % cell viability at varied drug concentrations. The biological activity of nanoparticles is determined by their structure, morphology, size, stability, and capacity to aggregate [10]. The cytotoxic action of Ag NPs has been extensively explored because of its possible impact on cellular uptake, penetration, and intracellular dispersion [12]. Previous studies have demonstrated that Ag NPs exhibit increased activity due to their typical high surface-to-volume ratio, which allows effective cellular absorption with cellular components, and have a detrimental impact on cellular signaling pathways [13–15, 17]. Ag NPs may interact with mitochondria, disrupting the electron transport chain and increasing ROS levels, as expected from their function. The oxidative stress generated by the Ag NP-mediated creation of ROS is similarly harmful to cells. The bioactive chemicals of fungal endophytes and Ag NPs display synergistic effects, as predicted by earlier studies, and so exhibit greater cytotoxicity [19, 22]. Previous studies evaluated the cytotoxic ability of fungus-derived silver nanoparticles against lung cancer (A549) cell lines [18, 22, 35]. Penicillium aculeatum and Botryosphaeria rhodina-mediated Ag NPs exhibit potential cytotoxicity (IC₅₀ = 48 and 40 µg/mL) against A549 cells [35, 39], respectively. Furthermore, silver nanoparticles demonstrate high anticancer activity with minimum toxicity, depending on the reducing and capping agents used to convert silver nitrate to silver [41, 42, 44].

Fig. 6.

Cytotoxicity of PV-Ag NPs against A549 lung cancer cells on MTT cell viability assay. Each value is presented as mean ± SD (n = 3). *p < 0.05 versus untreated controls

Fig. 8.

A Percentage of ROS generation of PV-Ag NPs against A549 lung cancer cells on DCFDA fluorescence assay, B Apoptotic effect of PV-Ag NPs post-exposed A549 cells under AO/EB dual staining. C Fluorescence microscopic images of PV-Ag NPs-treated A549 cells in AO/EB dual staining. Each value is presented as mean ± SD (n = 3). *p < 0.05 versus untreated controls

Mitochondrial membrane potential damage of PV-Ag NPs against A549 cells

In JC-1 staining following chaetocin treatment, A549 cells demonstrated an increased vulnerability to mitochondrial membrane potential (MMP) damage and a rise in apoptotic cell populations [24]. Notably, the levels of apoptosis were positively correlated with both the concentration and duration of PV-Ag NPs treatment. This suggests that the MMP damage pathway is a critical component in the apoptosis process [37]. The activation of caspase-3 and subsequent protein cleavage induced by PV-Ag NPs appears to drive the cell death mechanism in A549 cells. The observed decline in mitochondrial membrane integrity serves as a clear indicator of apoptosis [28]. The Fig. 7 illustrates the extent of mitochondrial membrane integrity damage in A549 cells following PV-Ag NPs treatment. Furthermore, quantitative analysis indicated that over 40% of the cells experienced mitochondrial damage in a dose-dependent manner, with significant MMP damage observed at a higher concentration (100 µg/mL) after 24 h of treatment. This data highlights the potential of PV-Ag NPs as a valuable material for the mechanisms of apoptosis and mitochondrial dysfunction-mediated cancer treatment [24, 27, 28].

Fig. 7.

Mitochondrial membrane damage potential of PV-Ag NPs against A549 cells. Each value is presented as mean ± SD (n = 3). *p < 0.05 versus untreated controls

PV-Ag NPs induced apoptosis in A549 cells

The investigation into the apoptosis mechanism of PV-Ag NPs treated cell morphology was conducted using the AO/Br dual staining assay [35]. Treatment with chaetocin resulted in notable structural and nuclear changes in A549 cells compared to control cells, highlighting the compound’s impact on cellular integrity. The AO/EB dual staining assay (Fig. 8) provided valuable insights into apoptotic activity [19]. In this assay, live cells emit green fluorescence, allowing for easy differentiation from apoptotic cells. Following PV-Ag NPs treatment, a significant reduction in the number of live cells was observed in the treated groups. Red cells were specifically noted in both the PV-Ag NPs and cisplatin treatment groups, indicating targeted effects. Further analysis using flow cytometry with Annexin V-FITC and PI staining demonstrated that PV-Ag NPs effectively induce apoptosis in A549 cells. The half-maximal inhibitory concentration (IC₅₀) value of 53.2 ± 1.5 µg/mL, as recorded in the MTT cell viability assay (Fig. 6), further supports the potency of PV-Ag NPs. Additionally, an increase in the percentage of apoptotic cells was documented 24 h post-exposure. Overall, these findings indicate that PV-Ag NPs treatment promotes cell death in A549 cells through an apoptotic mechanism, opening avenues for further research into its therapeutic potential [37, 43].

Nuclear damage efficacy of PV-Ag NPs against A549 cells

The apoptotic mechanism plays a crucial role in the changes observed in cellular morphology and biochemistry [38]. This study assessed the cytotoxic effects of PV-Ag NPs on nuclear alterations using the Hoechst staining technique (Fig. 9). The findings revealed that PV-Ag NP-treated cells exhibited 56.12 ± 0.24% of abnormal nuclei, while cisplatin-treated cells showed a higher percentage of 72.35 ± 0.14%. In contrast, control cells demonstrated only 4.1 ± 0.15% abnormal nuclei, indicating a significantly healthier cellular state. Microscopic analysis of control samples confirmed the presence of normal cell nuclei, which displayed minimal blue fluorescence. In comparison, PV-Ag NP-treated cells showed intense blue fluorescence and notable cytomorphological changes, including chromatin fragmentation, irregularity in nuclear structure, binucleation, and nuclear shrinkage [36, 38]. These observed intracellular changes strongly support the notion that apoptosis is a crucial pathway in cell death induced by PV-Ag NPs. Overall, the results from the Hoechst assay provide compelling evidence that the cytotoxic effects of PV-Ag NPs lead to significant cell death in A549 cells, highlighting their potential impact in therapeutic applications. Some fungal-derived compounds bind to tubulin, disrupting the microtubule network of cancer cell division mechanisms. A main example is Taxol (paclitaxel), originally discovered in endophytic fungi, which stabilizes microtubules and halts tumor growth. Extracts trigger cancer cell death by down-regulating anti-apoptotic proteins (like Bcl-2) and up-regulating pro-apoptotic factors (Bax). They also modulate cell signaling pathways (NF-kB and Akt), leading to cancer cells to self-destruct [39, 44, 47, 48].

Fig. 9.

Assessment of nuclear damage of A549 cells after treatment with PV-Ag NPs. Each value is presented as mean ± SD (n = 3). *p < 0.05 versus untreated controls

ROS generating effect of PV-Ag NPs against A549 cells

Chaetocin induces an apoptotic effect through ROS accumulation in A549 cells after treatment. The fluorescence of DCFH/DA was increased in PV-Ag NPs post-treatment, which confirmed the ROS generation in A549 lung cancer cells. Results suggested that PV-Ag NPs-induced cell death depends on the high level of ROS in a cellular region. In the control and fungal extract-treated groups, the low level of ROS production was not detectable in the initial period. In the treatment with PV-Ag NPs concentrations, a notable amount of ROS production appeared at 30 min. Compared to the control cells, PV-Ag NPs-treated A549 cells showed a rapid increase in intracellular ROS accumulation (Fig. 8A). At the apoptosis-leading PV-Ag NPs, a significant amount of intracellular ROS and free radicals is generated within 30 min of post-treatment. The free radical formation was accelerating cell death in the cellular DNA and mitochondrial membrane damage mechanisms [47].

Biocompatibility of PV-Ag NPs

The cytotoxicity of PV-Ag NPs was assessed using L929 fibroblast cell lines. The results exhibit that PV-Ag NPs are non-toxic at lower concentrations. The MTT cell viability assay showed a minimal level of cell death, even at the highest dose of 100 µg/mL (Fig. 10A). Importantly, fluorescence microscopy images exhibit no structural deformities or apoptotic cells in treated groups. In this case, commercial anticancer drug cisplatin-treated groups showed a high level of inhibition in cell proliferation and cellular spheroids. In the PV-Ag NPs treated group, no notable apoptotic cells were observed after 24 h. Interestingly, the anti-inflammatory properties of the fungal extract significantly contributed to the cytocompatibility of the PV-Ag NPs. The presence of fungal bioactive molecules thereby enhances biosafety. The fluorescence images of L929 cells showed significant viability and a high proliferation rate with PV-Ag NPs (Fig. 10B). The cell density and proliferation of the PV-Ag NPs treated group showed three-fold higher compare than cisplatin. The combination of fungal bioactive compounds and the green synthesis approach enhance the biocompatibility of PV-Ag NPs.

Fig. 10.

Cytotoxic effect of PV-Ag NPs and cisplatin for biosafety assessment against L929 fibroblast cells using MTT cell viability assay

Conclusion

In this study, we fabricated bioactive fungal extract-mediated PV-Ag NPs to enhance antibacterial and anticancer activities. These nanoparticles selectively release phytocompounds and silver ions at lung cancer cell sites, inducing mitochondrial damage and apoptosis. Our results demonstrate that PV-Ag NPs cause nuclear dysfunction and reactive oxygen species (ROS)-mediated cytotoxicity in A549 cells, showcasing their potent anticancer effects. The results of the AO/EB dual staining assay confirmed that the PV-Ag NPs lead to an apoptotic mechanism on A549 lung cancer cells at a minimum dose. Notably, these nanoparticles exhibit significant cytotoxicity against A549 cells and human pathogenic bacterial strains even at low concentrations. Finally, the in vitro cytotoxicity studies on L9292 fibroblast cells suggested that, PV-Ag NPs are highly biocompatible for normal cells. This research concluded that PV-Ag NPs are a promising material for both anticancer and antibacterial applications, with the potential to offer a low-cost therapeutic for cancer treatments.

Author contributions

The authors, SK and AAS, participated equally in this work by carrying it out. SK, JP, and PR are drafting the paper, reviewing, and editing. SK, AAS, and PR proposed the idea, outlined the work, validated the results, and revised the manuscript. WSD, NKS, EA, SA, RS, and ALKA assisted in the validation of the results, software, data curation, and authoring of the original publication. All authors have reviewed and approved the manuscript.

Funding

This work was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R317), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Data availability

The manuscript includes the necessary data. The fungal isolate used in this study is Penicillium verrucosum, with the GenBank accession number (PP732489). https://www.ncbi.nlm.nih.gov/nuccore/PP732489.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.