Materials
PDA (Potato Dextrose Agar), PDB (Potato Dextrose nutrient broth) and silver nitrate. All the experiments were performed by using Double distilled water.
Fungus used for screening
For the synthesis of silver nanoparticles two fungal species were used i.e. Aspergillus Niger, Fusarium oxysporum. The above fungus species were cultured and maintained in PDA (Potato Dextrose Agar) medium.
Isolation of test fungus Aspergillus niger
Soil sample were collected from an area of carpenter shop. The soil sample were taken from a depth of 5-10cm and kept in plastic bags until drying was performed immediately. After sampling in the laboratory. The soil samples were air dried at room temperature at 27⁰C for a week and grind it using a mortar pestle. Then soil sample were sieved with 0.5mm sieve to remove larger particles such as stone and plant debris in order to obtain a consistent soil particle size for isolation using the soil dilution technique.
Isolation of test fungus Fusarium oxysporum
The test fungus was isolated from decayed banana fruit in PDA (potato, dextrose, agar) and incubated at 28⁰C for a week. Individual fungal colonies were picked and further purified by sub culturing on PDA media.
Identification of fungus
The fungus was identified by cultural (mycelia, colony color, shape and size) and microscopic characteristics (macro and micro conidia and chlamydospores) by using Siefert’s key and Leslie’s Laboratory manual [46–50].
Maintenance of cultures
For maintaining the culture using appropriate medium. Fungus cultures were incubating in to the PDA plates. The plates were maintained at room temperature at 27⁰C for week for further use.
Synthesis of Silver nanoparticles
Production of biomass
To prepare the biomass for biosynthesis, the fungus culture obtained were inoculate in liquid broth for growth containing potato, dextrose and nutrient broth. The culture flasks were incubated on room temperature at 27⁰C. The biomass was harvested after 120 hours of growth. Sieving it through a plastic sieve followed by extensive washing with sterile double distilled water to remove any medium components from the biomass.
Synthesis of Silver nanoparticles
Typically, 10 gm of biomass (wet weight) were brought in to contact with 100 ml sterile doubled distilled water for 48 hours at 27⁰C in an Erlenmeyer flask and agitated 150 rpm. After incubation the cell filtrate was filtered by Whatman filter paper no. 1. After filtration the observed pH of the cell filtrate was 7.2. In to 80 ml of filtrate, a carefully weighed quantity of silver nitrate was added to the Erlenmeyer flask and incubated at room temperature in dark. Control containing cell free filtrate without silver nitrate was run simultaneously as standard with the experimental flask. Silver nanoparticles were concentrated by centrifugation of the reaction mixture at 11, 000 rpm. Cell free filtrate incubated with silver nitrate get change in color, was visually observed over a period of time. Silver nanoparticles (AgNPs) synthesized using biological methods, such as the fungus Fusarium oxysporum, have garnered significant attention for their medicinal properties. This biogenic synthesis method is considered eco-friendly and cost-effective compared to chemical and physical methods [51–54].
Antimicrobial Activity
Silver nanoparticles exhibit broad-spectrum antimicrobial properties. Silver nanoparticles (AgNPs) are renowned for their broad-spectrum antimicrobial properties, which make them effective against a wide variety of microorganisms, including bacteria, fungi, and viruses. These properties have significant implications for medical applications, such as wound dressings, coatings for medical devices, and as components of disinfectants [55–60].
Mechanisms of Antimicrobial Action
Disruption of Cell Membranes:
Attachment and Penetration
AgNPs can attach to the microbial cell membrane and penetrate it, leading to increased membrane permeability and eventual cell lysis.
Structural Damage
This interaction can cause structural changes in the membrane, including the formation of pores, which disrupts the integrity of the cell membrane and leads to cell death.
Generation of Reactive Oxygen Species (ROS):
Oxidative Stress
AgNPs can induce the production of ROS, such as hydrogen peroxide, superoxide anions, and hydroxyl radicals. These ROS can damage cellular components, including lipids, proteins, and DNA, leading to cell death.
Mitochondrial Damage
The oxidative stress caused by ROS can also affect the mitochondria, impairing their function and contributing to cell death.
Interaction with Cellular Proteins:
Enzyme Inhibition
AgNPs can interact with thiol groups in proteins and enzymes, leading to their inactivation. This inhibition can disrupt essential cellular processes, such as metabolism and replication.
Protein Denaturation
By binding to proteins, AgNPs can cause their denaturation, further disrupting cellular function and viability.
DNA Interaction:
DNA Damage
AgNPs can enter the cell and interact with DNA, causing structural damage and affecting replication and transcription processes. This damage can result in mutations and cell death.
Inhibition of Replication
By binding to DNA, AgNPs can inhibit the replication process, preventing the cell from proliferating [61–70].
Antimicrobial Spectrum
Bacterial Infections:
Gram-positive Bacteria
AgNPs are effective against Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae. They can penetrate the thick peptidoglycan layer and disrupt cellular functions.
Gram-negative Bacteria
AgNPs also show efficacy against Gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Their action is facilitated by the thin peptidoglycan layer and the presence of an outer membrane.
Fungal Infections:
AgNPs exhibit antifungal properties against various fungal species, including Candida albicans and Aspergillus niger. They can disrupt fungal cell membranes and inhibit fungal growth.
Viral Infections:
Viral Inhibition
AgNPs can inhibit viral replication and prevent viruses from entering host cells. They have shown efficacy against viruses such as HIV, hepatitis B, and influenza.
Viral Deactivation
AgNPs can bind to viral particles, deactivating them and preventing them from infecting host cells [71–74].
Anti-inflammatory Properties
Silver nanoparticles have shown potential in reducing inflammation. Silver nanoparticles (AgNPs) have shown significant potential in reducing inflammation, a beneficial property that can be leveraged in various medical applications [75–80].
Mechanisms of Anti-inflammatory Action
Inhibition of Pro-inflammatory Cytokines:
AgNPs can reduce the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). These cytokines are key mediators of the inflammatory response, and their reduction helps to alleviate inflammation.
Modulation of Immune Cells:
AgNPs can influence the activity of various immune cells, including macrophages, neutrophils, and lymphocytes, which play critical roles in the inflammatory process. By modulating these cells' activities, AgNPs help in reducing inflammation.
Inhibition of NF-κB Pathway:
The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway is a significant signaling pathway involved in the regulation of inflammation. AgNPs can inhibit the activation of the NF-κB pathway, thereby reducing the expression of inflammatory genes.
Reduction of Reactive Oxygen Species (ROS):
AgNPs can decrease the levels of ROS, which are chemically reactive molecules that contribute to inflammation by activating various inflammatory pathways. By reducing ROS levels, AgNPs can mitigate oxidative stress and associated inflammation.
Promotion of Anti-inflammatory Cytokines:
AgNPs can enhance the production of anti-inflammatory cytokines, such as interleukin-10 (IL-10). These cytokines help resolve inflammation and promote tissue repair and healing [81–89].
Anticancer Activity
Research indicates that silver nanoparticles (AgNPs) possess significant anticancer properties. These properties arise from their ability to induce apoptosis, generate reactive oxygen species (ROS), and disrupt critical cellular functions specific to cancer cells.
Mechanisms of Anticancer Action
Induction of Apoptosis:
Mitochondrial Pathway
AgNPs can disrupt the mitochondrial membrane potential, leading to the release of cytochrome c and activation of caspases, which are enzymes crucial for apoptosis (programmed cell death).
Death Receptor Pathway
AgNPs can activate death receptors on the cell surface, triggering the extrinsic pathway of apoptosis.
Generation of Reactive Oxygen Species (ROS):
Silver nanoparticles can induce the production of ROS within cancer cells. High levels of ROS cause oxidative stress, damaging cellular components such as DNA, proteins, and lipids, leading to apoptosis or necrosis.
DNA Damage:
AgNPs can interact directly with the DNA of cancer cells, causing structural damage and interfering with replication and transcription processes, leading to cell cycle arrest and apoptosis.
Inhibition of Cell Proliferation:
AgNPs can inhibit the proliferation of cancer cells by interfering with the cell cycle, causing cell cycle arrest at various phases (G0/G1, S, G2/M), which prevents the cells from dividing and growing.
Disruption of Cellular Functions:
AgNPs can disrupt essential cellular functions specific to cancer cells, including signal transduction pathways, protein synthesis, and metabolic processes, leading to cell death.
Anti-angiogenic Effects:
AgNPs can inhibit angiogenesis, the formation of new blood vessels from existing ones, which is crucial for tumor growth and metastasis. By inhibiting angiogenesis, AgNPs can starve the tumor of nutrients and oxygen, slowing its growth and spread [90–103].
Wound Healing
Silver nanoparticles have been extensively used in wound dressings due to their antimicrobial and anti-inflammatory properties. The antimicrobial properties of AgNPs help prevent infections in wounds, which is crucial for proper healing and reducing complications such as sepsis. By reducing inflammation, AgNPs help in alleviating pain, swelling, and redness at the wound site, creating a more conducive environment for healing. AgNPs have been shown to promote the proliferation of keratinocytes and fibroblasts, which are essential for tissue regeneration and wound closure. Studies have demonstrated that wound dressings containing AgNPs can accelerate the healing process, resulting in faster wound closure and reduced healing times.
These dressings are impregnated with silver nanoparticles, providing sustained antimicrobial activity. They are available in various forms, such as gauzes, foams, and hydrocolloids. These dressings have a coating of silver nanoparticles on their surface, offering direct antimicrobial contact with the wound bed. These dressings combine silver nanoparticles with other materials, such as hydrogels or alginates, to enhance their antimicrobial and moisture-retentive properties. AgNPs are particularly beneficial in the treatment of chronic wounds, such as diabetic ulcers, pressure ulcers, and venous leg ulcers, where infection and inflammation are major concerns. Silver nanoparticle-based dressings are extensively used in the management of burn wounds to prevent infections and promote healing. AgNP dressings are used post-surgery to reduce the risk of infections and aid in faster recovery.
The potential cytotoxic effects of AgNPs on human cells need to be evaluated to ensure their safe use. Studies suggest that the concentration and size of nanoparticles play a crucial role in their safety profile. Although rare, some individuals may experience allergic reactions to silver. Monitoring and addressing such reactions are important in clinical settings. The potential for microorganisms to develop resistance to silver should be monitored to ensure the long-term efficacy of silver-based dressings. Silver nanoparticles have proven to be highly effective in wound dressings due to their potent antimicrobial and anti-inflammatory properties. They help prevent infections, reduce inflammation, and promote faster healing, making them invaluable in the treatment of various types of wounds, including chronic, burn, and surgical wounds. While their benefits are well-documented, ongoing research is essential to optimize their use and ensure their safety and efficacy in clinical applications [104–105].
Antiviral Coatings:
AgNPs can be incorporated into coatings for medical devices, surfaces, and personal protective equipment (PPE) to reduce the transmission of viruses in healthcare settings and public spaces.
Topical Formulations:
AgNPs can be formulated into gels, creams, or ointments for topical application to prevent or treat viral infections, particularly in the case of skin or mucosal infections.
Pharmaceutical Formulations:
AgNPs can be included in pharmaceutical formulations, such as oral tablets, nasal sprays, or injectable solutions, to deliver antiviral agents directly to the site of infection.
Preventive Measures:
AgNPs can be used in the development of preventive measures, such as antiviral masks and air filters, to reduce the spread of respiratory viruses.
Safety and Efficacy Considerations
Cytotoxicity:
While AgNPs have demonstrated antiviral activity, their potential cytotoxic effects on human cells need to be carefully evaluated. Optimal dosing and delivery methods must be determined to minimize toxicity.
Resistance Development:
The potential for viruses to develop resistance to silver should be monitored. Combining AgNPs with other antiviral agents may help reduce the risk of resistance.
Regulatory Approval:
Comprehensive studies and clinical trials are required to gain regulatory approval for the use of AgNPs in antiviral applications. Ensuring their safety, efficacy, and quality is crucial for their successful implementation [105–112].
Anti-biofilm Activity
Silver nanoparticles can disrupt biofilms, which are structured communities of microorganisms that are resistant to conventional antibiotics. Silver nanoparticles (AgNPs) can disrupt biofilms, which are structured communities of microorganisms that exhibit high resistance to conventional antibiotics. Biofilms are a significant problem in medical and industrial contexts because they protect microorganisms from environmental stresses, including antibiotic treatment, making infections difficult to eradicate.
Mechanisms of Biofilm Disruption
Penetration of Biofilm Matrix:
AgNPs can penetrate the extracellular polymeric substance (EPS) matrix of biofilms, which is a protective layer composed of proteins, polysaccharides, and nucleic acids. This penetration allows AgNPs to reach and affect the microorganisms embedded within the biofilm.
Disruption of Biofilm Structure:
AgNPs can disrupt the structural integrity of biofilms by degrading the EPS matrix. This disruption can be due to the generation of reactive oxygen species (ROS) or direct interactions between AgNPs and matrix components, leading to the breakdown of the biofilm's physical structure.
Antimicrobial Activity Against Biofilm-Embedded Cells:
AgNPs exhibit strong antimicrobial properties against both planktonic (free-floating) and biofilm-embedded microorganisms. By interacting with bacterial cell membranes and internal structures, AgNPs can kill or inhibit the growth of biofilm-associated cells.
Inhibition of Biofilm Formation:
AgNPs can prevent the initial adhesion and aggregation of microorganisms on surfaces, thereby inhibiting the formation of biofilms. This is particularly important in preventing biofilm-related infections and contamination in medical devices and industrial systems.
Synergistic Effects with Antibiotics:
AgNPs can enhance the efficacy of conventional antibiotics against biofilms. They can disrupt the biofilm matrix and increase the permeability of microbial cells, allowing antibiotics to penetrate and act more effectively.
Wound Dressings:
AgNPs are used in wound dressings to prevent biofilm formation and promote wound healing. These dressings help manage chronic wounds and burns, where biofilms often complicate the healing process.
Medical Device Coatings:
Coating medical devices, such as catheters, implants, and prosthetics, with AgNPs can prevent biofilm formation and reduce the risk of device-associated infections. This is particularly important for indwelling devices that are prone to biofilm-related complications.
Dental Applications:
AgNPs can be incorporated into dental materials, such as composite resins, adhesives, and sealants, to prevent biofilm formation and dental plaque, thereby reducing the incidence of dental caries and periodontal diseases [113–120].
Characterization of silver nanoparticles
UV-Visible spectroscopy
The reaction mixture was subjected to UV-Vis Spectrophotometric Measurements (Model UV-1601 PC). According to this technique many molecules absorb ultraviolet or visible light. The percentage of transmittance light radiations determines when light of certain frequency passed through the samples. This spectrophotometer analyses records the intensity of absorbance or optical density (O.D) as a function of wavelength. Absorption is directly proportional to the concentration of the absorbing species (Beer’s law) [121].
Transmission Electron Microscope Analysis
This study was undertaken to know the morphology and particle size distribution of silver nanoparticles. In TEM there is an electron source at the top of the microscope, one-meter-long column is attached for vacuum, allows following down the electron. Electron gun, Electron lens, specimen and image forming system are different components of the microscope used for imaging. It has resolving power of 1nm and provide 2D image of the sample. TEM micrographs of the sample were taken using the JEOL JSM 1oocx instrument [122].