Physical methods, such as evaporation-condensation, spark discharge, and pyrolysis, offer rapid synthesis without hazardous chemicals but suffer from high energy consumption, low yield, and inconsistent particle distribution (Kruis et al., 2000; Zhang et al., 2016; Elsupikhe et al., 2015; Xu et al., 2020).

Chemical synthesis, which involves metal precursors, reducing agents, and stabilizers, follows either a “top-down” (mechanical grinding) or “bottom-up” (chemical reduction, electrochemical methods) approach ( Figure 1 ) (Amulyavichus et al., 1998; Mallick et al., 2004; Xu et al., 2020). While this method provides high yield, it is costly, involves toxic reagents (e.g., citrate, borohydride), and requires additional purification steps to prevent contamination and aggregation (Malik et al., 2002; Mallick et al., 2004). Chemical methods include laser ablation, lithography, electrochemical reduction, thermal decomposition, and sono-decomposition (Zhang et al., 2016). Despite their efficiency, chemical synthesis poses environmental and biological risks due to toxic byproducts (Gurunathan et al., 2015).

To address the limitations of chemical methods, biological synthesis has emerged as an eco-friendly and cost-effective alternative. This method utilizes bacteria, fungi, plant extracts, and biomolecules like amino acids and vitamins to produce AgNPs in a controlled manner (Gurunathan et al., 2013, 2014; Zhang et al., 2016). Early studies on bacterial bio-sorption of metals indicated the potential for nanoparticle synthesis, though initial results were aggregates rather than discrete nanoparticles (Mullen et al., 1989). Various microorganisms, including Pseudomonas stutzeri AG259, Lactobacillus species, Bacillus licheniformis, and Escherichia coli, as well as fungi like Fusarium oxysporum and Ganoderma neo-japonicum Imazeki, have been used for AgNP production (Zhang et al., 2016). Plant-based synthesis has been demonstrated with Allophylus cobbe, Artemisia princeps, and Typha angustifolia (Gurunathan et al., 2014; Gurunathan, 2015; Gurunathan et al., 2015). Additionally, biopolymers, starch, enzymes, and amino acids serve as reducing agents (Leung et al., 2010; Deepak et al., 2011; Kumar et al., 2014; Zhang et al., 2016).

Biological synthesis methods provide improved control over nanoparticle size and shape due to the presence of natural reducing and capping agents—such as polyphenols, proteins, and flavonoids—in plant extracts, which guide uniform nucleation and growth while eliminating the need for toxic chemicals or synthetic stabilizers (Gurunathan et al., 2014; Restrepo and Villa, 2021). The use of natural reducing agents allows for monodisperse, stable, and water-soluble nanoparticles (Thakkar et al., 2010; Zhang et al., 2016). Particle morphology influences bioactivity, with smaller, triangular nanoparticles exhibiting enhanced effects (Morones et al., 2005). Unlike chemical methods, biological synthesis allows optimization of reaction conditions to achieve desired nanoparticle characteristics (Zhang et al., 2016).

Green synthesis of AgNPs has emerged as an environmentally friendly alternative to conventional physical and chemical methods. However, several challenges hinder its large-scale application and industrial adoption. One major limitation is the dependence on plant materials that are often geographically restricted or seasonally available. For instance, Lithodora hispidula, used for Pd NP synthesis, is only found in limited regions such as Cyrenaica and southern Turkey (Turunc et al., 2017). Similarly, plants like coconut, Acacia, and Andean blackberry, which have been employed in synthesizing Ag or Cu nanoparticles, are region-specific and not easily accessible worldwide (Ying et al., 2022). Seasonal constraints further complicate sourcing; materials like cotton leaves, peach blossoms, and Trigonella trifoliata seeds must be harvested during narrow windows, which limits continuous production (Ying et al., 2022). Moreover, some raw materials require additional processing or extraction steps, such as the case with carboxymethyl cellulose and tea polyphenols, which increases complexity and cost (Wang et al., 2017). These factors challenge the feasibility, cost-effectiveness, and scalability of green synthesis in industrial contexts.

In addition to raw material constraints, the synthesis process itself presents technical and environmental limitations. Many green synthesis protocols require excessive energy input, long reaction durations, or strict environmental controls, such as high-temperature treatments or inert atmospheres. For example, AgNPs have been synthesized using Ferula persica root extract at 600°C for 3 hours (Nasiri et al., 2018), and Cystoseira baccata extract was stored at −24°C to retain its reactivity (González-Ballesteros et al., 2017). These conditions contradict the principle of sustainability and elevate production costs. Furthermore, the resulting nanoparticles often exhibit high variability in size, morphology, and crystallinity. AgNPs synthesized using Nigella arvensis leaf extract, for example, ranged from 5 to 100 nm, while NZVI particles from grape seeds varied between 63 and 381 nm (Gao et al., 2016; Chahardoli et al., 2018). This inconsistency limits reproducibility and standardization required for commercial and biomedical applications. In many cases, yields and metal ion conversion rates remain low—often below 50%—which diminishes economic viability (Ying et al., 2022). The limited understanding of the underlying biosynthetic mechanisms further complicates optimization, as many studies only infer the roles of plant extracts (e.g., reducing, capping, or chelating agents) without detailing specific chemical pathways (Ying et al., 2022). Collectively, these limitations highlight the need for more systematic research to improve the reliability, efficiency, and industrial compatibility of green synthesis methods.

The size of AgNPs is a critical determinant of their antimicrobial activity, as it directly affects their surface-area-to-volume ratio and interaction with bacterial cells. Smaller nanoparticles exhibit enhanced bioactivity due to their increased surface area, facilitating greater silver ion release and direct interaction with microbial membranes (Khan et al., 2021). Studies have shown that AgNPs attach to bacterial cell membranes, disrupting the lipid bilayer and increasing permeability, leading to cell death—an effect more pronounced with smaller nanoparticles (Li et al., 2013). The size of AgNPs also influences their toxicity; for instance, nanoparticles below 10 nm have been shown to induce more significant cytotoxic effects in mammalian cells than larger particles. For example, AgNPs exhibit strong potency, possess small average diameters of approximately 10 nm, and demonstrate cytotoxicity in human lung cells (Gliga et al., 2014). Furthermore, Fernández et al., 2019 examined the cytotoxic effects of 10 nm and 60 nm silver nanoparticles (AgNPs) in hepatic (HepG2) cells, a model chosen due to the liver’s known tendency to accumulate AgNPs. Their findings showed that 10 nm AgNPs could enter the nucleus, whereas 60 nm particles remained aggregated in the cytoplasm. Proteomic analysis revealed that nearly 50 proteins were altered by each nanoparticle size, but only four showed similar regulation patterns, suggesting that distinct cellular pathways were triggered depending on particle size (Fernández et al., 2019). Complementing this, Wu et al., 2019 studied AgNPs of various diameters (3.2, 20.7, 54.7, and 93.6 nm) in B16 mouse melanoma cells and found that nanoparticle size not only affected uptake efficiency but also dictated the endocytic pathways involved. These findings highlight the importance of nanoparticle size in determining both cellular entry routes and biological responses.

The morphology of AgNPs significantly affects their interaction with bacterial membranes and their overall antimicrobial properties. AgNPs can be synthesized in various shapes, including spherical, triangular, cubic, rod-shaped, and star-like structures (Khodashenas and Ghorbani, 2019). Studies have demonstrated that the shape of silver nanoparticles (AgNPs) plays a critical role in determining their antimicrobial efficacy. For instance, Alshareef et al. (2017) reported strong antibacterial activity of both rod- and spherical-shaped AgNPs. Similarly, Cheon et al. (2019) synthesized AgNPs in three shapes—spherical, disc-shaped, and triangular—and observed the highest bactericidal activity in spherical forms, followed by disc-shaped and then triangular nanoparticles. In contrast, other studies have highlighted the superior antibacterial properties of triangular nanoparticles due to their sharp edges, which enhance interactions with bacterial membranes and lead to mechanical disruption (Liao et al., 2019). These variations suggest that the antibacterial effectiveness of AgNPs may depend on multiple factors, including bacterial type, environment, and application. Additionally, comparative studies have shown that rod-shaped AgNPs penetrate bacterial biofilms more effectively than spherical particles, making them particularly useful for combating biofilm-associated MDR infections (Wang et al., 2021). Other studies have compared the antimicrobial activities of spherical, rod-shaped, and truncated triangular silver nanoplates against planktonic E. coli cells, revealing that truncated triangular AgNPs exhibit the strongest bactericidal effect. This enhanced activity is attributed to the greater number of facets on triangular nanoparticles, which allows for increased interaction with bacterial surfaces, leading to more extensive membrane damage (Pal et al., 2007). This difference is likely due to the higher aspect ratio of rod-shaped nanoparticles, which enhances their interaction with biofilms (Slomberg et al., 2013). This variation in antimicrobial activity is likely due to differences in silver ion release, which is influenced by the surface area of the nanoparticles. Consequently, they concluded that modifying the morphology of AgNPs can effectively regulate their antimicrobial efficacy.

Surface charge plays a crucial role in the stability and biological interactions of AgNPs. The zeta potential of AgNPs determines their colloidal stability, with highly positive or negative values preventing aggregation (Bélteky et al., 2019). Positively charged AgNPs exhibit stronger interactions with bacterial cell membranes, which are typically negatively charged due to the presence of lipopolysaccharides and teichoic acids (Franco et al., 2022). This electrostatic attraction enhances bacterial membrane penetration and increases antimicrobial activity. For example, Abbaszadegan et al. investigated the impact of AgNP surface charge on antimicrobial activity against Gram-positive (S. aureus, S. mutans, and S. pyogenes) and Gram-negative (E. coli and P. vulgaris) bacteria (Abbaszadegan et al., 2015). Their findings revealed that positively charged AgNPs exhibited the strongest bactericidal effect against all tested strains, while negatively charged AgNPs demonstrated the weakest activity. Neutral AgNPs displayed an intermediate level of antibacterial effectiveness. Functionalization of AgNPs with biocompatible polymers such as chitosan or polyethyleneimine further improves stability and bioavailability while reducing cytotoxicity to human cells (Liao et al., 2019).

To date, numerous nanomaterials have been developed with the potential to serve as transducing elements, owing to their superior electrical conductivity, as well as enhanced thermal and optical properties (Ventura-Aguilar et al., 2023). AgNPs exhibit a reduced melting point compared to bulk silver due to their high surface energy and increased atom mobility (Asoro et al., 2009). This property is particularly beneficial in antimicrobial coatings for medical devices, as it enables the formation of conductive and biocidal nanocomposites at lower processing temperatures (Abbas et al., 2024). The high electrical conductivity of AgNPs also facilitates their integration into biosensors for rapid detection of bacterial infections (Ventura-Aguilar et al., 2023).

AgNPs exhibit remarkable thermal conductivity, making them highly suitable for heat-based antimicrobial applications. In previous studies, the researchers explored their potential as multifunctional agents to enhance hyperthermia, directly eliminate cancer and bacterial cells, or function as photothermal therapy agents (Liu S. et al., 2023). The findings demonstrated the effectiveness of AgNPs in eradicating both breast cancer cells and bacteria within the breast tumor microenvironment. Recent studies also have highlighted the role of AgNP-based nanocomposites in improving heat transfer efficiency in antimicrobial surface coatings, and reducing bacterial colonization on biomedical implants (Sahoo et al., 2022).

AgNPs exhibit unique optical properties due to localized surface plasmon resonance (LSPR), where conduction electrons oscillate in response to incident light (Loiseau et al., 2019). This property makes AgNPs highly effective in biosensing and antimicrobial photodynamic therapy (Gabudean et al., 2011). This phenomenon enhances the generation of ROS, which contribute to bacterial cell membrane disruption and oxidative stress-induced cell death (Canaparo et al., 2020). Additionally, LSPR facilitates photothermal and photodynamic effects, where AgNPs absorb light energy and convert it into localized heat, further aiding in bacterial eradication (Liu H. et al., 2023). The plasmonic properties of AgNPs can also be tuned by modifying their size, shape, and surrounding environment, allowing for optimized antimicrobial effects (Vasil’kov et al., 2018). These optical characteristics make AgNPs promising candidates for developing light-activated antimicrobial therapies against multidrug-resistant bacteria.

AgNPs have been widely studied for their antibacterial properties. Green-synthesized AgNPs using Teucrium polium leaf extract exhibited strong antimicrobial effects against multiple bacterial strains, including Staphylococcus aureus, Bacillus subtilis, E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The antimicrobial activity was evaluated using both disk diffusion and broth dilution assays, showing significant inhibition of bacterial growth (Aljowaie and Aziz, 2025). Similarly, AgNPs synthesized from Teucrium Parvifolium seeds demonstrated high efficacy against E. coli O157:H7, Enterococcus faecalis, P. aeruginosa, and S. aureus using similar evaluation methods (Soltani et al., 2024).

Plant extracts have been widely documented for their diverse therapeutic applications (Abd El-Hafeez et al., 2018, 2022). Green-synthesized AgNPs using various plant extracts have shown significant antibacterial properties ( Table 1 ). AgNPs derived from Olive leaf wastes exhibited notable activity against Listeria monocytogenes, Bacillus cereus, S. aureus, E. coli, Yersinia enterocolitica, and Campylobacter jejuni (Alowaiesh et al., 2023). Similarly, AgNPs synthesized from Argyreia nervosa leaf extract effectively inhibited enteropathogenic E. coli (EPEC) (Parvathalu et al., 2023). AgNPs derived from Citrus limon zest extract demonstrated inhibitory effects against S. aureus, E. coli, and C. albicans via disk diffusion assays (Khane et al., 2022). Furthermore, Gardenia thailandica-synthesized AgNPs exhibited strong antibacterial activity against S. aureus, as confirmed by both disk diffusion and in vivo antibacterial studies in rats (Attallah et al., 2022). The efficacy of phytosynthesized AgNPs was further highlighted in a study by Hajizadeh et al. (2024), where AgNPs synthesized from Lepidium draba leaves exhibited potent antimicrobial activity against E. coli, K. pneumoniae, S. aureus, E. faecalis, and Candida albicans.

Biosynthesized AgNPs using bacterial strains have also shown effective antimicrobial action. AgNPs derived from Lactobacillus and Bacillus species demonstrated strong inhibition against P. aeruginosa and S. aureus, as confirmed by disk diffusion assays at concentrations of 10, 20, and 40 μg/mL (Al-Asbahi et al., 2024). Furthermore, Basheer et al. (2023) reported the biosynthesis of AgNPs using marine fungi (Penicillium simplicissimum, Aspergillus terreus, A. japonicus, and A. oryzae), which displayed significant antimicrobial effects against E. coli, K. pneumoniae, P. vulgaris, S. Typhi, E. faecalis, S. aureus (methicillin-resistant S. aureus (MRSA)), S. hominis, and S. epidermidis using the agar well diffusion assay at 2, 5, and 8 mM concentrations. Similarly, Shumi et al. (2023) synthesized AgNPs from Lippia abyssinica plant extract, which exhibited antimicrobial activity against S. aureus and E. coli at a concentration of 62.5 μg/mL using the agar well diffusion method.

AgNPs produced via chemical synthesis have also been assessed for their antibacterial potency ( Table 1 ). For instance, Baveloni et al. (2025) synthesized spherical AgNPs (~24.3 nm) that exhibited significant antimicrobial activity against S. aureus, P. aeruginosa, and E. coli using the broth microdilution assay at concentrations ranging from 6.74 to 117 μg/mL. Similarly, Iwuji et al. (2024) reported chemically synthesized AgNPs (~58.3 nm) demonstrating potent inhibition against the same bacterial species, further confirming their broad-spectrum efficacy.

Nanoparticle modifications have been explored to enhance antimicrobial properties. AgNPs conjugated with polyethylene glycol (PEG) and nystatin (AgNPs-PEG-NYS) exhibited superior antibacterial activity against S. aureus and E. coli compared to non-functionalized AgNPs, as evidenced by agar well diffusion assays (Ibraheem et al., 2024). Furthermore, AgNPs integrated with catecholamine-based polymers (PDA) displayed notable antibacterial activity against E. coli (Caniglia et al., 2024). Caniglia et al. (2024) synthesized AgNPs incorporated into catecholamine-based polymers (AgNPs-PDA) using a double potentiostatic method, which exhibited significant structural modifications in E. coli membranes, leading to bacterial inhibition. Additionally, Balciunaitiene et al. (2024) developed a polymer film embedded with biosynthesized AgNPs using Symphyti radix root extracts, demonstrating strong antibacterial activity against S. aureus, β-hemolytic streptococcus, S. epidermidis, E. faecalis, E. coli, K. pneumoniae, P. aeruginosa, P. vulgaris, B. cereus, and C. albicans.

Apart from their efficacy against bacteria, AgNPs have exhibited antifungal activity ( Table 1 ). AgNPs synthesized using Lepidium draba L. leaves demonstrated strong inhibitory effects against C. albicans (Hajizadeh et al., 2024). Furthermore, Tunç (2024) reported significant antifungal activity of chemically synthesized AgNPs and carboplatin-loaded AgNPs (AgNPs-Car) against C. albicans and C. tropicalis using broth microdilution assays. Similarly, Balciunaitiene et al. (2024) showed inhibition of C. albicans by AgNPs incorporated into a natural polymer film biosynthesized with Symphyti radix extract. In another green synthesis approach, Cao et al. (2021) evaluated lignin-stabilized AgNPs (L-AgNPs) and observed inhibition of C. albicans growth via disk diffusion. Broader antifungal activity was noted by Hasanin et al. (2021), who reported activity of AgNP-based nanocomposites (Ag-NC) against multiple fungal strains including C. albicans, A. niger, A. terreus, A. flavus, and A. fumigatus. Additionally, Renganathan et al. (2021) identified antifungal activity of AgNPs from Bauhinia tomentosa against C. albicans via molecular docking, indicating potential interactions with fungal protein targets. This may be indicated as an efficient approach develop new therapies to combat the emerging antifungal resistance (Khalifa et al., 2024a; Khalifa et al., 2024c).

One of the primary antimicrobial mechanisms of AgNPs involves their interaction with bacterial cell walls and membranes. AgNPs exhibit strong affinities for bacterial membranes due to their positive charge, which facilitates electrostatic interactions with negatively charged bacterial surfaces (Mikhailova, 2024). Upon attachment, AgNPs can cause structural disintegration of the cell membrane, leading to increased permeability and leakage of cytoplasmic contents. This process ultimately compromises bacterial viability and initiates cell death (Dakal et al., 2016). The antimicrobial efficacy of AgNPs is significantly influenced by the structural differences in bacterial cell walls. Gram-negative bacteria, such as E. coli, tend to be more vulnerable to AgNPs than Gram-positive bacteria like S. aureus. This is primarily due to variations in peptidoglycan composition and thickness. Gram-positive bacteria possess a substantially thicker peptidoglycan layer (~30 nm), which serves as a protective barrier, whereas Gram-negative bacteria have a thinner peptidoglycan layer (~3–4 nm) (Rai et al., 2012). The negatively charged peptidoglycan in Gram-positive bacteria can also bind silver ions, limiting their penetration and reducing their antimicrobial effectiveness (Feng et al., 2000). Conversely, Gram-negative bacteria are more susceptible to AgNPs due to their thinner cell wall and the presence of lipopolysaccharides (LPS), which not only contribute to membrane stability but also facilitate AgNP adhesion through electrostatic interactions. This enhanced attachment leads to greater bacterial inhibition, even at lower AgNP concentrations (Pal et al., 2007). Several studies confirm that AgNPs preferentially accumulate on the surface of Gram-negative bacteria due to LPS, increasing their antimicrobial susceptibility (Pal et al., 2007). These structural and compositional differences explain why S. aureus exhibits greater resistance, while E. coli is significantly inhibited by AgNPs, establishing a clear relationship between AgNP concentration and bacterial cell wall properties (Dakal et al., 2016).

AgNPs have been shown to induce oxidative stress in bacterial cells by generating ROS, including hydroxyl radicals (^•OH), superoxide anions (O₂ ^•−), and hydrogen peroxide (H₂O₂) (He et al., 2011). These ROS are highly reactive and can cause significant cellular damage by oxidizing lipids, proteins, and DNA. Silver ions (Ag⁺) interfere with the function of the respiratory electron transport chain by inhibiting key respiratory enzymes, leading to its uncoupling from oxidative phosphorylation. This disruption affects the efficiency of cellular respiration, ultimately impairing energy production (Holt and Bard, 2005; Dakal et al., 2016). The excessive accumulation of free radicals resulting from this process causes direct oxidative damage to the mitochondrial membrane, inducing necrosis and ultimately leading to cell death. Additionally, increased ROS levels contribute to the oxidation of essential biomolecules, including lipids, proteins, and DNA, further exacerbating cellular damage (Juan et al., 2021). Free radicals also interact with lipid molecules, which are abundant in cellular membranes, triggering lipid peroxidation. This process generates lipid hydroperoxides as an initial step in ROS formation, particularly affecting polyunsaturated fatty acids (Chandimali et al., 2025). AgNP-mediated ROS production also affects the activity of various antioxidant enzymes, including NADPH-dependent flavoenzyme, catalase, glutathione peroxidase, and superoxide dismutase, disrupting the balance between ROS generation and detoxification (Dakal et al., 2016).

Following penetration of the bacterial cell, AgNPs interfere with various intracellular processes. They can bind to essential enzymes and proteins, leading to the inhibition of critical metabolic pathways (More et al., 2023). Additionally, AgNPs can displace essential metal ions, such as zinc and iron, from bacterial proteins, thereby disrupting enzyme functions and cellular homeostasis (Girma, 2023).

Another critical antimicrobial mechanism of AgNPs is their ability to interact with bacterial nucleic acids. AgNPs can directly bind to bacterial DNA, causing structural distortions that hinder replication and transcription. Ag⁺ ions intercalate between purine and pyrimidine base pairs, disrupting the hydrogen bonds between the complementary DNA strands and thereby destabilizing the double-helix structure (Klueh et al., 2000). Additionally, AgNPs induce structural changes in DNA, causing it to transition from a relaxed to a condensed state, which ultimately inhibits its ability to replicate (Feng et al., 2000).

Furthermore, Ag⁺ has been shown to interact with functional groups in proteins, leading to their deactivation. Specifically, Ag⁺ ions bind to thiol (-SH) groups in membrane-associated proteins, forming stable Ag–S bonds that disrupt protein function (Fahim et al., 2024). These proteins play essential roles in transmembrane ATP production and ion transport across the cell membrane (Klueh et al., 2000). Both AgNPs and Ag⁺ ions can alter the three-dimensional structure of proteins, disrupt disulfide bonds, and block active binding sites, ultimately impairing bacterial proliferation and contribute to AgNP-mediated cytotoxicity (Lok et al., 2006).

Recent studies suggest that AgNPs can interfere with bacterial quorum sensing (QS) and signal transduction pathways (Aflakian and Hashemitabar, 2025). Quorum sensing is a critical communication mechanism that bacteria use to regulate gene expression and coordinate collective behaviors, including biofilm formation and virulence. AgNPs have been shown to inhibit quorum sensing by disrupting signaling molecules, thereby preventing the establishment of biofilms and reducing bacterial pathogenicity (Awadelkareem et al., 2023). Studies have demonstrated that AgNPs serve as effective anti-QS agents, inhibiting biofilm formation and reducing violacein production in Chromobacterium violaceum (Jagtap and Priolkar, 2013). Additionally, green-synthesized AgNPs have shown significant potential in managing microbial infections. Research indicates that AgNPs can interfere with the synthesis of QS signaling molecules by inhibiting the LasI and RhlI synthases, thereby disrupting bacterial communication and virulence regulation (Lahiri et al., 2021). Furthermore, AgNPs have been shown to downregulate quorum sensing-related genes. In P. aeruginosa, green-synthesized AgNPs exhibited a dose-dependent inhibition of pyocyanin production, a key virulence factor (Selem et al., 2022). Pyocyanin, a blue redox-active secondary metabolite, plays a crucial role in biofilm development and significantly contributes to bacterial evasion of the host immune system. By suppressing pyocyanin synthesis, AgNPs can weaken bacterial pathogenicity and enhance susceptibility to antimicrobial treatments (Awadelkareem et al., 2023). Beyond quorum sensing inhibition, AgNPs can also modulate bacterial signal transduction pathways by interfering with phosphorylation-based signaling cascades (More et al., 2023). Many bacterial regulatory systems rely on histidine kinases and response regulators to sense environmental changes and control adaptation (Capra and Laub, 2012). Furthermore, analyzing the phosphotyrosine profile of bacterial proteins in both Gram-positive and Gram-negative bacteria provides valuable insight into how AgNPs influence bacterial signal transduction pathways. These pathways regulate essential cellular functions, including growth and metabolism. The reversible phosphorylation of tyrosine residues in key protein substrates, such as RNA polymerase sigma factor (RNA pol σ factor), single-stranded DNA binding proteins (ssDBPs), and UDP-glucose dehydrogenase, is crucial for their activation (Mijakovic et al., 2006). Once phosphorylated, these proteins play significant roles in DNA replication, recombination, metabolism, and cell cycle regulation. Consequently, AgNP-mediated inhibition of protein phosphorylation disrupts enzymatic activity, ultimately hindering bacterial growth and survival (Dakal et al., 2016).

In addition to oxidative stress and metabolic disruption, AgNPs can trigger apoptosis-like responses in bacteria. Some studies have demonstrated that AgNPs activate bacterial self-destruction pathways, akin to programmed cell death in eukaryotic cells. Research has demonstrated that AgNPs can inhibit the growth of E. coli and trigger apoptosis-like cell death (Kim and Lee, 2021). However, the exact mechanism underlying AgNP-induced apoptosis-like death, as well as its potential link to DNA damage-inducible protein F (DinF), a key component of the SOS response, remains unclear (Kim and Lee, 2021).

One of the most effective methods for improving AgNP targeting is surface functionalization with biocompatible ligands, which facilitates selective bacterial adhesion and penetration (Fu et al., 2024). Among the various functionalization approaches, chitosan-coated AgNPs have demonstrated superior mucoadhesive properties, allowing for stronger electrostatic interactions with negatively charged bacterial membranes (Krishnaraj et al., 2022). For instance, Wang et al. developed a chitosan/oxidized konjac glucomannan hydrogel incorporating AgNPs for the treatment of irregular wounds. The hydrogel exhibited self-healing properties, strong tissue adhesion, and potent antibacterial activity (Wang et al., 2020). Another example is chitosan-coated AgNPs, which have proven effective in treating wounds infected with MRSA (Peng et al., 2017). Furthermore, research by Mostafa et al. (2022) demonstrated that chitosan-silver conjugates exhibit promising broad-spectrum anti-biofilm activity against B. subtilis, P. aeruginosa, S. aureus, and E. coli (Mostafa et al., 2022). Similarly, AgNPs conjugated with antimicrobial peptides (AMPs), such as LL-37, help overcome their inherent limitations. This combination shows promise as a potential therapeutic agent against antibiotic-resistant bacteria, particularly MRSA (Masimen et al., 2022). Additionally, functionalizing AgNPs with folic acid has demonstrated promising antibacterial activity against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria (Chowdhuri et al., 2015). These modifications enable AgNPs to achieve higher bacterial selectivity while reducing unintended cytotoxicity to human cells.

Encapsulation within biodegradable polymeric matrices represents another promising approach for controlled AgNP release and prolonged antimicrobial effects. Natural and synthetic biopolymers, including alginate, poly(lactic-co-glycolic acid) (PLGA), and gelatin, have been utilized as nanocarriers to enhance AgNP stability and mitigate toxicity. For example, a hydrogel incorporating tannic acid-stabilized AgNPs (TA-AgNPs/alginate) exhibited strong antibacterial activity against S. pyogenes, S. aureus, and P. aeruginosa. Additionally, it showed promising potential for treating complex wound biofilms (Srichaiyapol et al., 2022). Rugaie et al. (2022) developed a straightforward, single-step method to coat AgNPs using polymeric stabilizers, specifically polyvinylpyrrolidone (PVP) and ethyl cellulose (EC) (Rugaie et al., 2022). Their investigation demonstrated that these coated AgNPs effectively inhibited biofilm formation by clinical isolates of E. coli on urinary catheters. Notably, AgNPs coated with PVP exhibited significantly greater biofilm inhibition compared to those stabilized with EC. Furthermore, PLGA-encapsulated AgNPs ensure a controlled and sustained release of silver ions, offering prolonged and enhanced antimicrobial activity while reducing host toxicity (Stevanović et al., 2012). Moreover, gelatin-PVA-AgNPs hydrogel has been explored for wound healing applications, demonstrating accelerated tissue regeneration while maintaining potent antimicrobial activity (Bag et al., 2022). The incorporation of AgNPs into polymeric matrices not only enhances their therapeutic efficacy but also facilitates localized drug delivery, thereby reducing systemic toxicity.

Liposomal carriers have emerged as effective nanocarriers for improving AgNP bioavailability and stability. Liposomal encapsulation shields AgNPs from premature degradation and enhances their circulation time in the biological environment. Liposomes are spherical vesicles with a phospholipid bilayer capable of encapsulating various chemical compounds (Kumar et al., 2023). Their unique bilayer structure enables the efficient entrapment of both hydrophilic and hydrophobic drugs, making them a versatile and promising platform for drug delivery applications (Kumar et al., 2023). Previous research has shown nanoliposomes loaded with AgNPs exhibit potent broad-spectrum antimicrobial activity against various pathogens, including E. coli, S. enterica, P. aeruginosa, and S. aureus (Eid and Azzazy, 2014). Additionally, these formulations have shown potential in promoting wound healing. This finding is reinforced by studies indicating that encapsulating antimicrobial agents, such as AgNPs, within nanoliposomes enhances their stability and targeted delivery. Additionally, nanoliposomes have demonstrated the ability to transport encapsulated agents directly to target bacteria in both in vitro and in vivo settings (Mozafari et al., 2021). The use of liposomal nanocarriers not only enhances AgNP stability but also reduces toxicity by preventing direct interaction with mammalian cells.

In addition to passive targeting mechanisms, stimuli-responsive AgNP delivery systems offer an advanced strategy for spatiotemporal control over silver ion release. These systems are designed to respond to specific bacterial microenvironmental cues, such as pH variations, enzymatic activity, or oxidative stress levels. For example, a pH-responsive hydrogel has been developed to enable the controlled, pH-triggered release of AgNPs. This system is designed to detect changes in environmental pH and release AgNPs when the pH shifts from acidic to alkaline, a condition associated with pathogenic bacterial presence in wounds (Haidari et al., 2021). This innovative hydrogel shows promise as an effective material for treating infected wounds, demonstrating the ability to eliminate both Gram-negative and Gram-positive bacteria without causing toxicity to mammalian skin cells. Additionally, enzyme-responsive silver nanoparticle assemblies (ANAs) have been developed to selectively target MRSA (Zuo et al., 2020). These assemblies undergo a stable-to-collapsed transition upon encountering MRSA due to the decomposition of branched copolymers—used as macrotemplates in ANA synthesis—triggered by serine protease-like B (SplB) enzyme proteins. This structural transition significantly enhances the targeting affinity and efficiency of ANAs against MRSA. These smart nanoplatforms allow for on-demand silver release, minimizing toxicity while maximizing antibacterial efficacy.

A highly specific approach to AgNP targeting involves antibody-conjugated AgNPs, which are engineered to selectively bind to bacterial surface markers. This strategy enables highly targeted antimicrobial action while reducing off-target toxicity. For example, AgNPs functionalized with a specific antibody can be combined with laser radiation as an innovative treatment to selectively target resistant bacteria, particularly S. aureus, while minimizing effects on the normal microflora (Al-Sharqi et al., 2020). Similarly, AgNPs conjugated with BK510Lys endolysin at a concentration of 0.01 mg/mL, in a 2:1 ratio, at 40°C, and pH 5, exhibited a stronger inhibitory effect than AgNPs alone (0.5 µg/mL) against over 65% of the Gram-negative bacteria tested, indicating it highly specific alternative drugs for super-resistant Gram-negative bacteria (Ramírez Saenz et al., 2024). By harnessing the high specificity of monoclonal antibodies, antibody-functionalized AgNPs hold great potential for precision antimicrobial therapy against MDR pathogens.

Enhancing the antimicrobial activity of AgNPs can be achieved by functionalizing their surface with bioactive molecules, polymers, or targeting ligands. For instance, conjugating AgNPs with antimicrobial peptides, antibodies, or small molecules can improve specificity and reduce non-specific interactions (Al-Sharqi et al., 2020; Ramírez Saenz et al., 2024). Additionally, coating AgNPs with biocompatible polymers such as PEG can enhance stability and bioavailability while reducing toxicity (Srichaiyapol et al., 2022; Ibraheem et al., 2024).

Combining AgNPs with conventional antibiotics or natural antimicrobial agents may enhance their efficacy and prevent resistance development. Studies have shown that AgNPs can potentiate the effects of antibiotics by disrupting bacterial membranes and increasing drug uptake (Dove et al., 2023; More et al., 2023; Palau et al., 2023). For instance, recent studies have demonstrated that AgNPs conjugated with antibiotics such as amikacin (e.g., AgNPs_mPEG_AK) displayed enhanced antibacterial activity against MDR strains, including E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii (Palau et al., 2023). These hybrid nanomaterials achieved notable activity at lower antibiotic concentrations, suggesting a dose-sparing effect. Moreover, integrating AgNPs with plant-derived bioactive compounds, such as flavonoids and essential oils, could provide a dual mechanism of action, improving antimicrobial potency and reducing cytotoxicity (Xu et al., 2020). Additionally, AgNPs synthesized using plant extracts—such as those from Teucrium polium, Teucrium parvifolium, Lepidium draba L., and Moringa oleifera—have shown synergistic antimicrobial activity when paired with natural compounds like flavonoids, polyphenols, and essential oils. These green-synthesized AgNPs offer dual mechanisms: physical disruption of bacterial membranes and bioactive-mediated interference in bacterial metabolism, while also exhibiting reduced cytotoxicity compared to chemically synthesized counterparts (Aljowaie and Aziz, 2025; Soltani et al., 2024; Alowaiesh et al., 2023; Abdel-Rahman et al., 2022).

Developing advanced delivery systems, such as pH-responsive, enzyme-triggered, or temperature-sensitive nanocarriers, can help achieve controlled and targeted release of AgNPs. This approach can enhance antibacterial efficacy while minimizing exposure to healthy cells. For example, hydrogels or liposomes loaded with AgNPs have shown promising results in wound infections and biofilm-associated bacterial resistance (Mozafari et al., 2021; Wang et al., 2020). Additionally, nanocarriers designed for site-specific release can reduce the required dosage and mitigate potential cytotoxic effects.

The physicochemical properties of AgNPs, including size, shape, and surface charge, play a crucial role in their antimicrobial efficacy. Smaller nanoparticles exhibit greater surface area and enhanced bacterial interaction, while specific shapes, such as triangular or rod-shaped nanoparticles, have demonstrated improved antimicrobial effects compared to spherical ones (Liao et al., 2019; Rodrigues et al., 2024). Moreover, tuning the surface charge of AgNPs can influence their interaction with bacterial membranes, optimizing their antibacterial activity while reducing toxicity to mammalian cells (Bélteky et al., 2019).

To improve the biocompatibility and environmental sustainability of AgNPs, green synthesis methods utilizing plant extracts, fungi, or bacteria have been explored. These eco-friendly approaches reduce the use of toxic chemical agents and enhance the biological properties of AgNPs (Zhang et al., 2016; Xu et al., 2020). Future research should focus on optimizing these biosynthetic techniques to ensure reproducibility, scalability, and clinical applicability.

Although AgNPs exhibit broad-spectrum antimicrobial activity, there is a growing concern about bacterial adaptation and resistance. To mitigate this risk, researchers should investigate combination strategies, adaptive dosing regimens, and mechanisms to prevent bacterial efflux of silver ions. Additionally, integrating AgNPs with nanomaterials that disrupt bacterial communication systems, such as quorum sensing inhibitors, could reduce the likelihood of resistance development (Awadelkareem et al., 2023).

Despite extensive in vitro research, the clinical translation of AgNP-based antimicrobials remains limited. Future studies should focus on in vivo models to evaluate pharmacokinetics, biodistribution, and long-term safety. Clinical trials are necessary to validate their effectiveness against MDR bacterial infections while assessing potential side effects (Zhang et al., 2016; Xu et al., 2020). Regulatory guidelines must also be established to ensure the safe application of AgNPs in medical and pharmaceutical settings.