While picking a drug material for the treatment of certain disease types including cancer, hemocompatibility of that material should be kept in mind [25]. Kwon et al. demonstrated the minimization of the damage to membrane including hemolysis, potassium efflux, protein leakage and alterations in cell shape and membrane fragility by the use of AgNPs of specific shape and size [26]. Therefore, AgNPs have been evidenced to have great potential in anti-cancer activity since they show selective participation in the interruption of mitochondrial respiratory chain that leads to the production of reactive oxygen species (ROS) and disruption of adenosine triphosphate (ATP) synthesis, thus resulting in nucleic acid damage [27]. Various studies have established that temperature, pH of the solution, precursor concentration, the molar ratio of capping agent to that of precursor, the types of reducing agents, the strength of chemical interaction between the precursor and different crystallographic planes of AgNPs and the synthesis method determine the size and shape of the AgNPs [28]. Depending on the synthesis methods, a range of particle sizes and shapes (e.g., cubes, prisms, spheres, rods, wires, plates, etc.) can be obtained [29]. For instance, in the synthesis of AgNPs from the bacteria Xanthomonas oryzae, spherical as well as triangular and rod-shaped particles with an average size of 14.86 nm were obtained depending on the experimental conditions [30], whereas bacterial synthesis from Pseudomonas stutzeri AG259 resulted in triangular and hexagonal particles with an average size of 200 nm [31]. In the fungal synthesis from Fusarium acuminatum, spherical AgNPs in the range of 5–40 nm (average size 13 nm) were reported [32]. On the other hand, AgNPs synthesis from the fungus Trichoderma viride, spherical and sporadically rod-like particles in the range of 10–40 nm at 27 °C. Decreasing the temperature to 10 °C increased the particle size to 80–100 nm [15]. Studies have established that low reaction temperatures can facilitate the formation of two-dimensional nanostructures [33]. To underscore the range of sizes and shapes of AgNPs which plant-based green synthesis can yield, the study on Nelumbo nucifera can be used as an example. Synthesis using the leaf extract of this plant resulted in AgNPs with spherical, triangular, truncated triangular and decahedral morphologies. Sizes of the particles ranged between 25 and 80 mm averaging 45 mm [34]. Vilchis-Nestor et al. reported more spherical and larger AgNPs under ambient conditions with the increase in the concentration of the Camellia sinensis (green tea) extracts [35].

AgNPs efficiently interact with light through a phenomenon known as surface plasmon resonance (SPR) more efficiently than any known organic or inorganic chromophores. This strong interaction is a result of both the restriction of a large density of conducting electrons to smaller dimensions compared to the mean free path and the unique frequency dependence of the dielectric function in metallic silver. The combination of these two factors gives rise to SPR related properties. The size and shape of NPs and the dielectric function of the surrounding medium determines the frequency and resonance strength [36]. The light-interaction cross-sections of AgNPs is dependent on the photon electric field which can extend up to 10 times larger than the geometric cross-sections of the AgNPs. As such, these structures are capable of interaction with light photons that are not physically incident upon them ^[37][38][37,38]. Because of the unique optical characteristics of the AgNPs, non-invasive techniques like dark-field microscopy or surface-enhanced Raman Scattering can be utilized for the inspection of their tracking and cellular uptake [39]. Certain optical characteristics of AgNPs can be modified to obtain interesting results. For example, the absorption spectrum of AgNPs is tunable through careful optimization of the synthesis conditions to the near-infrared region. This eliminates the interference from tissue auto-fluorescence, resulting in NPs which have great promise in tumor-targeting and deep tissue-imaging [40].

One of the important electrical properties of AgNPs in a colloidal suspension is zeta potential which is influenced by the size of the NPs. AgNPs possess a large absolute value of zeta potential corresponding to a high surface electrical charge which can result in a strong repulsive force among particles to resist agglomeration [41]. Agglomeration needs to be suppressed at any cost to prevent the growth of the particles from nanoscale to microscale. It has been established that a high zeta potential value of around −25 mV ensures a large enough energy barrier for the stabilization of the nanosuspension. For example, Sankar et al. reported a zeta potential of about −26 ± 0.77 mV for the biosynthesis of AgNPs with an average size of 136 ± 10.09 nm from Origanum vulgare [42].

Biosynthesis of NPs may occur extracellularly or intracellularly. A widely utilized extracellular mechanism for AgNPs synthesis via bacteria employs NADH-dependent nitrate reductase-mediated reduction of silver ion (Ag⁺) by several bacteria species, such as Streptomyces sp. LK3 [51] and Bacillus licheniformis [52]. During reduction, nitrate ions (NO₃⁻) in silver nitrate (AgNO₃) are reduced to nitrite (NO₂⁻) by accepting two protons then releasing two electrons and water. Electrons released in this reaction are transferred to the Ag⁺ ion to form elemental silver (Ag⁰) [53]. This mechanism is possibly dependent on electron transport pathways and enzymatic metal reduction processes (Figure 2).

However, enzymes are not always necessary for bacterial synthesis of AgNPs. For example, non-enzymatic intracellular synthesis was reported in Lactobacillus A09, where Ag⁺ reduction occurred on the bacterial cell surface [53]. Gram-positive bacterial cell walls, such as those of Lactobacillus A09, contain numerous anionic surface groups. These groups can provide silver ion biosorption sites [55]. The pH of the surrounding medium decreased slightly after adsorption of Ag⁺ on Lactobacillus A09, suggesting competition between proton and silver ion binding to negatively charged sites [56]. Potentially an increase in pH can open bacteria cell wall monosaccharide rings and oxidize these moieties to open-chain aldehydes. At the same time, dissociation of protons from protonated anionic functional groups (–RH) creates negatively charged Ag⁺ adsorption sites on the cell surface. The two electron released while forming the aldehyde group from an alcohol can reduce Ag⁺ ions to elemental Ag⁰ [57]. The steps involved in the bacterial synthesis of AgNPs are mediated through glucose ring-opening (Figure 3).

Due to their inherent ability to efficiently secrete protein, fungi can produce substantially larger amounts of AgNPs than bacteria [58]. However, the exact mechanism by which fungi synthesize AgNPs is not completely understood. Mukherjee et al. hypothesized that the following steps may occur during synthesis in Verticillium model system: Ag⁺ ions interact electrostatically with negatively charged carboxylate groups in the mycelial cell wall and are trapped at the cell surface. Subsequently, trapped ions are reduced by specific fungal enzymes [46]. Enzymes involved in fungal NP synthesis are generally reductases [59]. For example, during synthesis of AgNPs by Aspergillus flavus, a 32-kDa reductase protein secreted by the fungal isolate, reduces Ag⁺ ions [60]. A schematic representation of AgNPs synthesis by fungi is presented in Figure 4.

Plant extracts have been used for AgNPs synthesis and can be superior to bacterial and fungal synthetic systems due to their availability, low toxicity and safety. A wide range of extracted phytochemicals can rapidly reduce silver ions. Reduction can be rapid compared to microbial fermentation [50], as this approach can avoid lengthy sterile cell-culture under controlled conditions [61]. Furthermore, plant extracts can act as both reducing and stabilizing agent during AgNPs synthesis. Approaches of reduction with plant extracts can be applied to other metallic NPs [62]. The plant source determines the properties of the AgNPs [63]. The concentration of individual phytochemicals and combinations of phytochemicals present vary considerably in extracts depending on the plant source. Therefore, AgNPs’ properties can be selected by controlling the extract composition [64]. The biosynthesis of AgNPs by plant extracts commonly involves mixing of an aqueous solution of silver nitrate with an aqueous plant extract. Typical reactions take place at room temperature and requires only a few minutes to complete [47].

Numerous phytochemicals have been identified that are capable of producing AgNPs. As examples, flavonoids, terpenoids, terpenes, flavones, phenolics, polysaccharides, saponins, tannins and alkaloids have all been used for AgNPs synthesis ^[47][65][47,65]. Phytochemical functional groups, such as hydroxyl, aldehyde, ketone, carboxyl and amino groups are capable of reducing Ag⁺ ions ^[50][66][50,66]. Due to the diversity of phytochemicals the exact mechanism of AgNPs synthesis varies. However, the chief mechanism is the reduction of Ag⁺ ions by specific functional groups. For example, Punica granatum peel extract contains the flavonoids- kaempferol and naringin along with their glycosides. All of these compounds have hydroxyl (–OH) groups that cause the reduction of the Ag⁺ ions, resulting in the formation of AgNPs [67]. A typical reduction is possible by the use of kaempferol as a reducing agent (Figure 5).

Interestingly, not all phytochemicals present in plant extracts capable of reducing other compounds remain active during Ag⁺ ions reduction and only certain phytochemicals are involved in this reduction. For example, phytochemical screening revealed the presence of phenols, glycosides, flavonoids, reducing sugars, resins, tannins, terpenoids, steroids, alkaloids and saponins in Anagallis tenella leaf extract (Table 1).

However, it was discovered that only flavonoids were involved in AgNPs synthesis [65]. Active phytochemicals associated with AgNPs synthesis in other plants are listed in Table 1. As stated earlier, the wide variety of phytochemicals present in different plants that contribute to AgNPs synthesis complicates this field of study. Each plant has a unique spectrum of phytochemicals that act as organic reducing agents [72].