Silver nanoparticles: synthesis, properties, toxicology, applications and perspectives

Currently, many methods have been reported for the synthesis of Ag-NPs by using chemical, physical, photochemical and biological routes. Each method has advantages and disadvantages with common problems being costs, scalability, particle sizes and size distribution. Among the existing methods, the chemical methods have been mostly used for production of Ag-NPs. Chemical methods provide an easy way to synthesize Ag-NPs in solution.

Monodisperse samples of silver nanocubes were synthesized in large quantities by reducing silver nitrate with ethylene glycol in the presence of polyvinylpyrrolidone (PVP) [14], the so-called polyol process. In this case, ethylene glycol served as both reductant and solvent. It showed that the presence of PVP and its molar ratio relative to silver nitrate both played important roles in determining the geometric shape and size of the product. It suggested that it is possible to tune the size of silver nanocubes by controlling the experimental conditions.

Spherical Ag-NPs with a controllable size and high monodispersity were synthesized by using the polyol process and a modified precursor injection technique [15]. In the precursor injection method, the injection rate and reaction temperature were important factors for producing uniform-sized Ag-NPs with a reduced size. Ag-NPs with a size of 17 ± 2 nm were obtained at an injection rate of 2.5 ml s⁻¹ and a reaction temperature of 100 °C. The injection of the precursor solution into a hot solution is an effective means to induce rapid nucleation in a short period of time, ensuring the fabrication of Ag-NPs with a smaller size and a narrower size distribution.

Nearly monodisperse Ag-NPs have been prepared in a simple oleylamine-liquid paraffin system [16]. It was shown that the formation process of Ag-NPs could be divided into three stages: growth, incubation and Oatwald ripening stages. In this method, only three chemicals, including silver nitrate, oleylamine and liquid paraffin, are employed throughout the whole process. The higher boiling point of 300 °C of paraffin affords a broader range of reaction temperature and makes it possible to effectively control the size of Ag-NPs by varying the heating temperature alone without changing the solvent. Otherwise, the size of the colloidal Ag-NPs could be regulated not only by changing the heating temperature, or the ripening time, but also by adjusting the ratio of oleylamine to the silver precursor.

Generally, the chemical synthesis process of the Ag-NPs in solution usually employs the following three main components: (i) metal precursors, (ii) reducing agents and (iii) stabilizing/capping agents. The formation of colloidal solutions from the reduction of silver salts involves two stages of nucleation and subsequent growth. It is also revealed that the size and the shape of synthesized Ag-NPs are strongly dependent on these stages. Furthermore, for the synthesis of monodispered Ag-NPs with uniform size distribution, all nuclei are required to form at the same time. In this case, all the nuclei are likely to have the same or similar size, and then they will have the same subsequent growth. The initial nucleation and the subsequent growth of initial nuclei can be controlled by adjusting the reaction parameters such as reaction temperature, pH, precursors, reduction agents (i.e. NaBH₄, ethylene glycol, glucose) and stabilizing agents (i.e. PVA, PVP, sodium oleate) [17–19].

For a physical approach, the metallic NPs can be generally synthesized by evaporation–condensation, which could be carried out by using a tube furnace at atmospheric pressure. However, in the case of using a tube furnace at atmospheric pressure there are several drawbacks such as a large space of tube furnace, great consumption energy for raising the environmental temperature around the source material and a lot of time for achieving thermal stability. Therefore, various methods of synthesis of Ag-NPs based on the physical approach have been developed.

A thermal-decomposition method was developed to synthesize Ag-NPs in powder form [20]. The Ag-NPs were formed by decomposition of a Ag¹⁺–oleate complex, which was prepared by a reaction with AgNO₃ and sodium oleate in a water solution, at high temperature of 290 °C. Average particle size of the Ag-NPs was obtained of about 9.5 nm with a standard deviation of 0.7 nm. This indicates that the Ag-NPs have a very narrow size distribution.

In another work Jung et al [21] reported an attempt to synthesize metal NPs via a small ceramic heater that has a local heating area. The small ceramic heater was used to evaporate source materials. The results showed that the geometric mean diameter, the geometric standard deviation and the total number concentration of NPs increase with heater surface temperature. The particle generation was very stable, because the temperature of the heater surface does not fluctuate with time. Spherical NPs without agglomeration were observed, even at high concentration with high heater surface temperature. The generated Ag-NPs were pure silver, when air was used as a carrier gas. The geometric mean diameter and the geometric standard deviation of Ag-NPs were in the range of 6.2–21.5 nm and 1.23 − 1.88 nm, respectively.

Tien et al [22] used the arc discharge method to fabricate Ag-NPs suspension in deionized water with no added surfactants. In this synthesis, silver wires (Gredmann, 99.99%, 1 mm in diameter) were submerged in deionized water and used as electrodes. The experimental results show that Ag-NPs suspension fabricated by means of arc discharge method with no added surfactants contains metallic Ag-NPs and ionic silver. With a silver rod consumption rate of 100 mg min⁻¹, yielding metallic Ag-NPs of 10 nm in size and ionic silver obtained at concentrations of approximately 11 ppm and 19 ppm, respectively.

More recently Siegel et al [23] reported on the development of an unconventional approach for the physical synthesis of gold-NPs and Ag-NPs. The notable metallic NPs were synthesized by direct metal sputtering into the liquid medium. The method, combining physical deposition of metal into propane-1,2,3-triol (glycerol), provides an interesting alternative to time-consuming, wet-based chemical synthesis techniques. From this method, both Au-NPs and Ag-NPs possess round shape with average diameter of about 3.5 nm with standard deviation of 1.5 and 2.4 nm, respectively. It was observed that the NPs size distribution and uniform particle dispersion remains unchanged for diluted aqueous solutions up to glycerol-to-water ratio 1:20.

In summary, the physical synthesis process of Ag-NPs usually utilizes the physical energies (thermal, ac power, arc discharge) to produce Ag-NPs with nearly narrow size distribution. The physical approach can permit producing large quantities of Ag-NPs samples in a single process. This is also the most useful method to produce Ag-NPs powder. However, primary costs for investment of equipment should be considered.

The photo-induced synthetic strategies can be categorized into two distinct approaches, that is the photophysical (top down) and photochemical (bottom up) ones. The former could prepare the NPs via the subdivision of bulk metals and the latter generates the NPs from ionic precursors. The NPs are formed by the direct photoreduction of a metal source or reduction of metal ions using photo-chemically generated intermediates, such as excited molecules and radicals, which is often called photosensitization in the synthesis of NPs [24, 25].

The direct photo-reduction process of AgNO₃ in the presence of sodium citrate (NaCit) was carried out with different light sources (UV, white, blue, cyan, green and orange) at room temperature [26]. It was shown that this light-modification process results in a colloid with distinctive optical properties that can be related to the size and shape of the particles. A simple and reproducible UV photo-activation method for the preparation of stable Ag-NPs in aqueous Triton X-100 (TX-100) was reported [27]. The TX-100 molecules play a dual role: they act as reducing agent and also as NPs stabilizer through template/capping action. In addition, surfactant solution helps to carry out the process of NPs growth in the diffusion controlled way (by decreasing the diffusion or mass transfer co–efficient of the system) and also helps to improve the NPs size distributions (by increasing the surface tension at the solvent–NPs interface).

In another study, the Ag-NPs were synthesized in an alkalic aqueous solution of AgNO₃/carboxymethylated chitosan (CMCTS) with UV light irradiation. CMCTS, a water-soluble and biocompatible chitosan derivative, served simultaneously as a reducing agent for silver cation and a stabilizing agent for Ag-NPs in this method [28]. It also revealed that the diameter range of as-synthesized Ag-NPs was 2–8 nm and they can be dispersed stably in the alkalic CMCTS solution for more than 6 months.

In summary, the main advantages of the photochemical synthesis are: (i) it provides the advantageous properties of the photo-induced processing, that is, clean process, high spatial resolution, and convenience of use, (ii) the controllable in situ generation of reducing agents; the formation of NPs can be triggered by the photo irradiation and (iii) it has great versatility; the photochemical synthesis enables one to fabricate the NPs in various mediums including emulsion, surfactant micelles, polymer films, glasses, cells, etc [25].

As mentioned above, when Ag-NPs are produced by chemical synthesis, three main components are needed: a silver salt (usually AgNO₃), a reducing agent (i.e. ethylene glycol) and a stabilizer or aping agent (i.e. PVP) to control the growth of the NPs and prevent them from aggregating. In case of the biological synthesis of Ag-NPs, the reducing agent and the stabilizer are replaced by molecules produced by living organisms. These reducing and/or stabilizing compounds can be utilized from bacteria, fungi, yeasts, algae or plants [29].

A facile biosynthesis using the metal-reducing bacterium, Shewanella oneidensis, seeded with a silver nitrate solution, was reported [30]. The formation of small, spherical, nearly monodispersed Ag-NPs in the size range from ∼2 to 11 nm (average size of 4 ± 1.5 nm) was observed. The Ag-NPs exhibit useful properties such as being hydrophilic, stable, and having a large surface area. This bacterially based method of synthesis is economical, simple, reproducible, and requires less energy when compared to chemical synthesis routes.

In another study, the use of the fungus Trichoderma viride (T. viride) for the extracellular biosynthesis of Ag-NPs from silver nitrate solution was reported [31]. In this regard T. viride proves to be an important biological component for extracellular biosynthesis of stable Ag-NPs. The morphology of Ag-NPs is highly variable, with spherical and occasionally rod-like NPs observed on micrographs. The obtained diameter of Ag-NPs was in the range of from 5 to 40 nm. In another study, stable Ag-NPs of 5–15 nm in size were synthesized by using an airborne bacteria (Bacillus sp.) and silver nitrate [32]. The biogenic NPs were observed in the periplasmic space of the bacterial cells, which is between the outer and inner cell membranes.

Also, the Ag-NPs were produced by using the Lactobcillus spp. as reducing and capping agent. Sintubin et al [33] were carried with different Lactobcillus species to accumulate and subsequently reduce Ag⁺. The result showed that only the lactic acid bacterial were confirmed to have the ability to produce Ag⁰. In addition, both particle localization and distribution inside the cell were dependent on Lactobcillus species. The mean diameter of the biogenic Ag-NPs produced by this method varied with the Lactobacillus spp. used. The smallest NPs were produced by L. fermentum and had a diameter of 11.2 nm. The recovery of silver and the reduction rate were pH dependent.

On the other hand, Naik et al [34] have demonstrated the biosynthesis of biogenic Ag-NPs using peptides selected by their ability to bind to the surface of silver particles. By the nature of peptide selection against metal particles, a 'memory effect' has been imparted to the selected peptides. The silver-binding clones were incubated in an aqueous solution of 0.1 mM silver nitrate for 24–48 h at room temperature. The silver particles synthesized by the silver-binding peptides showed the presence of silver particles 60–150 nm in size.

In summary, the biological method provides a wide range of resources for the synthesis of Ag-NPs, and this method can be considered as an environmentally friendly approach and also as a low cost technique. The rate of reduction of metal ions using biological agents is found to be much faster and also at ambient temperature and pressure conditions. In biological synthesis, the cell wall of the microorganisms pays a major role in the intracellular synthesis of NPs. The negatively charged cell wall interacts electrostatically with the positively charged metal ions and bioreduces the metal ions to NPs [35]. When microorganisms are incubated with silver ions, extracellular Ag-NPs can be generated as an intrinsic defense mechanism against the metal's toxicity. Other green syntheses of Ag-NPs using plant exacts as reducing agents have been performed [36, 37]. This defense mechanism can be exploited as a method of NPs synthesis and has advantages over conventional chemical routes of synthesis. However, it is not easy to have a large quantity of Ag-NPs by using biological synthesis. Characteristics of synthesis routes of the Ag-NPs are summarized in table 1.

The Ag-NPs have been demonstrated as an effective biocide against a broad-spectrum bacteria including both Gram-negative and Gram-positive bacteria [43], in which there are many highly pathogenic bacterial strains. Table 2 summarizes the exhibited antibacterial activities of Ag-NPs, the data were collected from recent publications.

In 2004 Sondi and Salopeck-Sondi [44] reported the antimicrobial activities of Ag-NPs against the growth of E. coli on Luria–Bertani agar plates. In this study, the E. coli bacterial strain served as a model of Gram-negative bacteria. Results showed that the growth inhibition of E. coli was dependent on the concentration of Ag-NPs and the initial concentration of cultivated bacteria. The growth inhibitory concentrations were found to be about 50–60 and 20 μg cm⁻³ for 10⁵ CFU and 10⁴ CFU of E. coli, respectively. Noticeably, the bacterial cells were damaged and destroyed along with the accumulation of Ag-NPs in the bacterial membrane. Morones et al [45] have also used different types of Gram-negative bacteria to test the antibacterial activities of Ag-NPs in the range of 1–100 nm. It was reported that the antibacterial activity of Ag-NPs against Gram-negative bacteria divided into three steps: (i) nanoparticles mainly in the range of 1–10 nm attach to the surface of the cell membrane and drastically disturb its proper functions, such as permeability and respiration; (ii) they are able to penetrate inside the bacteria and cause further damage by possibly interacting with sulfur- and phosphorus-containing compounds such as DNA; (iii) nanoparticles release silver ions, which will have an additional contribution to the bactericidal effect of Ag-NPs. In addition, Kim et al [46] have used a model of both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria to investigate the antibacterial activities of Ag-NPs. Their studies revealed that E. coli is inhibited at a low concentration of Ag-NPs (3.3 nM), and ten times less than the minimum inhibitory concentration on S. aureus (33 nM). In another report, Shrivastava et al [47] described the strong antibacterial potency of novel Ag-NPs in the range of 10–15 nm with increased stability against some strains of non-resistant and drug-resistant bacteria. It was concluded that the antibacterial effect is dose-dependent and is more pronounced against Gram-negative than Gram-positive bacteria; it was also independent of acquisition of resistance by the bacteria against antibiotics. It was also suggested that the major mechanism in which Ag-NPs manifested antibacterial properties was by anchoring to and penetrating the bacterial cell wall, and modulating cellular signaling by dephosphorylating putative key peptide substrates on tyrosine residues.

In a comparative study of the effect of the Ag-NPs in different shapes on the Gram-negative bacterium, Pal et al [48] have demonstrated that Ag-NPs undergo shape-dependent interaction with E. coli. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles, and with ionic silver.

Kvitek et al [49] have reported that the antibacterial activity of Ag-NPs is also dependent on surface modifications (surfactant/polymers). In their study, different types of surfactants/polymers (sodium dodecyl sulfate-SDS and polyoxyethylenesorbitane monooleate-Tween 80), and one polymer (polyvinylpyrrolidone-PVP 360) were used. These stabilized Ag-NPs were tested with some bacterial strains including S. aureus, E. faecalis, E. coli and P. aeruginosa, and other strains isolated from human clinical samples such as P. aeruginosa, methicillin-susceptible S. epidermidis, methicillin-resistant S. epidermidis, methicillin-resistant S. aureus, vancomycin-resistant E. faecium and K. pneumonia. The obtained results showed the minimum inhibitory concentrations (MICs) of Ag-NPs in the range of 1.69–13.5 μg ml⁻¹, depending on bacterial strains, and the use of surfactants/polymers. Specifically, the antibacterial activity of the Ag-NPs was significantly enhanced when modified by SDS where the MIC decreased under the 'magical value' of 1 μg ml⁻¹. Furthermore, Guzman et al [50] have reported the results of antibacterial activities of synthesized Ag-NPs against E. coli, P. aeruginosa and S. aureus around 14.38, 6.74, and 14.38 ppm, respectively.

In our studies, Ag-NPs were synthesized by different techniques and tested with several bacterial strains such as E. coli, S. aureus, V. cholera, etc. The low MICs were found against these bacterial strains [51–55]. Especially, oleic acid stabilized-Ag-NPs showed the MIC against E. coli as low as 1 μg ml⁻¹ [51].

Recently, the increasing number of drug-resistant bacteria has become a major challenge endangering human health. Ag-NPs have been also demonstrated as an effective biocide against these drug-resistant strains [43, 49, 56, 57]. In a study, Lara et al [56] have tested the antibacterial activities of commercial Ag-NPs (100 nm), and results revealed a minimum inhibitory concentration (on average) at 79.4 nM for drug-resistant bacteria such as Erythromycin-resistant Streptococcus. pyogenes (66.7 nM), ampicillin-resistant E. coli O157:H7 (83.3 nM) and multidrug-resistant P. aeruginosa (83.3 nM), and at 74.3 nM for drug-susceptible tested bacterial strains. This study also showed the MICs at 100 nM for methicillin-resistant S. aureus (MRSA), and at 200 nM for drug-susceptible S. aureus.

To date, there have been many studies on the effects of Ag-NPs against different bacterial strains. Although some articles proposed different ways to explain the growth inhibition and death of bacterial cells acted on by Ag-NPs [44, 46, 48], but the exact antibacterial mechanism of Ag-NPs has not been fully understood. In a recent review, Jones and Hoek [43] summarized three most common antibacterial mechanisms of Ag-NPs as follows: (i) uptake of free silver ions followed by disruption of ATP production and DNA replication, (ii) Ag-NPs and silver ion generation of reactive oxygen species (ROS) and (iii) Ag-NPs' direct damage to cell membranes. However, further investigations are still needed to demonstrate more clearly this mechanism, especially our question concerning the affinity of Ag-NPs to sulfur- and phosphorus-containing proteins of bacteria, and the effects of this affinity to the functions of bacterial proteins [54, 55].

Of course, with observed excellent antibacterial properties, Ag-NPs have been suggested as effective broad-spectrum biocides against a variety of drug-resistant bacteria, and a potential candidate for use in pharmaceutical and medical products in order to prevent the transmission of drug-resistant pathogens in different clinical environments [58].

Fungi are increasingly recognized as major pathogens in critically ill patients, especially nosocomial fungal infections [59]. Although the antibacterial activities of Ag-NPs are well-known, the antifungal activities of this material have not yet been studied adequately (table 2). This section aims to review the most important properties of Ag-NPs against common fungal strains.

Kim et al [60] studied the antifungal activities of Ag-NPs against a total of 44 strains of six fungal species from clinical isolates and ATCC strains of Trichophyton mentagrophytes (T. mentagrophytes) and Candida albanicans (C. albanicans). Results showed 80% inhibitory concentration (IC₈₀) from 1 to 7 μg ml⁻¹. The antifungal activity of Ag-NPs against C. albanicans could be exerted by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity [61]. In another publication, Roe et al [62] have tested the antifungal activity of plastic catheters coated with Ag-NPs (∼100 nm thick), and results showed that the growth inhibition was almost complete for C. albicans. More recently, Pamacek et al [63] investigated the antifungal activity of Ag-NPs prepared by the modified Tollens process. Results also revealed the minimum inhibition against C. albicans growth at 0.21 mg ⁻¹ using naked Ag-NPs, and 0.05 mg l⁻¹ using Ag-NPs modified with sodium dodecyl sulfate (SDS). Additionally, Ag-NPs effectively inhibited the growth of the tested yeasts at the concentrations below their cytotoxic limit against the tested human fibroblasts determined at a concentration equal to 30 mg l⁻¹ of Ag-NPs.

Other publications also reported MICs of Ag-NPs from 0.4 to 3.3 μg ml⁻¹ against C. albicans and C. glabrata adhered cells and biofilm [64], and at 10 μg ml⁻¹ against Trichophyton rubrum (T. rubrum) [65]. In summary, Ag-NPs have also been revealed as potential biocide against fungal strains, and could help to prevent fungal infections for protection of human health.

In recent years, there was an increase in reported numbers of emerging and re-emerging infectious diseases caused by viruses such as SARS-Cov, influenza A/H₅N₁, influenza A/H₁N₁, Dengue virus, HIV, HBV, and new encephalitis viruses etc. These viral infections are likely to break out into highly infectious diseases endangering public health [66].

Ag-NPs have shown effective activities against microorganisms including bacteria and fungi as mentioned above. However, the antiviral activities of Ag-NPs are still open questions to researchers. Very few papers have been found that investigate the effects of Ag-NPs against viruses (table 2). As the first report, Elechiguerra et al [67] have investigated the interaction between Ag-NPs and HIV-1. It was reported that Ag-NPs undergo a size-dependent interaction, with NPs exclusively in the range of 1–10 nm attached to the virus. It was also suggested that Ag-NPs interact with the HIV-1 virus via preferential binding to the exposed sulfur-bearing residues of the gp120 glycoprotein knobs, resulting in the inhibition of the virus from binding to host cells. This mechanism was then demonstrated by Lara et al [68]. In this article, it was reported that Ag-NPs exert anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or as an inhibitor of viral entry. The Ag-NPs bind to gp120 in a manner that prevents CD4-dependent virion binding, fusion, and infectivity, acting as an effective virucidal agent against cell-free virus (laboratory strains, clinical isolates, T and M tropic strains, and resistant strains) and cell-associated virus. Besides, Ag-NPs inhibit post-entry stages of the HIV-1 life cycle.

In another study, Lu et al [69] investigated the effects of Ag-NPs of different sizes (10, 50 and 800 nm) on the hepatitis B virus (HBV), and using a HepAD38 cell line as infection model. Their study showed that only Ag-NPs could inhibit production of HBV RNA and extracellular virions in vitro. Furthermore, Sun et al [70] have utilized Ag-NPs conjugated with (N-vinyl-2-pyrrolidone) (PVP), recombinant respiratory syncytial virus (RSV) fusion (F) protein, and bovine serum albumin (BSA) in order to study the inhibition of RSV infection in HEp-2 cell culture. The obtained results revealed that PVP-coated silver nanoparticles, which showed low toxicity to cells at low concentrations, inhibited RSV infection by 44%, a significant reduction compared to other controls. It was concluded that when the PVP-coated Ag-NPs mixed with RSV, they would bind to the G proteins on the viral surface and interfere with viral attachment to the HEp-2 cells resulting in the inhibition of viral infection. Monkeypox virus was tested with Ag-NPs in different sizes and surface coatings. The preliminary results showed that both polysaccharide-coated Ag-NPs (25 nm) and non-coated Ag-NPs (55 nm) exhibit a significant (P ⩽ 0.05) dose-dependent effect of test compound concentration on the mean number of plaque-forming units (PFU), and Ag-NPs of approximately 10 nm inhibit MPV infection in vitro [71].

De Gusseme et al [72] produced the bio-Ag-NPs (called biogenic Ag⁰), and tested the antiviral activities of both biogenic Ag⁰ and ionic Ag⁺ against murine norovirus 1. Remarkably, the obtained results showed that in the disinfection assay with ionic Ag⁺, only a small decrease in genomic copies was detected. Exposure to biogenic Ag⁰ did not result in a significant decrease in genomic copies as well. In contrast, the plaque assays demonstrated that the infectivity of MNV-1 was completely inhibited. It was suggested that the inactivation mechanism of MNV-1 is the interaction of biogenic Ag⁰ with (the thiol groups of) the MNV-1 capsid proteins, making the RNA accessible and rendering the virus particle noninfectious. This interaction might possibly be favored by smaller nanoparticles.

More recently, Xiang et al [73] investigated the inhibitory effects of Ag-NPs on H1N1 influenza A virus in vitro. Their study revealed that Ag-NPs have efficient inhibitory activity on H1N1 influenza A virus, which can rapidly inhibit H1N1 influenza A virus hemagglutination of chicken RBCs. In addition, Ag-NPs could also reduce H1N1 influenza A virus induced apoptosis toward MDCK cells. However, the authors also proposed to clarify how Ag-NPs inhibit H1N1 influenza A virus infectivity as well as the application of Ag-NPs to be an effective drug against influenza.

In summary of the antiviral effects of Ag-NPs, most publications have suggested that Ag-NPs could bind to outer proteins of viral particles, resulting in inhibition of binding and the replication of viral particles in cultured cells. Although the antiviral mechanism of Ag-NPs has not been fully known yet, Ag-NPs are still suggested as potential antiviral agents in the future [74].

Bioaerosols are airborne particles of biological origins including viruses, bacteria, fungi, which are capable of causing infectious, allergenic or toxigenic diseases. Particularly, indoor air bioaerosols were found to accumulate in large quantities on filters of heating, ventilating, and air-conditioning (HVAC) systems [76]. It is found that outdoor air pollution and insufficient hygiene of an HVAC installation often resulted in the low quality of indoor air. Moreover, the organic or inorganic materials deposited on the filter medium after air filtration contribute to microbial growth. The WHO estimated that 50% of the biological contamination present in indoor air comes from air-handling systems, and the formation of harmless micro-organisms such as bacterial and fungal pathogens was found in air filters. It is important to note that most of these pathogens produce mycotoxins which are dangerous to human health. To reduce the microbial growth in air filters, the integration of antimicrobial Ag-NPs in air filters has been proposed and developed.

The antimicrobial effect of Ag-NPs on bacterial contamination of activated carbon filters (ACF) was studied [76]. The results showed that Ag-deposited ACF filters were effective for the removal of bioaerosols. The antibacterial activity analysis of Ag-coated ACF filters indicated that two bacteria of Bacillus subtilis and E. coli were completely inhibited within 10 and 60 min, respectively. It was found that silver deposition did not influence the physical properties of ACF filters such as pressure drop and filtration efficiency, however, the adsorptive efficacy was decreased by silver deposition. Therefore, the authors also suggested that the amount of Ag-NPs on the ACF filters needs to be optimized to avoid excessive reduction of their adsorptive characteristics and to show effective antimicrobial activity.

In a recent work [77], the authors generated Ag-coated CNT hybrid nanoparticles (Ag/CNTs) using aerosol nebulization and thermal evaporation/condensation processes and studied their applicability to antimicrobial air filtration. CNT and Ag-NPs aerosols mixed together and attached to each other, forming Ag/CNTs. The antimicrobial activity of Ag/CNT-coated filters was tested against two bacteria of Gram-positive S. epidermidis and Gram-negative E. coli. It was found that when Ag/CNTs were deposited on the surface of an air filter medium, the antimicrobial activity against tested bacterial bioaerosols was enhanced, compared with the deposition of CNTs or Ag-NPs alone, whereas the filter pressure drop and bioaerosol filtration efficiency were similar to those of CNT deposition only. It was reported that the surface area of Ag-NPs was enhanced by CNTs. This is the main reason for the higher antimicrobial filtration efficacy of Ag/CNTs compared with that of pure Ag-NPs.

Polymer air filters made of polypropylene and silver nitrate (AgNO₃) were examined for bacterial survival [78]. This study showed that the addition of antibacterial AgNO₃ agent to filters was effective in preventing bacteria from colonizing filters. The presence of an antimicrobial AgNO₃ compound in the air filters caused a decrease in the amount of bacteria, which was observed in the case of both Gram-negative and Gram-positive strains of Micrococcus luteus, Micrococcus roseus, B. subtilis, Pseudomonas luteola. The clear reduction in living bacterial cells in silver treated filters made the technology of antimicrobial filter treatment really necessary for the future.

4.2.1. Drinking water disinfection.

4.2.2. Groundwater and biological wastewater disinfection.

4.3.1. Silver-nanoparticle-embedded antimicrobial paints.

4.3.2. Antimicrobial surface functionalization of plastic catheters.

4.3.3. Antimicrobial gel formulation for topical use.

4.3.4. Antimicrobial packing paper for food preservation.

4.3.5. Silver-impregnated fabrics for clinical clothing

To understand the toxicity of different NPs in vitro, Braydich-stolle et al [94] have assessed the suitability of a mouse spermatogonial stem cell line to assess toxicity of silver (Ag-15 nm), molybdenum (MoO₃-30 nm), and aluminum (Al-30 nm) NPs in the male germ line. Results showed a concentration-dependent toxicity for all types of tested NPs. Ag-NPs were the most toxic (5–10 μg ml⁻¹), and reduced mitochondrial function drastically and increased membrane leakage. This cell line was suggested as a valuable model with which to assess the cytotoxicity of NPs in the germ line in vitro. In addition, Hussain et al [95] have used the in vitro rat-liver-derived cell line (BRL 3A) to evaluate the acute toxic effects of metal/metal oxide NPs including silver (Ag; 15, 100 nm), molybdenum (MoO₃; 30, 150 nm), aluminum (Al; 30, 103 nm), iron oxide (Fe₃O₄; 30, 47 nm), and titanium dioxide (TiO₂; 40 nm). Results showed that mitochondrial function decreases significantly in cells exposed to Ag-NPs at 5–50 μg ml⁻¹. Fe₃O₄, Al, MoO₃ and TiO₂ had no measurable effect at lower doses (10–50 μg ml⁻¹), while there was a significant effect at higher levels (100–250 μg ml⁻¹). Lactate dehydrogenase (LDH) leakage significantly increased in cells exposed to Ag-NPs (10–50 μg ml⁻¹). Other NPs have been tested to displayed LDH leakage only at higher doses (100–250 μg ml⁻¹). The study showed the significant depletion of reduced glutathione (GSH) level, reduced mitochondrial membrane potential and increase in reactive oxygen species (ROS) levels, which suggested that cytotoxicity of Ag (15, 100 nm) in liver cells is likely to be mediated through oxidative stress. In addition, Calson et al [96] have investigated evaluating size-dependent cellular interactions of biologically active Ag-NPs (Ag-15, Ag-30 nm, and Ag-55 nm). After 24 h of exposure, results showed that viability metrics significantly decreased with increasing dose (10–75 μg ml⁻¹) of Ag-15 and Ag-30 nm. With a more than ten-fold increase of ROS levels in cells exposed to 50 μg ml⁻¹ Ag-15 nm, it was suggested that the cytotoxicity of Ag-15 nm is likely to be mediated through oxidative stress; this mechanism was then supported by the study on human hepatoma cells [97]. Moreover, Arora et al [98] have studied the interaction of synthesized Ag-NPs with HT-1080 (human fibrosarcoma) and A431 (human skin/carcinoma) cells in vitro. Results showed that a concentration of Ag-NPs was safe in the range from 1.56 to 6.25 μg ml⁻¹, and some effects appeared when concentrations >6.25 μg ml⁻¹. In another study, the authors have also investigated the in vitro interactions of 7–20 nm spherical Ag-NPs with primary fibroblasts and primary liver cells isolated from Swiss albino mice. Upon exposure to Ag-NPs for 24 h, IC₅₀ values for primary fibroblasts and primary liver cells were 61 and 449 μg ml⁻¹, respectively. It was suggested that although Ag-NPs seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage [99]. In relation to the genotoxicity of Ag-NPs following exposure to mammalian cells, Ahamed et al [100] have examined the DNA damage response to polysaccharide surface functionalized (coated) and non-functionalized (uncoated) Ag-NPs in two types of mammalian cells: mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF). Results showed that the different surface chemistry of Ag-NPs induce different DNA damage response: coated Ag-NPs exhibited more severe damage than uncoated Ag-NPs. AshaRani et al [101] have investigated the cytotoxicity and genetoxicity of starch coated Ag-NPs to normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251). The results indicated mitochondrial dysfunction, induction of ROS by Ag-NPs which in turn set off DNA damage and chromosomal aberrations. A possible mechanism of toxicity was proposed which involves disruption of the mitochondrial respiratory chain by Ag-NPs leading to production of ROS and interruption of ATP synthesis, which in turn causes DNA damage. It was anticipated that DNA damage is augmented by deposition, followed by interactions of Ag-NPs to the DNA leading to cell cycle arrest in the G2/M phase. Notably, Greulich et al [102] have reported the influence of spherical Ag-NPs (diameter about 100 nm) on the biological functions of human mesenchymal stem cells (hMSCs). Results showed a concentration-dependent activation of hMSCs at nanosilver levels of 2.5 μg ml⁻¹, and cytotoxic cell reactions occurred at Ag-NPs concentrations above 5 μg ml⁻¹. Kittler et al [103] have demonstrated that the toxicity of Ag-NPs increases during storage because of slow dissolution under release of silver ions. The NPs could release up to 90% of their weight depending on the functionalization as well as on the storage temperature. The release of silver was suggested as a considerably increased toxicity of Ag-NPs which had been stored in dispersion for several weeks toward human mesenchymal stem cells due to the increased concentration of silver ions. In another publication, Hackenberg et al [104] also reported cytotoxic effects of Ag-NPs (46 ± 21 nm) at concentrations of 10 μg ml⁻¹ for all test exposure periods to hMSCs. Interestingly, Kawata et al [105] have reported the in vitro toxicity of Ag-NPs at non-cytotoxic doses to HepG2 human hepatoma cells. Results showed that Ag-NPs accelerate cell proliferation at low doses (<0.5 mg/l⁻¹). However, only Ag-NPs exposure exhibited a significant cytotoxicity at higher doses (>1.0 mg l⁻¹) and induced abnormal cellular morphology, displaying cellular shrinkage and acquisition of an irregular shape. Moreover, Park et al [106] have showed cytotoxicity to mouse peritoneal macrophage cell line (RAW264.7) by increasing sub G1 fraction, which indicates cellular apoptosis. Ag-NPs decreased intracellular glutathione level, increased nitric oxide secretion, increased TNF-α in protein and gene levels, and increased gene expression of matrix metalloproteinases (MMP-3, MMP-11 and MMP-19). It was suggested that Ag-NPs were ionized in the cells to cause cytotoxicity by a Trojan-horse type mechanism. To investigate the effects of Ag-NPs on skin, Zanette et al [107] have performed their study on the human-derived keratinocyte HaCaT cell line model. Ag-NPs caused a concentration- and time-dependent decrease of cell viability, with IC₅₀ values of 6.8 ± 1.3 μM (MTT assay) and 12 ± 1.2 μM (SRB assay) after 7 days of contact. Results also demonstrated that on HaCaT keratinocytes, a relatively short time of contact with Ag-NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag-NPs internalization. Recently, Asare et al [108] have investigated the cytotoxic and genotoxic effects of nanoparticles such as Ag-NPs (20 nm) and submicron- (200 nm) size, and titanium dioxide nanoparticles (TiO₂-NPs; 21 nm) in testicular cells. The results suggested that silver nano- and submicron-particles (Ag-NPs) are more cytotoxic and cytostatic compared to TiO₂-NPs, causing apoptosis, necrosis and decreased proliferation in a concentration- and time-dependent manner. The 200 nm Ag-NPs in particular appeared to cause a concentration-dependent increase in DNA-strand breaks in NT2 cells, whereas the latter response did not seem to occur with respect to oxidative purine base damage analyzed with any of the particles tested. There are some more publications demonstrating the cytotoxicity and genotoxicity of Ag-NPs tested in vitro to peripheral blood mononuclear cells (PBMCs) [109]; mouse lymphoma cell line (L5178Y thymidine kinase (tk)^+/−-3.7.2C cells) and human bronchial epithelial cells (BEAS-2B) [110]; monocytic cell line THP-1 (ATCC 202) [111]; human mesenchymal stem cells [104]; RAW 264.7 murine peritoneal macrophage cell line, L929 mouse fibroblast, D3 murine embryonic stem cell line and mouse embryonic fibroblasts (MEF-LacZ) [112]; human lung carcinoma epithelial-like cell line [113]; primary testicular cells [108]; human monocytic cell line [114]; and human intestinal cell line [115].

Generally, in in vitro tests, the mechanism of Ag-NPs-mediated cytotoxicity is mainly based on the induction of reactive oxygen species (ROS). Particularly, exposure to Ag-NPs causes reduction in GSH, elevated ROS levels, lipid peroxidation and increased expression of ROS responsive genes, it also leads to DNA damage, apoptosis and necrosis. The cytotoxicity and genotoxicity of Ag-NPs are size-, concentration- and exposure time-dependent.

The most important question here is on the real impact of Ag-NPs to human health and animals. There are several in vivo studies on cytotoxicity and genotoxicity of Ag-NPs reported to demonstrate this question (table 3). Due to the ultra-small sizes of Ag-NPs, a great mobility is conferred in different environments, and humans are easily exposed via routes such as inhalation, ingestion, skin, etc. Ag-NPs can translocate from the route of exposure to other vital organs and penetrate into cells. For inhalation toxicity of Ag-NPs, Ji et al [116] have investigated the inhalation toxicity of Ag-NPs on Sprague-Dawley rats over a period of 28 days. The rats were exposed to the Ag-NPs for 6 h per day, 5 days per week, for a total of 4 weeks. Results showed that the male and female rats did not show any significant changes in body weight relative to the concentration of Ag-NPs during the 28-day experiment. There were also no significant changes in the hematology and blood biochemical values in either the male or female rats. Whereas, some investigators have reported that lungs are major target tissues affected by prolonged inhalation exposure to Ag-NPs [117]. In another publication, Lee et al [118] have reported Ag-NPs exposure modulated the expression of several genes associated with motor neuron disorders, neurodegenerative disease, and immune cell function, indicating potential neurotoxicity and immunotoxicity associated with Ag-NPs exposure. Minimal pulmonary inflammation or cytotoxicity of mice was found after 10 days of Ag-NPs exposure [119]. For gastrointestinal toxicology caused by Ag-NPs exposure via ingestion, Kim et al [120] have tested the oral toxicity of Ag-NPs (60 nm) over a period of 28 days in Sprague-Dawley rats. Results showed that the male and female rats did not show any significant changes in body weight relative to the doses of Ag-NPs during the 28-day experiment. But, some significant dose-dependent changes were found in the alkaline phosphatase and cholesterol values in either the male or female rats, seeming to indicate that exposure to over more than 300 mg of Ag-NPs may result in slight liver damage. It was suggested that Ag-NPs do not induce genetic toxicity in male and female rat bone marrow in vivo. There are some publications found concerning the studies on toxicology of Ag-NPs to organism via skin exposure or injection [121, 122].

Generally, very few papers on the in vivo toxicology of Ag-NPs were found, so further investigation is needed in this field to evaluate exactly the real impact of Ag-NPs in commercial products to humans and animals. Furthermore, the impact of Ag-NPs to environment and ecology has been discussed in detail by Ahamed et al [8]. In this review, the authors have indicated that Ag-NPs produce reproductive failure, developmental malformations and morphological deformities in a number of non-mammalian animal models. Common causes of Ag-NPs-induced toxicity include oxidative stress, DNA damage and apoptosis.

Due to the potent activities of Ag-NPs, they can be promisingly used in treating infectious pathogens and preventing microbial infections. Very recently, our research group demonstrated an excellent disinfectant ability of colloidal Ag-NPs for the prevention of gastrointestinal bacterial infections [55]. The reports revealed that the Ag-NPs colloid showed an enhancement of antibacterial activity and long-lasting disinfectant effect as compared to conventional chloramin B (5%) disinfection agent. Additionally, the Ag-NPs displayed long-term antibacterial effect as compared with two other disinfectants of sodium hypochlorite (NaClO) and phenol (C₆H₅OH) [124]. With observed advantages such as long-lasting effect and enhanced bactericidal activity, the Ag-NPs are promising for environmental treatments contaminated with gastrointestinal bacteria and other infectious pathogens. More importantly, the powerful disinfectant activity of Ag-NPs will open a new generation of silver-containing disinfection products for controlling and preventing further outbreak of diseases (e.g. diarrhea and cholera). However, the emerging questions on the disinfectant ability of Ag-NPs against viral infections (e.g. A-H5N1 influenza virus, enterovirus 71, Rota virus etc.) need to be clarified. Further investigations on evaluation of disinfectant efficiency of silver-based NPs for real environmental contaminations are needed.

Ag-NPs can be promisingly used in core/shell magnetic disinfectant systems for effective disinfection of drinking water. The magnetic disinfectant includes magnetic oxide NPs as the core (i.e. Fe₃O₄) and Ag-NPs as the shell [125]. Importantly, these core/shell nanostructures can be successfully removed from the medium by using an external magnetic field, which provides a mechanism to prevent uncontrolled waste disposal of these potentially hazardous nanostructures. This indicates that the nanocomposite disinfectant can be recovered from water solution for reuse through magnetic separation and the waste and possible contamination of the environment by disinfectant are avoided [126]. Fe₃O₄–Ag and Fe₃O₄–SiO₂–Ag core/shell nanocomposites exhibited excellent antibacterial effect and enhanced stability against bacterial pathogens [127, 128]. The main characteristic of magnetic disinfectant is its effectiveness against waterborne pathogens using low concentrations of silver, which makes it economic to use. Hence, the magnetic disinfectant can be possibly used in disinfection and biomedical applications where they can be exploited for a targeted delivery of an antimicrobial agent and its subsequent removal by means of an external magnetic field.

A novel concept is proposed to synthesize a new class of composites featuring magnetic, molecular sieve and metallic NPs properties. These multi-functional materials have potential applications as recyclable catalysts, disinfectants and sorbents. The magnetic property enables effective separation of the spent composites from complex multiphase systems for regeneration and recycling, safe disposal of the waste and/or recovery of loaded valuable species. The zeolite molecular sieve provides a matrix which supports a remarkably new, simple, efficient and economical method to make stable, supported Ag-NPs by silver ion exchange and controlled thermal reduction [129]. Each component has a specially designed function, which makes this novel composite practically operational, sustainable, economical and environmentally friendly [130]. A new class of magnetic zeolite composites with surface-supported Ag-NPs as sorbents for mercury removal was tested. The produced nanocomposite has potential applications as a catalyst, a bacterial disinfectant for municipal water, or a sorbent for the removal of elemental mercury from the flue gases of coal-fired power plants, a pressing environmental concern. Hence, the Ag-NPs embedded in complex composite systems can be promisingly used as sorbent and catalyst for removal of environmental pollution.

Nanosilver-containing solutions are of particular interest to industry for surface disinfection applications. Disinfection methods include spraying, wiping, dipping in solutions. Nanosilver-based disinfectant solutions are very useful for decontamination of surfaces, tools in kindergartens, schools, workplaces, for different types of equipment and computers, toys, all kinds of furniture and the like, in industrial and domestic, and public utilities. This will open new opportunities to develop nanosilver-based consumer products for surface disinfection such as spray bottles for providing long-lasting protection, disposable wipes for disinfecting hands and to maintain daily personal hygiene, etc. In this regard, nanosilver-containing products are very promising for surface disinfection of contaminated environmental areas and/or the product can be sprayed on surfaces and materials in order to inhibit the pathogen development and to improve human health and safety.

As mentioned in section 5, it has been concluded that Ag-NPs have the potential to cause health and ecotoxicity issues in a concentration- and size-dependent manner. In order to overcome this problem, new approaches for development of silver-containing nanocomposites were proposed. A recent approach is to develop the hybrid nanostructure between carbon-based nanomaterials such as carbon nanotubes (CNTs) [130] or graphene (Grp) [131] and Ag-NPs. These silver-decorated nanohybrids constitute an interesting class of antibacterial materials. By decorating Ag-NPs on carbon nanomaterials, the toxicity of Ag-NPs will be reduced, thus helping in preventing potential contamination to the environment [132]. It is noteworthy that these silver-decorated nanohybrids are very different from isolated and dispersed colloidal Ag-NPs, and it appears that the strong interactions between the Ag-NPs and the CNTs or Grp surfaces make the Ag-NPs less toxic due to the fact that they are not able to release themselves into the environment. Another important class of antibacterial silver-based nanomaterials is composed of inorganic–inorganic and organic–inorganic nanocomposites. The silver-based nanocomposite on inorganic support (i.e., Ag/TiO₂) [133] or that on polymer matrix (i.e. Ag/PVA) [17] are advanced functional materials composed of Ag-NPs dispersed inside the polymeric matrix and/or coated by polymer/inorganic carrier, thus forming a core/shell structure. This nanocomposite structure inhibited effectively the release of silver ions from NPs, thus reducing the generation of ROS and toxicity of Ag-NPs [134–136]. As a result, these nanocomposites can be environmentally friendly when used for developing novel products applications.