**3. Mechanism of nanoparticle synthesis using plants and microbes**

There are three main phases in the synthesis of metal nanoparticles from plants and plant extracts. Initially, an activation phase takes place during which metal ions are reduced from mono or divalent oxidation states to zero-valent states, followed by nucleation of the reduced atoms. This step is immediately followed by a growth phase where small neighbouring nanoparticles coalesce into larger particles with greater thermodynamic stability while further biological reduction occurs. As growth proceeds nanoparticles aggregate to form various shapes such as: cubes, spheres, triangles, hexagons, pentagons, rods and wires [68]. Lastly, a termination phase follows in which nanoparticles acquire the most energetically favourable conformation, which ultimately determines the final shape of the particles **(Figure 4)** [69]. This step is largely influenced by the ability of the plant extract to stabilise the resulting nanoparticles. For example, the high surface energy of nanotriangles results in their decreased stability. Such nanoparticles would then acquire a more stable morphology such as a truncated triangle to minimise Gibbs free energy unless the stability is supported by the given extracts. It can be tentatively suggested that a similar mechanism occurs by the use of bacterial extracts since proteins and metabolites may also participate in Ag+ reduction as previously stated.

Several controlling factors affect the synthesis and morphology of derived nanoparticles. Several researchers have associated these variations with the choice of adsorbate and catalyst used in the synthetic process [29, 70]. However, reaction parameters have also been shown to strongly affect the synthesis of nanoparticles from biological extracts.

Studies have revealed that the pH of a reaction solution strongly influences the formation of the produced nanoparticles. Variances in reaction pH tend to induce variability in the shape and size of the produced nanoparticles. Lower acidic pH values tend to produce larger particles when compared to higher pH values. In a study employing *Avena sativa* (oat) biomass for the production of gold nanoparticles, larger particles (25–85 nm) where formed at pH 2 whilst smaller particles (5–20 nm) were formed at pH 3 and 4 [71]. The researchers suggested that at pH 2, fewer functional groups were available for particle nucleation resulting in aggregation of the particles. A similar finding was observed in the synthesis of gold nanoparticles from the bacterium *Rhodopseudomonas capsulate.* At an increased pH of 7, spherical particles in the range of 10–20 nm in size were observed. In contrast, lowering the reaction pH to 4 resulted in the formation of nanoplates [63].

Temperature is an important factor in any synthesis. With respect to nanoparticle formulation with the use of biological entities, temperature elevation has demonstrated catalytic behaviour by increasing the reaction rate and efficiency of nanoparticle formation. For example, a study on the influence of reaction temperature in the synthesis of AgNPs from neem leaf extracts suggested that temperature elevation (10–50°C) was correlated with enhanced reduction of Ag+ [72]. It was also noted that smaller sized AgNPs were produced at 50°C, similar to the finding of Kaviya *et al.* in the production of AgNPs from *Citrus sinensis* peel extracts using varying temperatures [73]. Similarly, this trend was observed in the production of AgNPs from the spent culture supernatants of *Escherichia coli* [61]. The authors tentatively suggested that the increased reaction rate might be because of temperature on a key enzyme participating in nanoparticle synthesis. However, the study importantly revealed that temperature elevation above 60°C contrastingly favoured the production of larger sized particles. The reason for this observation was reported as follows: at high temperatures, kinetic energy of the molecules increase resulting in rapid reduction of Ag<sup>+</sup> (facilitating reduction and nucleation), to the

*Schematic representation of nanoparticle synthesis using a plant extract. Adapted from [35].*

detriment of secondary reduction on the surface of nascent particles in the growth phase. However, higher temperatures beyond the optimum are thought to increase the growth of the crystal around the nucleus, resulting in the production of larger particles [48, 61].

Temperature has also been demonstrated to affect the structural form of nanoparticles. For example, AgNP synthesis using *Cassia fistula* extracts resulted in the formation of Ag nanoribbons at room temperature whilst spherical AgNPs

**263**

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

inhibiting the coalescence of adjacent nanoparticles.

display improved bactericidal activities [42, 80].

spherically shaped AgNPs [41].

promising alternative in this regard.

**3.1 Anti-microbial properties of silver nanoparticles**

make comparison between published data inapplicable [88].

were formed at temperatures above 60°C [74]. High temperatures in the study were thought to alter the interaction of plant biomolecules with the faces of Ag,

formation, has been observed to derive AgNPs with desired physical attributes. Recent studies on sunlight driven AgNP synthesis using *Allium sativum* (garlic extract) and *Andrachnea chordifolia* ethanol leaf extract revealed that sunlight rapidly enhanced nanoparticle formation to produce spherical AgNPs with average diameters of 7.3 nm and 3.4 nm, respectively [75, 76]. In addition, this use of sunlight has also been used in AgNP synthesis from *Bacillus amyloliquefaciens* CFS to produce circular and triangular crystalline AgNPs with an average diameter of 14.6 nm [77]. A variety of literature reports on the synthesis of AgNPs with differing morphologies. Understanding the effects of these morphological characteristics on bioactivity is therefore an important consideration when deriving nanoparticles for therapeutic purposes. Characteristically, AgNPs are small (1–100 nm) and therefore possess a large surface area that facilitates their interaction with bacterial cell membranes [41, 78]. However, it has been suggested that within this confined size range, AgNPs present a size-dependent inhibition spectrum. Martinez-Castanon *et al*. reported that AgNPs of 7 nm in size had minimum inhibitory concentration (MIC) values of 6.25 μg ml−1 and 7.5 μg ml−1 for *E. coli* and *Staphylococcus aureus*, respectively. In contrast, larger nanoparticles (29 nm) capped with the same reducing agent displayed higher MIC values for the respective strains [79]. These results are in accordance with other studies that report nanoparticles of ˂ 10 nm in size

Sunlight irradiation, a recently reported primary energy source for nanoparticle

The interaction of AgNPs of varying shapes with *E. coli* cells has unveiled that shape plays an important factor in bioactivity. Pal *et al*. reported that at a low Ag content of 1 μg, truncated triangular nanoparticles showed nearly complete inhibition of *E. coli* cells, whilst spherical nanoparticles with a total silver content above 12.5 μg displayed a reduction in colony forming units. Rod-shaped particles and AgNO3 presented inferior activities when compared to truncated triangular and

Considering these factors and the aforementioned factors affecting synthesis of nanoparticles, it can tentatively be suggested that the fine tuning of reaction parameters such as pH or temperature may be applied in producing AgNPs with these desired physical attributes. However, the use of sunlight irradiation provides a

There exists an abundance of literature reporting on antimicrobial activities of biologically derived AgNPs [81–84]. Most of these studies utilise the disc diffusion assay [85] or agar well diffusion assay [86] to establish inhibitory effects. Positive indication of inhibitory activities are visualised by zones of inhibition on a microbial lawn. Veersamy *et al*. reported zones of inhibition of *S. aureus* and *E. coli* to be 15 mm and 20 mm respectively for AgNPs (20 μg ml−1) derived from mangosteen leaf extracts [48]. Similarly, Logeswari *et al*. reported zones of inhibition of AgNPs synthesised from various plant extracts against several bacterial strains [81]. Although diffusion techniques are preferred amongst researchers, they seem to be labour-intensive. In addition, many researchers do not establish the initial concentration of AgNP solution prior to antimicrobial evaluation [82, 87]. Such disparities

Determination of minimum inhibitory concentration (MIC) by the broth microdilution or macrodilution method [89, 90] is easy to access and provides accurate

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

#### *Green Synthesis of Metal Nanoparticles for Antimicrobial Activity DOI: http://dx.doi.org/10.5772/intechopen.94348*

*Novel Nanomaterials*

**262**

particles [48, 61].

**Figure 4.**

detriment of secondary reduction on the surface of nascent particles in the growth phase. However, higher temperatures beyond the optimum are thought to increase the growth of the crystal around the nucleus, resulting in the production of larger

*Schematic representation of nanoparticle synthesis using a plant extract. Adapted from [35].*

Temperature has also been demonstrated to affect the structural form of nanoparticles. For example, AgNP synthesis using *Cassia fistula* extracts resulted in the formation of Ag nanoribbons at room temperature whilst spherical AgNPs

were formed at temperatures above 60°C [74]. High temperatures in the study were thought to alter the interaction of plant biomolecules with the faces of Ag, inhibiting the coalescence of adjacent nanoparticles.

Sunlight irradiation, a recently reported primary energy source for nanoparticle formation, has been observed to derive AgNPs with desired physical attributes. Recent studies on sunlight driven AgNP synthesis using *Allium sativum* (garlic extract) and *Andrachnea chordifolia* ethanol leaf extract revealed that sunlight rapidly enhanced nanoparticle formation to produce spherical AgNPs with average diameters of 7.3 nm and 3.4 nm, respectively [75, 76]. In addition, this use of sunlight has also been used in AgNP synthesis from *Bacillus amyloliquefaciens* CFS to produce circular and triangular crystalline AgNPs with an average diameter of 14.6 nm [77].

A variety of literature reports on the synthesis of AgNPs with differing morphologies. Understanding the effects of these morphological characteristics on bioactivity is therefore an important consideration when deriving nanoparticles for therapeutic purposes. Characteristically, AgNPs are small (1–100 nm) and therefore possess a large surface area that facilitates their interaction with bacterial cell membranes [41, 78]. However, it has been suggested that within this confined size range, AgNPs present a size-dependent inhibition spectrum. Martinez-Castanon *et al*. reported that AgNPs of 7 nm in size had minimum inhibitory concentration (MIC) values of 6.25 μg ml−1 and 7.5 μg ml−1 for *E. coli* and *Staphylococcus aureus*, respectively. In contrast, larger nanoparticles (29 nm) capped with the same reducing agent displayed higher MIC values for the respective strains [79]. These results are in accordance with other studies that report nanoparticles of ˂ 10 nm in size display improved bactericidal activities [42, 80].

The interaction of AgNPs of varying shapes with *E. coli* cells has unveiled that shape plays an important factor in bioactivity. Pal *et al*. reported that at a low Ag content of 1 μg, truncated triangular nanoparticles showed nearly complete inhibition of *E. coli* cells, whilst spherical nanoparticles with a total silver content above 12.5 μg displayed a reduction in colony forming units. Rod-shaped particles and AgNO3 presented inferior activities when compared to truncated triangular and spherically shaped AgNPs [41].

Considering these factors and the aforementioned factors affecting synthesis of nanoparticles, it can tentatively be suggested that the fine tuning of reaction parameters such as pH or temperature may be applied in producing AgNPs with these desired physical attributes. However, the use of sunlight irradiation provides a promising alternative in this regard.

#### **3.1 Anti-microbial properties of silver nanoparticles**

There exists an abundance of literature reporting on antimicrobial activities of biologically derived AgNPs [81–84]. Most of these studies utilise the disc diffusion assay [85] or agar well diffusion assay [86] to establish inhibitory effects. Positive indication of inhibitory activities are visualised by zones of inhibition on a microbial lawn. Veersamy *et al*. reported zones of inhibition of *S. aureus* and *E. coli* to be 15 mm and 20 mm respectively for AgNPs (20 μg ml−1) derived from mangosteen leaf extracts [48]. Similarly, Logeswari *et al*. reported zones of inhibition of AgNPs synthesised from various plant extracts against several bacterial strains [81]. Although diffusion techniques are preferred amongst researchers, they seem to be labour-intensive. In addition, many researchers do not establish the initial concentration of AgNP solution prior to antimicrobial evaluation [82, 87]. Such disparities make comparison between published data inapplicable [88].

Determination of minimum inhibitory concentration (MIC) by the broth microdilution or macrodilution method [89, 90] is easy to access and provides accurate

information with respect to microbial susceptibility. Moreover, MIC values are reported in various concentration units such as μg ml−1, μg l−1 or ppm thereby facilitating comparison between publications [53]. These methods are therefore attractive for AgNP bioactivity analysis. Furthermore, determination of MICs is an important consideration for any therapeutic agent in development to assess their toxicity at the specified concentration range. As previously mentioned, the antimicrobial effects of AgNPs are well established. However, a relatively confined amount of studies has been conducted to elucidate their mechanisms of antimicrobial action. These mechanisms are poorly understood and have failed to achieve consensus amongst researchers. Despite this, three common mechanisms of bactericidal activity have been proposed by various studies. These include the uptake of Ag+ (1), generation of reactive oxygen species (ROS) (2) and cell membrane disruption (3) (**Figure 5**) [91].

Since Ag<sup>+</sup> are known to possess antibacterial activities, their release from AgNPs may potentially aid to the bioactivity of the nanoparticles. It is therefore fitting to consider the mechanistic action of Ag+ on bacterial cells.

The NADH–ubiquinone reductase has been established as one of the major targets for Ag+ . Specifically, the binding of Ag<sup>+</sup> to this enzyme may be responsible for their bactericidal effect even at minute concentrations [92]. Later, Dibrov *et al*. reported the binding of Ag+ to transport proteins leads to the leakage of protons and ultimately induces the collapse of the proton motive force [93]. Such interactions with transport proteins may be attributed to the strong affinity of Ag<sup>+</sup> to thiol groups found on cysteine residues of these molecules [94]. Ag<sup>+</sup> has also been reported to inhibit phosphate uptake and additionally causes an efflux of intracellular phosphate [95]. It has also been hypothesised that the antimicrobial effect of Ag<sup>+</sup> is correlated with the disruption of DNA replication. DNA molecules in a relaxed conformation can be replicated effectively. However, when Ag<sup>+</sup> are present in bacterial cells, DNA molecules enter a condensed form and replicating ability diminishes which ultimately leads to cell death [8].

#### **Figure 5.**

*Interactions of AgNPs with bacterial cells: (1) release of Ag+ and generation of ROS; (2) interaction with cell membrane proteins; (3) accumulation in cell membrane and disruption of permeability; (4) entry into the cell and release of Ag<sup>+</sup> , leading to generation of ROS and damage of cellular DNA. In turn, generated ROS may affect DNA, cell membrane and membrane proteins whilst released Ag+ may affect cell membrane proteins and DNA. Adapted from [91].*

**265**

**Figure 6.**

*AgNPs [80, 100].*

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

ticles inside the cell also play a role in the generation of ROS. Ag+

membrane are capable of ROS generation by acting as electron acceptors whilst those present inside the cell more likely to interact with thiol groups of respiratory chain enzymes as previously stated, or scavenging superoxide dismutase enzymes [98]. The effect of ROS scavengers on *E. coli* cells was reported by Inoue *et al*.. Specifically, ROS such as superoxide anions, hydroxyl radicals, hydrogen peroxide and singlet oxygen contributed to the bactericidal activity against *E. coli* [99]. According to literature, the bactericidal effect of AgNPs may also be the result of damage to the outer membrane of bacterial cells. Previous studies by Sondi and Salopek-Sondi suggested that treatment of *E. coli* cells with AgNPs induced changes in the membrane morphology (**Figure 6a**). This resulted in increased membrane permeability and shifts in normal transport through the plasma membrane [100]. Morones *et al*. hypothesised that these mechanisms could explain the number of nanoparticles found inside *E. coli cells* (**Figure 6b**). AgNPs with oxidised surfaces were also reported to induce the formation of holes on the surface of *E. coli* cells and portions of the cellular surface were observed to be eaten away [101]. The attachment and penetration of AgNPs has also been observed in *P. aeruginosa* (**Figure 6c**),

The mechanism of AgNP adhesion and penetration of bacterial cell membranes remains to be elucidated. Literature reports indicate that electrostatic interactions between positively charged particles and negatively charged cell membranes is essential for the bioactivity of these particles [102, 103]. However, this strategy does

*Transmission electron micrographs of (a) E. coli cell after 1 h treatment with 50 μg cm−3 AgNPs; (b) E. coli cell after 30 min treatment with 100 μg ml−1 AgNPs (c) P. aeruginosa cells after 30 min treatment with 100 μg ml−1*

The exposure of bacterial cells to AgNPs leads to the generation of ROS [96]. Naturally, ROS are metabolic by-products of respiring beings. Whist low levels of these species are skilfully controlled by various antioxidant defence mechanisms, high levels of ROS results in oxidative stress which is detrimental to any living organism. Metals can serve as catalysts and produce ROS in an oxygen containing environment [97]. AgNPs are therefore likely to catalyse reactions with oxygen leading to the production of excess free radicals. Kim *et al*. demonstrated the generation of free radicals from AgNPs by means of spin resonance measurements. Toxicity of AgNPs and AgNO3 diminished upon addition of an antioxidant suggesting that the mechanism of action against bacterial strains was associated with the formation of free radicals from AgNPs. The generation of excess free radicals attack membrane lipids resulting in the breakdown of the membrane and cause damage to

from nanoparticles attached to the membrane and nanopar-

released on the

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

DNA [1].

The release of Ag<sup>+</sup>

*V. cholera* and *S. typhus* [80].

*Novel Nanomaterials*

Since Ag<sup>+</sup>

targets for Ag+

consider the mechanistic action of Ag+

which ultimately leads to cell death [8].

*Interactions of AgNPs with bacterial cells: (1) release of Ag+*

*affect DNA, cell membrane and membrane proteins whilst released Ag+*

reported the binding of Ag+

information with respect to microbial susceptibility. Moreover, MIC values are reported in various concentration units such as μg ml−1, μg l−1 or ppm thereby facilitating comparison between publications [53]. These methods are therefore attractive for AgNP bioactivity analysis. Furthermore, determination of MICs is an important consideration for any therapeutic agent in development to assess their toxicity at the specified concentration range. As previously mentioned, the antimicrobial effects of AgNPs are well established. However, a relatively confined amount of studies has been conducted to elucidate their mechanisms of antimicrobial action. These mechanisms are poorly understood and have failed to achieve consensus amongst researchers. Despite this, three common mechanisms of bactericidal activity have

reactive oxygen species (ROS) (2) and cell membrane disruption (3) (**Figure 5**) [91].

may potentially aid to the bioactivity of the nanoparticles. It is therefore fitting to

The NADH–ubiquinone reductase has been established as one of the major

for their bactericidal effect even at minute concentrations [92]. Later, Dibrov *et al*.

and ultimately induces the collapse of the proton motive force [93]. Such interactions with transport proteins may be attributed to the strong affinity of Ag<sup>+</sup>

reported to inhibit phosphate uptake and additionally causes an efflux of intracellular phosphate [95]. It has also been hypothesised that the antimicrobial effect of Ag<sup>+</sup> is correlated with the disruption of DNA replication. DNA molecules in a relaxed

rial cells, DNA molecules enter a condensed form and replicating ability diminishes

*membrane proteins; (3) accumulation in cell membrane and disruption of permeability; (4) entry into the cell* 

*, leading to generation of ROS and damage of cellular DNA. In turn, generated ROS may* 

are known to possess antibacterial activities, their release from AgNPs

on bacterial cells.

to transport proteins leads to the leakage of protons

(1), generation of

to

has also been

are present in bacte-

to this enzyme may be responsible

 *and generation of ROS; (2) interaction with cell* 

 *may affect cell membrane proteins and* 

been proposed by various studies. These include the uptake of Ag+

. Specifically, the binding of Ag<sup>+</sup>

thiol groups found on cysteine residues of these molecules [94]. Ag<sup>+</sup>

conformation can be replicated effectively. However, when Ag<sup>+</sup>

**264**

**Figure 5.**

*and release of Ag<sup>+</sup>*

*DNA. Adapted from [91].*

The exposure of bacterial cells to AgNPs leads to the generation of ROS [96]. Naturally, ROS are metabolic by-products of respiring beings. Whist low levels of these species are skilfully controlled by various antioxidant defence mechanisms, high levels of ROS results in oxidative stress which is detrimental to any living organism. Metals can serve as catalysts and produce ROS in an oxygen containing environment [97]. AgNPs are therefore likely to catalyse reactions with oxygen leading to the production of excess free radicals. Kim *et al*. demonstrated the generation of free radicals from AgNPs by means of spin resonance measurements. Toxicity of AgNPs and AgNO3 diminished upon addition of an antioxidant suggesting that the mechanism of action against bacterial strains was associated with the formation of free radicals from AgNPs. The generation of excess free radicals attack membrane lipids resulting in the breakdown of the membrane and cause damage to DNA [1].

The release of Ag<sup>+</sup> from nanoparticles attached to the membrane and nanoparticles inside the cell also play a role in the generation of ROS. Ag+ released on the membrane are capable of ROS generation by acting as electron acceptors whilst those present inside the cell more likely to interact with thiol groups of respiratory chain enzymes as previously stated, or scavenging superoxide dismutase enzymes [98]. The effect of ROS scavengers on *E. coli* cells was reported by Inoue *et al*.. Specifically, ROS such as superoxide anions, hydroxyl radicals, hydrogen peroxide and singlet oxygen contributed to the bactericidal activity against *E. coli* [99]. According to literature, the bactericidal effect of AgNPs may also be the result of damage to the outer membrane of bacterial cells. Previous studies by Sondi and Salopek-Sondi suggested that treatment of *E. coli* cells with AgNPs induced changes in the membrane morphology (**Figure 6a**). This resulted in increased membrane permeability and shifts in normal transport through the plasma membrane [100]. Morones *et al*. hypothesised that these mechanisms could explain the number of nanoparticles found inside *E. coli cells* (**Figure 6b**). AgNPs with oxidised surfaces were also reported to induce the formation of holes on the surface of *E. coli* cells and portions of the cellular surface were observed to be eaten away [101]. The attachment and penetration of AgNPs has also been observed in *P. aeruginosa* (**Figure 6c**), *V. cholera* and *S. typhus* [80].

The mechanism of AgNP adhesion and penetration of bacterial cell membranes remains to be elucidated. Literature reports indicate that electrostatic interactions between positively charged particles and negatively charged cell membranes is essential for the bioactivity of these particles [102, 103]. However, this strategy does

#### **Figure 6.**

*Transmission electron micrographs of (a) E. coli cell after 1 h treatment with 50 μg cm−3 AgNPs; (b) E. coli cell after 30 min treatment with 100 μg ml−1 AgNPs (c) P. aeruginosa cells after 30 min treatment with 100 μg ml−1 AgNPs [80, 100].*

not validate the adhesion and penetration abilities of negatively charged nanoparticles [104]. The researchers argued that although the particles were negatively charged, interactions between the particles and building elements of the membrane are likely to have occurred causing structural changes and degradation of the membrane. Morones *et al*. proposed that the interaction of AgNPs and bacterial membranes could be attributed to the strong affinity of the particles to sulphur containing proteins present on the membrane [80]. These interactions are thought to be conserved in the interaction of Ag<sup>+</sup> and thiol groups on respiratory enzymes and transport proteins [80, 91].

Sondi and Salopek-Sondi [104] further reported that damage to *E. coli* cell membranes might also occur due to the incorporation of AgNPs into their membrane structure. Scanning electron microscopy revealed the formation of "pits" on the surface of the membrane [100]. Similar findings were observed by [102]. Amro *et al*. [109] additionally reported the formation of irregularly shaped "pits" on the outer membrane of *E. coli* cells through the progressive release of lipopolysaccharide molecules. This release of LPS molecules was induced by metal depletion in the cells [105]. A membrane with such morphological changes would display a high increase in permeability, rendering the cell incapable of regulating proper transport through the membrane as previously described.

Although these studies have been conducted on Gram-negative bacteria, AgNPs have also been reported to exert inhibitory activities against Gram-positive bacteria which differ from their counterparts based on differences in cell wall structure [106]. It can be tentatively suggested that AgNPs may form interactions with Grampositive bacteria through surface proteins present on the cell wall. Once penetrated, the mechanisms of bacterial activity are conserved with that of Gram-negative bacteria.

A relatively confined amount of literature focuses on the mechanisms of antifungal activity exerted by AgNPs. However, based on the studies that have been reported, it seems that inhibition of fungal growth by AgNPs may be the result of damage to fungal cellular membranes. Kim *et al*. demonstrated the effect of AgNPs on *Candida albicans*. Transmission electron microscopy (TEM) analysis revealed that the treatment of cells with AgNPs lead to the formation of "pits" on the cell membrane which ultimately disrupts membrane potential [107]. A similar finding was made by Nasrollahi *et al*. who reported that AgNP incubation with *C. albicans* led to damage of the cell membrane [108]. Endo *et al*. reported that disruption of membrane integrity inhibits the normal budding process of daughter cells. Therefore, the authors suggested that AgNPs exert their inhibitory activity by inhibiting the budding of daughter cells due to the destruction of the cell membrane [109].

AgNPs may also disrupt antioxidant defences in fungal cells. Eukaryotic cell studies suggest that AgNPs directly interact with gluthathione, gluthathione reductase or enzymes responsible for maintaining proper levels of gluthathione [110]. With respect to fungal cells, it has been hypothesised that Ag<sup>+</sup> largely affect the function of membrane bound enzymes such as those in the respiratory chain. It has also been reported that exposure of fungal cells to Ag<sup>+</sup> led to the loss of DNA replication ability. This results in the deactivation of ribosomal subunit protein expression and synthesis of non-functional enzymes and cellular proteins [111].

From these findings it can be tentatively suggested that bactericidal mechanisms of AgNPs are conserved in their inhibition of fungal cells. In summary, AgNPs exert their antimicrobial effects by releasing Ag<sup>+</sup> , disrupting the cell membrane/wall, generating ROS and inhibiting proper DNA replication.

**267**

*Green Synthesis of Metal Nanoparticles for Antimicrobial Activity*

The unique physico-chemical and biological properties of AgNPs have extremely promising industrial and medical applications, as previously mentioned. However, there exists a dearth of knowledge regarding the effects of prolonged exposures to nanoparticles on human health and the environment [112]. It is therefore imperative to establish the *in vitro* and *in vivo* cytotoxic effect of AgNPs in mind for

Human contact with nanoparticles occurs in the form of intravenous injection, oral administration, inhalation and dermal contact [113]. Injection of AgNPs *in vivo* results in short circulation times and broad tissue distribution. Target sites often include the liver (main target), spleen, lungs and kidneys [114]. Inhalation studies suggest that AgNPs become deposited in the olfactory mucosa and olfactory nerves which can potentially induce impairment and dysfunction of brain cells [115] in addition to immunotoxicity [116]. With regard to oral administration, migration of AgNPs to the gastrointestinal tract promotes dissolution of the particles which

that these ions interact with sulphur leading to the formation of sulphur containing Ag granules in the intestinal epithelium [118]. The authors suggested that during

or AgCl salt. They further added that this formation might influence their uptake

are yet to be elucidated [118, 119]. Reports on the exposure of workers to low doses

Many researchers have demonstrated the cytotoxic effects of AgNPs *in vitro*, however there is still a lack of consistent and reliable data amongst publications. For example, in a recent review, Kim and Ryu (2013) attributed oxidative stress, apoptosis and genotoxicity to be the main *in vitro* outcome of AgNP exposure [120]. Later, Gliga *et al*. identified a major drawback of this review, highlighting that the AgNPs were different in each study, *i.e.* synthesised by different techniques, of varying size distributions and coatings, tested on different cell lines under different cell culture conditions and often without the use of appropriate controls [121]. Additionally, Hackenberg *et al*. reported cytotoxicity of human mesenchymal stem cells at a concentration of 10 μg ml−1 AgNPs (˂50 nm), whereas Samberg *et al*. reported no toxicity of progenitor human adipose-derived stem cells at concentrations up to 100 μg ml−1 AgNPs (10–20 nm) [122, 123]. To determine the effect of size on cytotoxicity, Liu *et al*. compared the cytotoxicity of AgNPs ranging in size from 5 to 50 nm on four different cell lines (A549, HepG2, MCF-7 and CGC-7901) and reported that 5 nm AgNPs were most toxic [124]. On the contrary, Kim *et al*. reported the enhanced release of lactate dehydrogenase (LDH) and reduced cell viability in the presence of 100 nm sized AgNPs when compared to smaller AgNPs (10–50 nm) [125]. It can be noted that the variation in parameters in these studies makes it difficult to observe trends and come to accurate assumptions. To achieve some consensus in this regard, Gliga *et al*. studied the cytotoxic effect of varying sized AgNPs capped by various agents on the normal bronchial epithelial cell line (BEAS-2B). They reported that 10 nm sized AgNPs induced cytotoxicity

of Ag dust indicated no significant changes in health status.

[117]. A recent study on oral exposure to Ag+

give rise to particle formation, possibly in the form of Ag2S

, however the effects of Ag salts on the intestine

indicated

*DOI: http://dx.doi.org/10.5772/intechopen.94348*

**3.2 Cytotoxicity of silver nanoparticles**

therapeutic purposes.

subsequently releases Ag<sup>+</sup>

intestinal digestion, Ag+

*3.2.2* In vitro *studies*

and reduce the toxic effects of Ag<sup>+</sup>

*3.2.1* In vivo *studies*
