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11.1 Introduction

In recent years, the accelerate expansion of nanotechnology has led to the production of various commercially available nano-sized materials, such as silver nanoparticles (AgNPs) (Ahmed et al. 2008), which received considerable attention due to their potent antimicrobial activities (Durán et al. 2010). AgNPs can be synthesized by traditional chemical methods, which are considered aggressive to the environment and to human health. Biogenic syntheses of AgNPs have been emerging as an attractive “green” alternative to synthesize nanomaterials for diverse applications (Bansal et al. 2012).

AgNPs have been used in many commercial products (Hood 2004; Wijnhoven et al. 2009) with different chemical and physical properties (Wise et al. 2010; AshaRani et al. 2008). The increased uses of AgNPs in several applications led to a concern regarding the toxicity of these nanoparticles and the safe use of these nanomaterials, as discussed in diverse subareas of nanotechnology, such as nanobiotechnology and nanomedicine (Brayner 2008; Panda et al. 2011). In fact, nanoparticles may have higher toxicity than bulk materials (Donaldson et al. 1999; Xiong et al. 2011). Nanoparticle toxicity direct impacts human health and the environment, and more investigations are needed in this topic (Nel et al. 2006; Lewinski et al. 2008; Ju-Nam and Lead 2008).

Important studies have demonstrated that size and the chemical nature of nanoparticles coating can lead to different toxic effects at the cellular, subcellular, and biomolecular levels, such as genes and proteins (Gurr et al. 2005; Chi et al. 2009). Most of the in vitro assays to characterize the toxicity of AgNPs have been carried out in different cellular models, such as human lung fibroblasts (AshaRani et al. 2009). The majority of the studies reported an increase in the oxidative stress and severe lipid peroxidation, as observed in the case of fish brain tissue upon exposure to nanomaterials (Oberdörster 2004). In general, the proposed mechanisms for these effects consider the induction of reactive oxygen species (ROS) formation by AgNPs, which is associated with DNA damage, apoptosis, and necrosis (Arora et al. 2008; Kim et al. 2009a, b; Foldbjerg et al. 2011). In vitro exposure to AgNPs showed a reduction in glutathione levels and the observation of lipid peroxidation (Arora et al. 2008; Kim et al. 2009a, b). In this scenario, complete elucidation of the toxicity mechanisms of AgNPs is of paramount importance, and more studies based on the cytotoxicity and genotoxicity of AgNPs are still necessary. In this regard, many important reviews described the methodologies currently available for in vitro and in vivo genotoxicological studies of nanomaterials, including AgNPs (Ng et al. 2010; Johnston et al. 2010; Rico et al. 2011; Gonzalez et al. 2011; de Lima et al. 2012; Doak et al. 2012). Therefore, the aim of this chapter is to present and discuss recent progress on the synthesis of AgNPs by using environmentally friendly approaches, which minimizes negative impacts of nanoparticles on human health, and also the evaluation of the toxicity of AgNPs, highlighting the necessity of more studies in this exciting area.

11.2 Biogenic Silver Nanoparticles: Preparation and Characterization

Traditional chemical methods of synthesis of metallic nanoparticles, in particular AgNPs, employ toxic reagents, release harmful byproducts to the environment, consume a lot of energy, and use expensive chemicals. Moreover, chemical syntheses result in the absorbance of toxic chemicals on the surface of nanoparticles (Bansal et al. 2012). In contrast, microorganisms and plants extracts are considered interesting nanofactories in the fabrication of metallic nanoparticles, in a novel concept of environmentally friendly synthesis of nanomaterials (Antony et al. 2011; Bai et al. 2011; Kumar et al. 2012a). In this context, the use of plant extracts and microorganisms in the synthesis of NPs has been emerged as a biocompatible, “green,” and exciting approach. In fact, biogenic synthesis of NPs has been gained a tremendous attention in recent years, since it is cost-effective and environmentally friendly. Either uni- or multicellular organisms have been shown immense potential for the synthesis of NPs, since they are able to synthesize AgNPs either intracellulary or extracellulary (Birla et al. 2009). Biogenic synthesis of inorganic NPs employs mild experimental conditions, such as pH, pressure, and temperature, leading to the formation of NPs coated with capping layer formed by proteins and/or lipids that confer physiological solubility and stability for the nanoparticles (Banu et al. 2011; Fayaz et al. 2011; Li et al. 2011). In this scenario, this section highlights recent developments on the biogenic syntheses of AgNPs by bacteria, yeast, fungi, algae, and plant extracts. The most common techniques used to characterize the obtained biogenic AgNPs are UV-visible spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy (Kora et al. 2012).

11.2.1 Bacterial Synthesis

Recently, many bacterial species have been successfully used to extracellularly synthesize AgNPs (Bai et al. 2011; Bansal et al. 2012). Biogenic synthesis of AgNPs by bacteria is carried out by the incubation of silver ions with cell filtrates of bacteria at room temperature for minutes–hours.

Incubation of bacterial culture isolated of Pseudomonas stutzeri AG259 with silver ions led to the formation of AgNPs with controlled size and distinct morphology within the periplasmic space of the bacteria (Klaus et al. 1999; Joerger et al. 2000; Klaus-Joerger et al. 2001). Recently, marine actinomycetes strain of Streptomyces albidoflavus was used to synthesize either extracellular or intracellularly AgNPs (Prakasham et al. 2012). The obtained AgNPs showed high stability in aqueous solution. This stabilization could be attributed to the secretion of proteins by the bacterium in the reaction mixture. Moreover, biogenic synthesized AgNPs revealed antimicrobial activities against both Gram-negative and Gram-positive bacterial strains (Prakasham et al. 2012). Fayaz et al. (2011) reported the preparation of stable AgNPs through the exposition of cell-free extract of the bacterium Geobacillus stearothermophilus to AgNO3 solution. The stability of AgNPs was attributed to the presence of capping proteins on nanoparticle surface, as evidenced by FTIR and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The formation of AgNPs was confirmed by the detection of the characteristic optical absorption band in the UV-visible region (plasmon band at ca. 420 nm). TEM analysis of the AgNPs revealed that the particles have spherical shape and polydispersed pattern, with an average size of dimensions of 5–35 nm. Moreover, X-ray diffraction pattern confirmed that NPs are face-centered cubic in shape and FTIR indicated the presence of reducing enzymes and capping proteins on the NPs surface (Fayaz et al. 2011).

Nowadays, several different bacteria have been extensively used to synthesize AgNPs. Table 11.1 summarizes some important and recent studies based on the preparation of AgNPs by different organisms, including bacteria.

Table 11.1 List of important organisms that synthesize silver nanoparticles (AgNPs)
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11.2.2 Yeasts Synthesis

Compared with other microorganisms, such as bacteria and fungi, there are a few reports describing the use of yeasts to produce AgNPs. In fact, yeasts can be successfully employed to synthesize metallic NPs, and this approach should be more investigated. Extracellular synthesis of AgNPs was observed in silver-tolerant yeast strains MKY3 when challenged with 1 mM silver ions, in the log phase of yeast growth (Kowshik et al. 2003). Recently, biosynthesis of AgNPs by using yeast biomass was reported (Mourato et al. 2011). Extremophilic yeast strain was isolated from acid mine drainage in Portugal. Exposition of washed yeast cells with Ag ions produced AgNPs with diameter size smaller than 20 nm (Mourato et al. 2011).

11.2.3 Fungal Synthesis

Mycosynthesis is the synthesis of nanoparticles by fungi. The term “mycosynthesis” was used for the first time by Ingle et al. (2008) describing the synthesis of nanoparticles by Fusarium acuminatum. Overall, fungi possess some important advantages as a potent microorganism for the synthesis of AgNPs. It is relatively easy to culture fungi, the NPs synthesis is mostly extracellular, and the synthesized NPs have a good polydispersivity, size, and stability (Gade et al. 2010a; Birla et al. 2009).

Rai et al. (2009) proposed the term “Myconanotechnology” to include research carried out on nanoparticles synthesized by fungi. Many fungal species have been explored for the production of different metal nanoparticles of different shapes and sizes. Chen et al. (2003) reported the extracellular formation of AgNPs using Phoma species. Some soil-borne fungi like Aspergillus fumigatus are able to synthesize AgNPs when the cell extract was challenged with aqueous silver ions (Bhainsa and D’souza 2006). Among different fungal genera used for the synthesis of NPs, the genus Fusarium was extensively used. Ahmad et al. (2003) used for the first time the Fusarium oxysporum for the synthesis of AgNPs and opened a new avenue in the field of nanobiotechnology. Durán et al. (2005) studied the extracellular production of metal NPs by several strains of the fungus F. oxysporum. Similarly, other Fusarium species like F. oxysporum strain 5115 (Mohammadian et al. 2007), F. semitectum (Basavaraja et al. 2008), F. acuminatum (Ingle et al. 2008), F. solani (Ingle et al. 2009), and F. culmorum (Bawaskar et al. 2010) have been successfully used for the synthesis of AgNPs.

Recently, Kumar et al. (2012a) reported the mycogenic synthesis of AgNPs by Aspergillus tamari. Ag ions were reduced by the fungal extracellular filtrate leading to the formation of AgNPs, after 30 min of incubation. SEM images revealed that the nanoparticles were spherical with size ranging from 25 to 50 nm. UV-visible spectrophotometry confirmed the presence of the characteristic plasmon band at 420 nm; X-ray diffraction confirmed the crystalline face-centered cubic of AgNPs. Moreover, FTIR indicated that AgNPs were coated with proteins secreted by the fungus, allowing stabilization of AgNPs in aqueous solution (Kumar et al. 2012a). Narayanan and Sakthivel (2010) discussed in detail the biogenic syntheses of metallic nanoparticles, including AgNPs by microbes, in particular by fungi. Table 11.1 summarizes recent important papers based on the mycosynthesis of AgNPs.

11.2.4 Algae Synthesis

The unicellular algae Chlamydomonas reinhardtii was used as a model system to investigate the role of cellular proteins in the synthesis of AgNPs (Barwal et al. 2011). Cell-free extract (in vitro) of C. reinhardtii and in vivo cells produced AgNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm, respectively. The authors have identified several cellular proteins related to the synthesis of AgNPs by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), such as ATP synthase, superoxide dismutase, carbonic anhydrase, and ferredoxin-NADP+ reductase. This elegant paper provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of AgNPs (Barwal et al. 2011).

Shukla et al. (2012) reported the formation of AgNPs by the reduction of silver ions, at room temperature, with the addition of agar extracted from the red algae Gracilaria dura. AgNPs were found to be spherical in shape, with a good polydispersivity with average size of 6.0 ± 2 nm, as reveled by TEM analysis. Similarly, Govindaraju et al. (2008) reported the formation of several metallic nanoparticles, including AgNPs from Spirulina platensis.

11.2.5 Plants Synthesis

Rai et al. (2008) stated that plants have emerged as a simple, cost-effective and eco-friendly system to rapidly synthesize NPs. In this scenario, Persimmon (Diospyros kaki) leaf extract led to the formation of AgNPs of 15–90 nm size (Song and Kim 2008). Krishnaraj et al. (2010) reported the rapidly biosynthesis of AgNPs (within 30 min) using leaf extract of Acalypha indica and their activity on water-borne bacterial pathogens. Antibacterial activity of biogenically synthesized AgNPs showed effective inhibitory activity against water-borne pathogens, viz., Escherichia coli and Vibrio cholera (Krishnaraj et al. 2010).

Gade et al. (2010b) reported the green synthesis of AgNPs by Opuntia ficus-indica. They evaluate antibacterial activity of synthesized AgNPs against E. coli and S. aureus, in combination with commercially available antibiotics, such as ampicillin, gentamicin, kanamycin, streptomycin, and vancomycin. Antibacterial activities of the antibiotics increased due to combination with AgNPs, since AgNPs are known to possess antimicrobial properties. The authors also proposed a mechanism for the biogenic synthesis of AgNPs, which involved the biomolecule quercetin (Gade et al. 2010b). Recently, Kumar et al. (2012b) reported the reduction of silver ions to AgNPs by aqueous extract of Terminalia chebula, within only 20 min. The formation of AgNPs was confirmed by surface plasmon resonance at 452 nm using UV-visible spectrophotometer. In addition, high-resolution TEM (HR-TEM) revealed the formation of anisotropic nanostructures of pentagons, spherical and triangular-shaped AgNPs, with diameter less than 100 nm. Selected area electron diffraction pattern (SAED) confirmed the crystalline nature of AgNPs. Atomic force microscopy (AFM) images indicated the presence of coating comprised by oxidized polyphenols on the surface of AgNPs. Indeed, in biogenic synthesis of AgNPs by plant extracts, polyphenols act as reducing agent and also as a capping material leading to stabilization of AgNPs in aqueous solution (Kumar et al. 2012b). Narayanan and Sakthivel (2011) published detailed and updated review article based on the biogenic synthesis of metallic nanoparticles, including AgNPs by plant extracts and algae. Table 11.1 summarizes some relevant and recent studies based on green synthesis of AgNPs by plant extracts.

11.3 Toxic Effects of Silver Nanoparticles Administrated In Vitro and In Vivo

In recent years, nanotechnology has been rapidly expanding leading to important impacts on different areas such as health, environment, and economy. In fact, the commercialization of products comprised by nanomaterials is now a reality. Among commercially available nanocompounds, AgNPs are the most used (Ahmed et al. 2008), mainly due to their potent microbicidal activity (Durán et al. 2010). The rapid development of nanotechnology has been leading to an increasing concern related to possible toxic effects of nanomaterials, including human health and environmental impacts (Brayner 2008; Panda et al. 2011). In this scenario, the detailed investigation of nanoparticles toxicity has been emerging as an important area of research. The great interest of the scientific community by toxicological evaluations of nanomaterials is relatively new, and it has been increasing in recent years. However, detailed studies on nanoparticles toxicity, including AgNPs, are still limited and relatively unexplored (de Lima et al. 2012). Therefore, the great appeal of nanotechnology can be considered the better evaluation of the cytotoxicity and genotoxicity of nanoparticles, in particular, biogenic synthesized metallic nanoparticles. In this scenario, this section summarizes a survey of recent evaluations of cytotoxicity and genotoxicity of AgNPs, in special biogenic synthesized AgNPs, and highlights the necessity of more studies in the field.

11.3.1 Culture Cells

Vivek et al. (2012) reported the in vitro cytotoxicity of biogenic AgNPs synthesized by plant extract of Annona squamosa. The AgNPs synthesized by plant leaf extract showed to be spherical in shape with average diameter ranging from 20 to 100 nm. The in vitro cytotoxicity of these biogenic AgNPs were evaluated against normal epithelial cells (HBL-100) and human breast cancer cell (MCF-7). The authors reported a dose-dependent cytotoxicity effect. The inhibitory concentrations (IC50), in which 50 % of cells die, were found to be 50 μg/mL, 30 μg/mL and 80 μg/mL, 60 μg/mL for AgNPs against MCF-7 and normal HBL-100 cells at 24- and 48-h incubation, respectively (Vivek et al. 2012). Moreover, it was also observed an induction of apoptosis. Overall, the results revealed cytotoxic effects of biosynthesized AgNPs against breast cancer cell line, in comparison with normal breast cell line, indicating that these nanoparticles might be used in cancer treatments.

A similar work describes the preparation of biogenic AgNPs by plant extract of Iresine herbstii (Dipankar and Murugan 2012). The obtained AgNPs were found to be face-centered cubic in shape, with size ranging from 44 to 64 nm, and capped with plant components (Dipankar and Murugan 2012). The in vitro cytotoxicity of plant-mediated AgNPs was evaluated towards HeLa cancer cells with the trypan blue assay. AgNPs exhibited a potent toxic effect (CL50 of 51 μg/mL) on cancer cells with 88 % death of HeLa cells upon treatment with 300 μg/mL of AgNPs (Dipankar and Murugan 2012).

However, the toxicity of AgNPs towards culture cells is still under study. Several studies did not observe toxicity on human culture cells treated with capped AgNPs (diameters ranging from 6 to 80 nm) below 10 mg/mL (Hussain et al. 2005; AshaRani et al. 2009; Lu et al. 2010; Foldbjerg et al. 2011). An interesting paper investigated the toxicity of biogenic AgNPs (size of 25–45 nm) synthesized by Alternaria alternata towards human lymphocytes using comet assay (Sarkar et al. 2011). The authors reported that up to 50 mg/mL of biogenic AgNPs, no DNA damage was observed. DNA damage was only reported upon incubation of human lymphocytes with over 300 mg/mL of AgNPs.

On contrary, some papers described different degrees of toxicity upon incubation of cell cultures with AgNPs, at concentrations up to 50 mg/mL (de Lima et al. 2012). For example, culture of human mesenchymal stem cells incubated with 0.1 mg/mL of albumin-capped AgNPs (average size 46 nm) (Hackenberg et al. 2011) or human glioblastoma cells starch incubated with capped AgNPs (sizes 6–20 nm) (AshaRani et al. 2008) showed genotoxicity up to 50 mg/mL of AgNPs. Another interesting work showed that albumin-coated AgNPs (average size 70 nm) were found to be more genotoxic on mouse peritoneal macrophage cell line (genotoxicity at around 2 mg/mL) (Park et al. 2010) in comparison with AgNPs capped with polysaccharides (average size 25 nm), on mouse embryonic stem and fibroblasts cells, which exhibited genotoxicity at 50 mg/mL (Ahmed et al. 2008). In general and with a few exceptions, human culture cells were observed to be less sensitive compared to mouse culture cells, upon treatment with capped AgNPs, independently of the chemical nature of the capping (de Lima et al. 2012). Obviously, this tendency should be further investigated.

11.3.2 Calf Thymus DNA

It was observed that the genotoxicity of AgNPs alone (sizes 20–50 nm) was weak on calf thymus DNA (ctDNA); however, in the presence of detergent (cetylpyridinium bromide), the NPs showed significant genotoxicity, mainly owing to the presence of the surfactant (Chi et al. 2009). This result is important from an environmental point of view, since considerable amounts of AgNPs are eliminated to water (rivers, oceans) and probably readily interacting with surfactant presented in the water, leading to a strong genotoxicity owing to the formation of AgNPs–detergent interactions (Chi et al. 2009).

11.3.3 In Vivo Assays

Many papers have reported in vivo toxicity studies of AgNPs by different routes of administration (Tian et al. 2007; Vlachou et al. 2007; Trop et al. 2006; Samberg et al. 2010). For example, Rahman et al. (2009) reported that intraperitoneal administration of AgNPs may lead to alterations of gene expression, suggesting the neurotoxic effects of these nanoparticles. Furthermore, several reports describe oral administration of AgNPs (Cha et al. 2008; Kim et al. 2009a, b). After oral exposure, AgNPs and/or silver ions are assumed to translocate from the gut into the blood, systemically inducing liver damage. However, it is necessary to carefully investigate these statements, since it is necessary to distinguish whether the systemic distribution of silver is due to the presence of AgNPs or silver ions in the liver, and this topic deserves more studies, as stated by Johnston et al. (2010). Concerning to the possible oxidation of AgNPs to silver ions, an in vivo study based on intravenous administration of AgNPs on mouse supported the antiplatelet properties of these nanoparticles (AgNPs are considered to have antiplatelet properties) (Shrivastava et al. 2009). It was suggested that there is a low possibility of oxidation of AgNPs into silver ions inside the platelets due to the high silver ionization potential (735 kJ mol−1), since this high ionization potential is difficult to reach in an intracellular environment. In view of this fact the observed antiplatelet property effects are more likely to be due to AgNPs rather than to silver ions. It is noticed that this same effect was observed in human platelets (patients with type 2 diabetes mellitus) (Shrivastava et al. 2009).

In a recent study, the mycosynthesis of AgNPs by filamentous fungus Cochliobolus lunatus, their characterization, and larvicidal effects were reported (Salunkhe et al. 2011). Toxic effects of biogenic AgNPs towards Aedes aegypti and Anopheles stephensi, which are responsible for diseases of public health importance, were investigated. Potent mortality effects on second, third, and fourth instar larvae of A. aegypti and A. stephensi were found to correlate with concentrations of AgNPs. The observed larvicidal activity of mycogenic AgNPs was related to the penetration of nanoparticles through larvae membrane. Moreover, toxicity studies were also carried out against nontarget fish species Poecilia reticulata. Common organisms found in this kind of habits are A. aegypti and A. stephensi. Interestingly, mycogenic AgNPs did not exhibit noticeable effects on P. reticulata after either 24 or 48 h of exposure at the level of LC50 and LC90 values against fourth instar larvae of A. aegypti and A. stephensi. These results indicate that biogenic AgNPs might be used as a potent larvicidal agent, with no toxic effects to the environment (Salunkhe et al. 2011).

Interestingly, recent studies suggested that in vivo toxicity of AgNPs is dependent on the organism. In this scenario, in vivo studies demonstrated that mice are more sensitive to capped AgNPs in comparison with fish. Indeed, some papers showed that AgNPs capped with starch (8–15 nm), or with bovine serum albumin (10–20 nm) (AshaRani et al. 2008), or with polyvinyl alcohol (5–35 nm) (AshaRani et al. 2011) were no genotoxic towards zebra fish embryos up to nanoparticles concentration of 25 mg/mL. Genotoxicity was observed by increasing the Nanoparticles concentration to values over 100 mg/mL, in special in the case of albumin-capped AgNPs, which lead to embryos apoptosis and also abnormalities (AshaRani et al. 2008). However, at low nanoparticle dose, such as 1 mg mL−1, this effect was found to be very discrete (Ordzhonikidze et al. 2009).

Very few studies investigated the pulmonary toxicity of AgNPs. As stated by Johnston et al. (2010), there is limited information available related to this topic. Studies based on the investigation of pulmonary toxicity of AgNPs are important since proinflammatory and oxidative potentials of AgNPs within the lung are known to drive nanoparticles toxicity (Sung et al. 2009; Hyun et al. 2008; Ji et al. 2007). Aspects such as exposure time to nanoparticles, their concentrations, routes of applications, and particle sizes are extremely important to define nanoparticle toxicity. AgNP translocation and their potential accumulation within secondary targets, such as liver, spleen, and brain, following pulmonary exposure, were reported. However, Johnston et al. (2010) stated that, until now, it is not possible to confirm that the silver content of any cell distribution is due to AgNPs or to silver ions, since the methods used are not sensible enough to discriminate between them. It is known that in biological systems both forms of silver can exist owing to degradation and/or metabolism of the nanoparticles, indicating that further studies are required in this field.

Moreover, several applications of AgNPs are based on dermatological uses, such as the production of wound dressing containing AgNPs. In this regards, it is important to further investigate possible toxic effects associated with administration of AgNPs on either wounded skin or normal skin. It is important to clearly define the hazards associated with dermal exposure to AgNPs, since liver has been suggested as a secondary target for nanoparticles toxicity (de Lima et al. 2012 and reference therein).

11.4 Aspects That Determine Nanoparticles Cytotoxicity and Genotoxicity

Several aspects including size, surface area, surface reactivity, composition, surface charge, and capping molecules are known to influence cell toxicity. In general, reactivity of nanomaterials in biological medium is directly related to the observed toxic effects of nanoparticles, and particle size controls the reactivity of these nanomaterials (Brown et al. 2001; Duffin et al. 2007). Indeed, small nanoparticles can cause several side effects in lung, due to their higher ability for cell invasion (Oberdörster et al. 2005). Hence, smaller sized particles were found to be more toxic than bigger sized particles (Panda et al. 2011; Gaiser et al. 2012). However, some exception was reported, as in the case of nonmetallic nanoparticles, such as polymeric nanoparticles. Toxicity of chitosan nanoparticles indicated that smaller particles might be used more safely than larger particles, at higher concentrations, and probably others factors could be involved in nanoparticle toxicity (Lima et al. 2010). Overall, in the case of metallic nanoparticles such as AgNPs, it can be observed a tendency that small particles have higher reactivity and thus produce higher toxicity (Ordzhonikidze et al. 2009; Kim et al. 2011). However, it must be noted that particle size is not the only factor that determines nanoparticle toxicity, since important parameters such as synthesis procedures, presence and nature of capping agents, surface charge, and aggregation are also important (Panda et al. 2011; Suresh et al. 2012). In fact, Suresh et al. (2012) investigated the cytotoxicity of effects of AgNPs with different surface coatings on two different cell lines. Figure 11.1 shows the cytotoxicity of mouse macrophage and lung epithelial cells treated with four different types of AgNPs: (a) poly(diallyldimethylammonium) chloride-AgNPs; (b) biogenic AgNPs, synthesized by bacteria Shewanella oneidensis; (c) uncoated-AgNPs (colloidal-AgNPs); and (d) oleate-AgNPs. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay measured mitochondrial integrity and oxidative metabolism and is an indirect measure of cell viability. All different AgNPs tested showed a dose-dependent cytotoxicity in both cell lines. However, depending on the type of AgNPs different cytotoxic parameters were obtained. For example, in the case of mouse macrophage cells, the inhibitory concentration values were found to be 0.1, 0.125, 1.1, and 4.9 μg/mL for poly(diallyldimethylammonium) chloride-AgNPs, biogenic AgNPs, oleate-AgNPs, and uncoated-AgNPs, respectively. For lung epithelial cells, the inhibitory concentration values were found to be 0.45, 0.7, 1.6, and 6.3 μg/mL for poly(diallyldimethylammonium) chloride-AgNPs, biogenic AgNPs, oleate-AgNPs, and uncoated-AgNPs, respectively. It can be concluded that uncoated-AgNPs were the less toxic to both cell lines, followed by olate-AgNPs, biogenic AgNPs, and poly(diallyldimethylammonium) chloride-AgNPs (Suresh et al. 2012). These results reveal the impact of synthesis procedure and surface coating in determining nanoparticle toxicity.

Fig. 11.1
figure 1

Results of MTT assay of the mouse macrophage and lung epithelial cells exposed to the four different types of silver nanoparticles at various concentrations: (a) poly(diallyldimethylammonium) chloride-AgNPs, (b) biogenic AgNPs, (c) uncoated-AgNps, and (d) oleate-Ag nanoparticles NPs. Reproduced from Suresh et al. 2012 by permission of American Chemical Society

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11.5 Comments and Future Perspectives

It can be concluded that biogenic synthesis of metallic NPs, in particular AgNPs, has been emerging as an important and fruitful domain of nanotechnology. Despite the fact that several papers have successfully described different routes to biogenically synthesize AgNPs, as stated in this chapter, the mechanisms involved in the biogenic production of AgNPs have not yet been elucidated. Therefore, elucidation of the mechanistic aspects of some biological systems in the synthesis of nanoparticles needs more detailed studies (Durán et al. 2011).

Moreover, by reviewing the actual literature based on AgNPs and toxicity, it can be concluded that there are few studies based on cytotoxicity and genotoxicity of biogenic AgNPs, in comparison with chemically synthesized AgNPs. The lack of more studies based on toxicity of biogenic AgNPs can be understood by considering that biogenic synthesis of metallic nanoparticles is a relatively new methodology (de Lima et al. 2012). By analyzing and comparing published papers on toxic effects of AgNPs, the determination of AgNPs toxicity (both cytotoxicity and genotoxicity) is a complex issue, since many different parameters play important roles. In fact, controversial papers showing different effects of toxicity of AgNPs can be understood by considering several parameters, such as different methodologies to synthesize the nanoparticles, nanoparticles size, shape, presence and nature of capping agents, surface area and charge, and finally, the diverse kinds of toxic evaluation tests employed (de Lima et al. 2012). The uses of different organisms and/or culture cells to investigate AgNPs toxicity make the comparison between different studies a difficult and complex task, leading to inconclusive results in some cases. In this context, it is necessary to elaborate standard protocols to carefully analyze nanoparticles toxicity in order to decrease the possible discrepancies related to final conclusions and comparison between different works.

Therefore, with the present information, it is premature to conclude whether biogenic nanoparticles are less genotoxic/cytotoxic compared with chemically synthesized nanoparticles. However, some considerations and tendencies can be postulated with the actual literature, such as the lower toxicity of AgNPs compared with silver ions (Ordzhonikidze et al. 2009; Kim et al. 2011; Griffitt et al. 2008; Park and Choi 2010; Panda et al. 2011; Gaiser et al. 2012). Panda et al. (2011) investigated the induction of cellular death of cultured cells of Allium cepa incubated with silver ions (Ag+), silver complexes (AgCl), capped biogenic AgNPs (AgNP-biogenic), and commercially uncapped AgNPs from Sigma (AgNP-Sigma), and they found that the induction of cell death followed the order: Ag+ ions > colloidal AgCl > AgNP-commercial from Sigma > AgNP-biogenic, showing that the less toxic form of silver was biogenic synthesized capped AgNPs. Similar results were reported for biogenic AgNPs for different applications, such as wound healing antileishmaniases, antifungal, and antibacterial effects (Melo et al. 2011; Marcato and Durán 2011; Rossi-Bergmann et al. 2012; Marcato et al. 2012a, b).

Taken together, it can be assumed that biogenic synthesis of AgNPs has been emerging as an attractive and important route to prepare nanoparticles for several applications. In general, biogenic AgNPs are less toxic in comparisons with chemically synthesized nanoparticles. This chapter highlights the necessity of more mechanist studies on the biogenic synthesis of AgNPs, in order to optimize the green synthesis process, and more studies based on the toxicity of AgNPs, in particular, genotoxicity studies of biogenic AgNPs.