**2.1 Physical and chemical method**

There are various physical and chemical approach widely used for the synthesis of silver nanoparticles, such as reduction in solution, (Goia, 1998) chemical and photochemical reactions in reverse micelles, (Taleb, 1997) thermal decomposition of metal compounds, (Esumi, 1990) radiation assisted, (Henglein, 2001) electrochemical, (Rodriguez-Sanchez, 2000) sonochemical, (Zhu, 2000) microwave assisted process (Isabel Pastoriza-Santos, 2002).

Fig. 1. Applications of metal nanoparticles in various fields (G.Thirumurugan, 2011).

synthesis deserves merit.

**2. Synthesis of silver nanoparticles** 

**2.1 Physical and chemical method** 

properties.

The synthesis of monodispersed metal nanoparticles with different size and shape has been challenge in nanotechnology. Although various physical and chemical methods are extensively used to produce monodispersed nanoparticles, the stability and the use of toxic chemicals is the subject of paramount concern. Moreover, the use of toxic chemicals on the surface of nanoparticles and non-polar solvents in the synthesis procedure limits their applications in clinical and pharmaceutical field (Oghabian, 2010) Therefore, development of clean, biocompatible, non-toxic and eco-friendly methods for silver nanoparticles

In this chapter, we will discuss an overview of silver nanoparticle preparation involving physical, chemical method and biological method, mechanism and advantages and disadvantages of the above methods. We provide various antibacterial mechanisms of silver nanoparticles to reduce antibiotic resistance and incorporation of SNPs on cotton fabrics and conjugation of SNPs on pharmaceutical compounds. Finally, we will discuss site-specific, antibacterial drug delivery of SNPs due to its unique surface modification, photo-thermal

There are various physical and chemical approach widely used for the synthesis of silver nanoparticles, such as reduction in solution, (Goia, 1998) chemical and photochemical reactions in reverse micelles, (Taleb, 1997) thermal decomposition of metal compounds, (Esumi, 1990) radiation assisted, (Henglein, 2001) electrochemical, (Rodriguez-Sanchez, 2000) sonochemical, (Zhu, 2000) microwave assisted process (Isabel Pastoriza-Santos, 2002). Most frequently preparation of silver nanoparticles is carried out by chemical reduction method. Borohydrate, citrate, ascorbate, and elemental hydrogen are commonly used reductants for the synthesis of silver nanoparticles. The reduction of metal ions (Me+) like silver (Ag+ or gold (Au+) in aqueous solution generally yields colloidal metal with particle diameters of several nanometers (Wiley, 2005). Initially, the reduction of various complexes with metal (Ag+) ions leads to the formation of metal atoms (Ag0), which is followed by agglomeration into oligomeric clusters (Kapoor, 2002). These clusters eventually lead to the formation of colloidal Metal particles (Kapoor, 2002). For example, while formation of colloidal silver particles, when the colloidal particles are much smaller than the wavelength of visible light, the solutions have a yellow color with an intense band in the 380–400 nm range and other less intense or smaller bands at longer wavelength in the absorption spectrum (Cao, 2002). This band is attributed to collective excitation of the electron gas in the particles, with a periodic change in electron density at the surface (surface Plasmon absorption), (Gutiérrez, 1993).

The synthesis of silver nanoparticles in this project will be based on a wet chemical method. The starting point of the synthesis is the production of a silver nitrate (AgNO3) solution. When silver nitrate is dissolved it splits into a positive silver ion (Ag+) and a negative nitrate ion (NO3-). In order to turn the silver ions into solid silver, the ions have to be reduced by receiving an electron from a donator. A flowchart illustrating the reduction of the silver ions by addition of an electron can be seen in Equation 1. The flowchart of Equation 2 illustrates the reduction of (Ag+) in a solution of ethanol. After the silver germ has been formed it starts to grow and continue the growth until the equilibrium between the final nanoparticles and the (Ag+) of the solution is reached (Chou, 2005).

$$\text{Ag}^+\_{\text{(aq)}} + \text{e}^- \to \text{Ag}\_{\text{(s)}} \tag{1}$$

$$\text{2Ag}^+\_{\text{(aq)}} + \text{C}\_2\text{H}\_5\text{OH} + \text{H}\_2\text{O} \rightarrow 2\text{Ag}\_{\text{(s)}} + \text{C}\_2\text{H}\_5\text{O}\_2^- + 3\text{H}^+ \tag{2}$$

The chemical reaction is the sodium borohydride reduction of silver nitrate:

$$\text{AgNO}\_3 + \text{NaBH}\_4 \rightarrow \text{Ag} + \frac{1}{2}\text{H}\_2 + \frac{1}{2}\text{B}\_2\text{H}\_6 + \text{NaNO}\_3 \tag{3}$$

The preparation of silver nanoparticles in briefly, A 10-mL volume of 1.0 mM silver nitrate was added dropwise (about 1 dropsecond) to 30 mL of 2.0 mM sodium borohydride solution that had been chilled in an ice bath. The reaction mixture was stirred vigorously on a magnetic stir plate. The solution turned light yellow after the addition of 2 mL of silver nitrate and a brighter yellow, when all of the silver nitrate had been added. The entire addition took about three minutes, after which the stirring was stopped and the stir bar removed. The clear yellow colloidal silver is stable at room temperature stored in a transparent vial for as long as several weeks or months. Reaction conditions including stirring time and relative quantities of reagents (both the absolute number of moles of each reactant as well as their relative molarities) must be carefully controlled to obtain stable yellow colloidal silver. A large excess of sodium borohydride is needed both to reduce the ionic silver and to stabilize the silver nanoparticles. The possibility of aggregation during the synthesis, colloidal silver solution turns darker

Silver Nanoparticles: Real Antibacterial Bullets 411

Bio-recovery of silver metals from solution, a process referred to as "biosorption", occurs by either active or passive mechanisms. Active metal transformation processes require viable microbes, enzymatically catalyzing the alteration of the metal, leading to sequestration or concentration. One possible (passive) role of the microorganisms is in providing a multitude of nucleation centers; establishing conditions for obtaining highly disperse nanoparticle systems. In addition, they slow down aggregation, or entirely prevent it by immobilizing the particles, and providing viscous medium (*Sun,* 2002). Thus produced nanoparticles have highly intricate architectures and are ordered during assembly. In some cases, the particles have a well-defined shape formed within an arrow size range and have orientational and geometrical symmetry (Sarikaya, 1999). In case of silver nanoparticle production, the resistance conferred by bacteria to silver is determined by the *'sil'* gene in plasmids (Silver, 2003) while a nitrate-dependent reductase and a shuttle quinine extracellular process were reported for the reduction of silver ions by several Fusarium oxysporum strains (Duran, 2005). The extract of unicellular green algae Chlorella vulgaris was used to synthesize single-crystalline Ag nanoplates at room temperature (Jianping Xie, 2007). Proteins in the extract provide dual function of Ag+ reduction and shape-control in the nano silver synthesis. The carboxyl groups in aspartic and or glutamine residues and the hydroxyl groups in tyrosine residues of the proteins were suggested to be responsible for the Ag+ ion reduction (Jianping Xie, 2007). Carrying out the reduction process by a simple bifunctional tripeptide Asp-Asp-Tyr-OMe further identied the involvement of these residues. This synthesis process gave small Ag nanoplates with low polydispersity in good yield (>55%) (Gole, 2001). Balaji, 2009 reported FTIR spectroscopic studies on silver nanoparticles obtained from the fungus, Cladosporium cladosporioides. Their study conrmed that the carbonyl groups from the amino acid residues and peptides of proteins have strong ability to bind silver. The proteins could possibly forma coat covering the metal nanoparticles to prevent their agglomeration and aid in its stabilization in the medium. Hence, the biological molecules could possibly function in the formation and stabilization of the silver nanoparticles in aqueous medium. Gole, 2001 reported that proteins can bind to silver nanoparticles either through free amine groups in the proteins and possibly play a role in stabilization of the silver nanoparticles by surface-bound proteins. Moreover, Vigneshwaran, 2006 explained the synthesis of metal nanoparticles by using fungal mycelium, in the a rotary shaker, metal ions in solution were adsorbed on the surface of the mycelia through interactions with chemical functional groups such as carboxylate anion, carboxyl and peptide bond of proteins, and hydroxyl of saccharides (Lin, 2005) found on the mycelia. The mycelia, matted together, was more immobile, and more capable of binding Me+ than that of the external cellular substances that distributed in the inter-mycelial space, then most of the Me+ was *in situ* reduced to Me0 by reducing sugars from the saccharides (Gole, 2001) on the mycelia. In the mean time, stronger adsorptive groups such as the carbonyl group on the extracellular substances could further adsorb the particles located on the surface of the mycelia, resulting in capping these nanoparticles, while rocking. When other Me+ in the solution was rocked on to this overlay and was bound and reduced to Me0 on the surface of the layer and these Me0 might be possibly further coated with the other extra cellular substances; this process was repeated continuously until these substances

distributing in the inter-mycelial space was used up.

Plant extracts from live alfalfa, the broths of lemon grass, geranium leaves and others have served as green reactants in silver nanoparticle synthesis (Shankar, 2004; Gardea-Torresdey,

yellow, violet, and then grayish. Adsorption of borohydride plays a key role in stabilizing growing silver nanoparticles by providing a particle surface charge as shown in the schematic diagram in Figure 2. There must be enough borohydride to stabilize the particles as the reaction proceeds. However, later in the reaction too much sodium borohydride increases the overall ionic strength and aggregation will occur (Van Hyning, 2001). The aggregation can also be brought about by addition of electrolytes such as NaCl. Nanoparticles are kept in suspension by repulsive electrostatic forces between the particles owing to adsorbed borohydride (Fig. 2). Salt shields the charges allowing the particles to clump together to form aggregates.

Fig. 2. Repulsive forces separate Ag nanoparticles (NP) with adsorbed borohydride (G.Thirumurugan, 2011).

#### **2.2 Biological method of silver nanoparticle synthesis**

Though various chemical and biochemical methods are being explored for silver nanoparticle production (Wiley, 2005), microbes, plants are also very effective in this process. Various microbes (Sharma, 2006; Mann, 1984; Beveridge, 1997), plants (Shankar, 2004; Gardea-Torresdey, 2002) are known to reduce the metals [Table 1], most of them are found to be spherical particles as reported earlier (Chen, 2003; Ahmad, 2005). Extracts from microbes act both as reducing and capping agents in metal nanoparticles synthesis. The reduction of metal ions by combinations of biomolecules found in these extracts such as enzymes or proteins, amino acids, polysaccharides, and vitamins (Jagadeesh*,* 1981) is environmentally benign, yet chemically complex.

yellow, violet, and then grayish. Adsorption of borohydride plays a key role in stabilizing growing silver nanoparticles by providing a particle surface charge as shown in the schematic diagram in Figure 2. There must be enough borohydride to stabilize the particles as the reaction proceeds. However, later in the reaction too much sodium borohydride increases the overall ionic strength and aggregation will occur (Van Hyning, 2001). The aggregation can also be brought about by addition of electrolytes such as NaCl. Nanoparticles are kept in suspension by repulsive electrostatic forces between the particles owing to adsorbed borohydride (Fig. 2). Salt shields the charges allowing the

Fig. 2. Repulsive forces separate Ag nanoparticles (NP) with adsorbed borohydride

Though various chemical and biochemical methods are being explored for silver nanoparticle production (Wiley, 2005), microbes, plants are also very effective in this process. Various microbes (Sharma, 2006; Mann, 1984; Beveridge, 1997), plants (Shankar, 2004; Gardea-Torresdey, 2002) are known to reduce the metals [Table 1], most of them are found to be spherical particles as reported earlier (Chen, 2003; Ahmad, 2005). Extracts from microbes act both as reducing and capping agents in metal nanoparticles synthesis. The reduction of metal ions by combinations of biomolecules found in these extracts such as enzymes or proteins, amino acids, polysaccharides, and vitamins (Jagadeesh*,* 1981) is

**2.2 Biological method of silver nanoparticle synthesis** 

environmentally benign, yet chemically complex.

particles to clump together to form aggregates.

(G.Thirumurugan, 2011).

Bio-recovery of silver metals from solution, a process referred to as "biosorption", occurs by either active or passive mechanisms. Active metal transformation processes require viable microbes, enzymatically catalyzing the alteration of the metal, leading to sequestration or concentration. One possible (passive) role of the microorganisms is in providing a multitude of nucleation centers; establishing conditions for obtaining highly disperse nanoparticle systems. In addition, they slow down aggregation, or entirely prevent it by immobilizing the particles, and providing viscous medium (*Sun,* 2002). Thus produced nanoparticles have highly intricate architectures and are ordered during assembly. In some cases, the particles have a well-defined shape formed within an arrow size range and have orientational and geometrical symmetry (Sarikaya, 1999). In case of silver nanoparticle production, the resistance conferred by bacteria to silver is determined by the *'sil'* gene in plasmids (Silver, 2003) while a nitrate-dependent reductase and a shuttle quinine extracellular process were reported for the reduction of silver ions by several Fusarium oxysporum strains (Duran, 2005). The extract of unicellular green algae Chlorella vulgaris was used to synthesize single-crystalline Ag nanoplates at room temperature (Jianping Xie, 2007). Proteins in the extract provide dual function of Ag+ reduction and shape-control in the nano silver synthesis. The carboxyl groups in aspartic and or glutamine residues and the hydroxyl groups in tyrosine residues of the proteins were suggested to be responsible for the Ag+ ion reduction (Jianping Xie, 2007). Carrying out the reduction process by a simple bifunctional tripeptide Asp-Asp-Tyr-OMe further identied the involvement of these residues. This synthesis process gave small Ag nanoplates with low polydispersity in good yield (>55%) (Gole, 2001). Balaji, 2009 reported FTIR spectroscopic studies on silver nanoparticles obtained from the fungus, Cladosporium cladosporioides. Their study conrmed that the carbonyl groups from the amino acid residues and peptides of proteins have strong ability to bind silver. The proteins could possibly forma coat covering the metal nanoparticles to prevent their agglomeration and aid in its stabilization in the medium. Hence, the biological molecules could possibly function in the formation and stabilization of the silver nanoparticles in aqueous medium. Gole, 2001 reported that proteins can bind to silver nanoparticles either through free amine groups in the proteins and possibly play a role in stabilization of the silver nanoparticles by surface-bound proteins. Moreover, Vigneshwaran, 2006 explained the synthesis of metal nanoparticles by using fungal mycelium, in the a rotary shaker, metal ions in solution were adsorbed on the surface of the mycelia through interactions with chemical functional groups such as carboxylate anion, carboxyl and peptide bond of proteins, and hydroxyl of saccharides (Lin, 2005) found on the mycelia. The mycelia, matted together, was more immobile, and more capable of binding Me+ than that of the external cellular substances that distributed in the inter-mycelial space, then most of the Me+ was *in situ* reduced to Me0 by reducing sugars from the saccharides (Gole, 2001) on the mycelia. In the mean time, stronger adsorptive groups such as the carbonyl group on the extracellular substances could further adsorb the particles located on the surface of the mycelia, resulting in capping these nanoparticles, while rocking. When other Me+ in the solution was rocked on to this overlay and was bound and reduced to Me0 on the surface of the layer and these Me0 might be possibly further coated with the other extra cellular substances; this process was repeated continuously until these substances distributing in the inter-mycelial space was used up.

Plant extracts from live alfalfa, the broths of lemon grass, geranium leaves and others have served as green reactants in silver nanoparticle synthesis (Shankar, 2004; Gardea-Torresdey,

Silver Nanoparticles: Real Antibacterial Bullets 413

Fig. 3. Various mechanisms of bacterial resistance against antibacterials (G. Thirumurugan, 2011). Therefore, an alternative way to overcome the antibiotic and drug resistance of various micro organisms is needed desperately, especially in medical devices, pharmaceutical etc. The nano size allowed expansion of the contact surface of silver with the microorganisms, and this nano scale has applicability for medical devices and pharmaceutical by surface coating agents. Kim et al., 2007 studied antibacterial mechanism of silver nanoparticles for certain microbial species. The peptidoglycan layer is a specific membrane feature of bacterial species and not mammalian cells. Therefore, if the antibacterial effect of silver nanoparticles is associated with the peptidoglycan layer, it will be easier and more specific to use silver nanoparticles as an antibacterial agent. Sondi and Salopek-Sondi, 2007 reported that the antibacterial activity of silver nanoparticles on Gram-negative bacteria was dependent on the concentration of Ag nanoparticle, and was closely associated with the formation of 'pits' in the cell wall of bacteria. Then, Ag nanoparticles accumulated in the bacterial membrane caused the permeability, resulting in cell death and they reported degradation of the membrane structure of micro organism with silver nanoparticles. Kim et al., 2007 suggested that the antimicrobial mechanism of Ag nanoparticles is related to the formation of free radicals and subsequent free radical–induced membrane damage. The free radicals may be derived from the surface of silver nanoparticles and be responsible for the antibacterial activity. In proteomic and biochemical studies, nano molar concentrations of AgNPs have killed E.coli cells within minutes possibly due to immediate dissipation of the proton motive force (Lok, 2006). This action is similar to that found for antibacterial activities of Ag+ ions (Dibrov, 2002). For example, low concentrations of Ag+ ion result in massive proton leakage through the Vibrio cholerae membrane (Dibrov, 2002). This proton leak might be happening from either any Ag+ -modied membrane protein or any Ag+-modied phospholipids

2002). Using plants for nanoparticle synthesis can be advantageous over other biological processes because it eliminates the elaborate process of maintaining cell cultures and can also be suitably scaled up for large-scale nanoparticle synthesis (Shenton, 1999)
