titanium calcium phosphate films. Here, it was found that the surface with high roughness promoted the retention E. coli bacteria onto the surface (Sukhorukova et al., 2015). Longer retention time enhanced antibacterial property of the films with higher roughness.
1.10.5 Synthesis Methods and Stabilizing Agents
The choice of synthesis methods and stabilizing agents is very crucial in the fabrication of antimicrobial NMs, since these factors also can influence the properties of the NMs to a major extent. As stated, during synthesis, NMs are synthesized by different methods such as laser ablation, mechanical milling, chemical etching, melt mixing, sputtering, and other chemical methods such as thermolysis, microemulsion, and sol–gel. However, the NMs that are synthesized through the chemical or physical methods are unstable, have surface‐attached toxic materials, and are formed along with toxic by‐products. Considering Ag NPs' synthesis, the process involves a reducing agent such as sodium borohydride or sodium citrate with capping agent such as polyethyl glycol. On the other hand, the biological synthesis methods employ biological sources such as microorganisms and plants. In the case of microbial biosynthesis, the microbes exert a bio‐reduction process to reduce and accumulate the metallic ions to avoid the metal‐related toxicity. The mechanism involves the reduction of metal ions inside the cell through intracellular reducing species and outside using their different extracellular metabolites. Plants also contain a number of reducing agents such as proteins, flavonoids, and other water‐soluble biomolecules (Singh et al., 2018). Green synthesis methods improve the stability of NMs with no hazardous by‐products. Further, they provide a biocompatible coating over NMs, which not only improves the biocompatibility but also increases surface area with reactive groups, which can improve the interaction with biological environment (Singh, Garg, Pandit, Mokkapati, & Mijakovic, 2018). For example, Sudhasree et al. (2014) showed that nickel NPs prepared from Desmodium gangeticum were monodispersed. The green synthesized NPs were found to possess high antibacterial activity against Klebsiella pneumonia, Pseudomonas aeruginosa, and Proteus vulgaris whereas the chemically synthesized nickel NPs had the least effect on the same microbes. Apart from enhancing the antimicrobial property, it also improved the biocompatibility as observed from biocompatibility studies using LLC PK1 (epithelial cell lines) (Sudhasree et al., 2014). However, the choice of a particular synthesis method depends on the nature of NM required and the type of applications.
Antimicrobial property is directly proportional to the surface area, surface charge, and the extent of interaction or contact of NMs with bacterial cell. In this regard, understanding the effect of stabilizing agents on NMs is also important. The stabilizing agents reduce agglomeration and provide net charge over NMs. Hence, these stabilizing agents to an extent determine the toxicity or antimicrobial property of the NMs. It was observed from a past study that the Ag NPs stabilized with chitosan and citrate enhanced the antimicrobial property of Ag NPs against multidrug‐resistant bacteria (S. aureus and K. pneumonia). The study also showed that effect of stabilizing agents chitosan and citrate over the microbes was at basal level (Cavassin et al., 2015). Similarly, Ag NPs coated with 11‐mercaptoundecanoic acid exhibited higher toxicity over P. aeruginosa in comparison to Ag NPs coated with citrate. Citrate capping provides net negative charge to the Ag NPs, which results in electrostatic repulsion between negatively charged bacterial cell and Ag NPs. In the case of 11‐mercaptoundecanoic acid capped Ag NPS, the particles agglomerated over the surface of the hydrophilic P. aeruginosa, which facilitated the release of Ag+ ions near the proximity of cell and improved the toxicity of the NPs toward the bacteria (Dorobantu et al., 2015). In another study, El Badawy et al. (2010) studied the toxicity of Ag NPs coated with different capping agents such as citrate, polyvinylpyrrolidone, and branched polyethyleneimine. The coating provided Ag NPs with a range of surface charge from highly negative to highly positive. Among the different coatings, citrate coating showed the least toxicity against Bacillus species. The surface potential of the citrate capped Ag NPs was found to be −38 mV, which was in line with the surface charge of Bacillus species (−37 mV). The electrostatic repulsion between the negatively charged citrate capped Ag NPs and bacteria was the probable reason for the least toxic effect of citrate capped NPs. In accordance with that, highly positively charged branched polyethyleneimine‐coated Ag NPs (+40 mV) showed the highest toxicity whereas uncoated Ag NPs (−22 mV) and polyvinylpyrrolidone‐coated Ag NPs (−10 mV) exerted toxicity above the citrate capped Ag NPs (El Badawy et al., 2010). This clearly reveals that the nature and the structure of stabilizing agents affect the toxicity and the bactericidal potential of the NMs, which should be taken into account while fabricating NMs for antimicrobial properties.
1.10.6 Environmental Conditions
Various environmental factors have shown influence over the antimicrobial property of NMs. Among different environmental conditions, temperature causes significant effect on the antimicrobial property because of its influence on ROS production rate. The stimulation of ZnO NMs with temperature resulted in the generation of electrons at the active site. Later the electrons interact with oxygen to generate ROS, which enhances the antimicrobial efficiency of ZnO NMs (Saliani, Jalal, & Goharshadi, 2015). Next to temperature, pH is another very common factor that has a strong influence over the antimicrobial efficacy of NMs. In the case of ZnO NMs, with decrease in pH the dissolution rate of the NMs increases, thereby increasing its antimicrobial properties. Adding to pH, the osmotic pressure of the medium also influences the behavior of NMs, such as the aggregation, surface charge, and solubility of NMs (Saliani et al., 2015). A study of ZnO in five different mediums suggested that the antimicrobial activity of NMs is mainly caused by the Zn2+ ions and the complexes of zinc. Additionally, by supplying the nutrients and other substances, the medium improves the tolerance of bacteria to the NMs (Li, Zhu, & Lin, 2011). Another study showed that stirring conditions of ZnO NPs during the synthesis phase has influence over their antimicrobial property against Gram‐positive (Bacillus subtilis) bacteria, Gram‐negative bacteria (E. coli), and fungus (Candida albicans) (Khan et al., 2016). Schematic of the key factors that contribute to the antimicrobial property of NMs is given in Figure 1.6 as described by Jagadeeshan and Parsanathan (2019).
Figure 1.6 Schematic of the key factors that contribute to the antimicrobial property of NMs.
1.11 Influence of Size on the Antibacterial Activity and Mechanism of Action of Nanomaterials
It is understood from the definition and previous discussions of NMs that size is the predominant factor of any NM, which lies between the atomic and bulk zone of the same composition. Along with size, ion composition and active biomolecules or functional group on the surface also contribute to the interaction. As have discussed, the properties of the material at the nanoscale differ significantly from the bulk material, which affects its interaction with the biological system. With the development of NMs, several new opportunities have emerged due to their reduced size with increased number of particles contributing to the high surface area‐to‐volume ratio. The reduced size of the NMs may promote the interaction of bacterial cells with the surface of material and cause membrane damage with subsequent bacterial cell death. Similarly, in the case of in vivo; applications, the size of the materials plays a crucial role in the kinetics of adsorption, distribution, metabolism, and excretion of the NMs. In order to study the effect of size on the antibacterial activity and toxicity of the material, several studies have been conducted.
In one of the studies, Raghupathi, Koodali, and Manna (2011) showed that the antibacterial activity of the ZnO NPs varied significantly with the particle size. The authors studied the antibacterial activity of NPs' size ranging from 12 to 307 nm against S. aureus. They found that the particles with size more than 100 nm at a concentration
