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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 of 6 mM were merely acting as bacteriostatic whereas particles of size 12 nm at the same concentration not only limited the growth of the bacteria but also killed them completely. Here, the mechanism of action involved ROS production and the accumulation of nano‐sized particles in the cytoplasm of S. aureus (Raghupathi et al., 2011).

      In another study, size‐dependent antimicrobial activity of cobalt ferrite core/shell NPs was demonstrated. Antimicrobial property of three different sizes of NPs (1.65, 5, and 15 nm) was studied against Saccharomyces cerevisiae and Candida parapsilosis. Notably, against S. cerevisiae 1.65 nm exhibited 12 and 25% higher killing than 5 and 15 nm particles, respectively. Similarly, in the case of C. parapsilosis, the same trend was reported whereas 1.6 nm particles showed 15 and 44% higher killing efficiency in comparison to 5 and 15 nm particles, respectively. The antimicrobial activity of the cobalt ferrite NPs at size of 7–8 nm was suggested to be due to intracellular diffusion with subsequent interaction with cell membrane causing oxidative stress and finally DNA damage. It was also suggested that with decrease in size, the cobalt content of the shell might have increased, which in turn improved the interaction or binding efficiency of particle with bacterial cell (Žalnėravičius et al., 2016).

      Morones et al. (2005) investigated the effect of size of silver NPs in the range of 1–100 nm against four different Gram‐negative bacteria (E. coli, V. cholera, P. aeruginosa, and Scrub typhus). High‐angle annular dark‐field (HAADF) scanning transmission electron microscopy (STEM) technique showed that silver NP in range of 1–10 nm was able to attach to bacterial cell membrane, which altered its permeability and respiration. Further the NPs that have penetrated caused intracellular damage by interacting with sulfur and phosphorous‐containing substances such as proteins and DNA. Through this study the author confirmed that the size of silver NPs does play a crucial role in the antibacterial effect (Morones et al., 2005).

Later, Adams et al. (2014) demonstrated a size‐dependent antimicrobial activity of the palladium NPs with a small difference in the particle size (<1 nm). Three different self‐contained sub‐10 nm particles (2.0 ± 0.1, 2.5 ± 0.2, and 3.0 ± 0.2 nm) were tested against Gram‐negative and Gram‐positive bacteria (i.e. E. coli and S. aureus). In the case of E. coli it was observed that smallest particles had higher bacterial killing effect followed by medium‐sized and larger particles. Interestingly, in the case of S. aureus, middle‐sized particles exhibited higher effect than the smallest and larger particles. This study clearly suggested that even a small difference in size (i.e. <1 nm) affects the antimicrobial property of the NPs, which also depends on the strains used (Adams et al., 2014).

      In a similar study, the effect of size was explained in terms of change in diameter of carbon nanotubes: a well‐known antibacterial material. In this study, the antibacterial activity of single‐walled (SWCNTs) and multi‐walled carbon nanotubes (MWCNTs) with outer diameter of about 0.9 and 30 nm respectively was considered for assessing the effect of size. Scanning electron microscopy studies showed that E. coli cells attached to SWCNTs exhibited higher degree of cellular damage than those attached to MWCNTs. It was also observed that the E. coli cells treated with SWCNTs got inactivated (80 ± 10%) at a higher percentage than those treated with MWCNTs (24 ± 4%). Similarly, a metabolic activity study also suggested that the cells attached to SWCNTs had lesser metabolic activity than the cells with MWCNTs. Further, the measurement of cytoplasmic content efflux and gene expression of stress and DNA‐related products of CNTs‐treated bacterial

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