# Doped Zinc Oxide Nanoparticles: Synthesis, Characterization and Potential Use in Nanomedicine
## 4 Use of Doped ZnO NPs in the Biomedical Field
### Antimicrobial Agents  
Pure ZnO nanoparticles have been extensively used as antibacterial agents due to their photo-oxidizing and photocatalytic properties [216]. Its antimicrobial properties can be tailored in several ways: parameters like morphology, size, concentration, surface defects and functionalization all contribute to change the performances of ZnO nanoparticles in this field [216]. Despite the mechanisms through which ZnO induces bacterial and fungi death being still under debate, there are three main hypotheses, summarized in Figure 9.  
The first one deals with ROS generation. As a matter of fact, ZnO is able to generate reactive oxygen species as a consequence of its photoexcitation from UV light in aqueous media [220]. The idea is that the electromagnetic radiation gives electrons lying in the valence band (VB) sufficient energy to jump to the conduction band (CB). The hole left in the VB can react with water, generating a hydroxyl  
Figure 8: Fluorescence microscopy images of different cell lines exposed to differently Fe-doped ZnO nanoparticles (no nanoparticles (NPs) are present in the control samples). The green signal is related to Zn\({}^{2+}\) free ions (FluoZin3-AM binds to ions and have a green fluorescent emission), the blue signal is related to the presence of cell nuclei. Less doped ZnO nanoparticles lead to a higher concentration of free zinc ions. Adapted from ref. [204]. radical (\({}^{\bullet}\)OH) and H\({}^{+}\), while the CB electron can react with oxygen to generate a peroxide radical [231]. At this point, the following reactions can occur:  
\[{}^{\bullet}\mathrm{O}_{2}+\mathrm{H}^{+}\to\mathrm{HO}_{2}{}^{\bullet}, \tag{4}\]  
\[\mathrm{HO}_{2}{}^{\bullet}+\mathrm{H}^{+}+\mathrm{e}^{-}\to\mathrm{H}_{2} \mathrm{O}_{2}, \tag{5}\]  
This leads to the formation of hydrogen peroxide, which can destroy bacteria by penetrating their membrane. If the generated ROS exceeds the capability of the bacterium to produce reducing agents able to contrast this phenomenon, oxidative stress phenomena are induced [232]. The second mechanism is based on the release of harmful zinc cations. Indeed, ZnO nanoparticles can dissolve releasing Zn\({}^{2+}\) ions into the environment, affecting the metabolism of bacteria and leading to dysfunctions of their enzymatic systems [216]. A work from [PERSON] et al. reported evidences of these effects [219]. ZnO nanoparticles, bulk ZnO and a solution of Zn\({}^{2+}\) ions were analyzed against _E. coli_, finding similar toxicity curves and confirming the negative influence that zinc cations may have on bacterial viability. However, this work further confirmed that ZnO toxicity is subjected to several intrinsic conditions like the size, porosity, and concentration of the particles, coupled with external parameters like pH and the composition of the environment [216]. Finally, another possible mechanism that explains the antibacterial action of ZnO is the absorption of the nanoparticles into the bacteria membrane, leading to a physical disruption of the cell wall and ultimately to the microorganism's death [233]. This is possible because ZnO nanoparticles typically present a positive Z-potential, i.e., a positively charged surface, while the bacteria surface is characterized by a negative charge. Therefore, ZnO NPs are attracted toward the bacterium, damaging its outer membrane. Moreover, if the particles are smaller than 10 nm, NPs' internalization into the bacterium can also occur, damaging the internal components of the cell [232]. Additionally, it is worth mentioning that a different behavior can be found for different kinds of microorganisms. Actually, among bacteria there exist two main typologies, i.e., Gram-positive and Gram-negative ones, which show a different external cell membrane structure [216]. Therefore, it is clear that tests aimed at proving the antimicrobial mechanism of ZnO nanostructures should be performed on both these biological systems: Gram-negative bacteria such as _Escherichia coli_ (_E.