# Doped Zinc Oxide Nanoparticles: Synthesis, Characterization and Potential Use in Nanomedicine
## 4 Use of Doped ZnO NPs in the Biomedical Field
### Biological Behavior  
One of the main problems related to the use of ZnO in biomedical applications is its dissolution in aqueous/biological media [223]. ZnO is in fact stable at a basic pH, while it can easily dissolve in an acid environment [224]. The pH of a biological medium depends on several parameters and can change over time. Therefore, it is possible to have a certain degree of ZnO dissolution, which can be even enhanced when the dimensions of the nanoparticles under analysis are highly reduced, exposing a large area to the liquid environment [224]. The dissolution leads to the release of zinc cations (Zn\({}^{2+}\)) which have been proven to be harmful for cells, damaging the cellular zinc homeostasis and, consequently, leading to lysosome and mitochondria damage and cell death [223]. Hence, particular attention should be paid to the control of ZnO dissolution, in order to limit cytotoxicity. For the chemical design of ZnO-based systems with increased zinc cation stabilities in media, specifically used in medical applications, one successful approach is represented by the methodology based on electrochemical equilibria in aqueous solutions [225]. An alternative approach to improve the biocompatibility and zinc cation stability of ZnO nanoparticles is to cover their surface with inorganic [226; 227; 228] or organic [140; 229; 230] coating materials. However, this approach requires additional synthesis steps and a general increase of the nanoparticles' size, which may represent a limitation for specific applications. The best situation would be to prepare ZnO nanoparticles that are intrinsically stable in a biological medium, without the addition of any external layer. This task can be addressed once again by including a dopant atom in the crystal lattice of ZnO. Actually, Fe doping has been proposed as a stabilizer of ZnO in aqueous media. [PERSON] et al. [128] demonstrated that the inclusion of Fe ions into ZnO nanoparticles successfully decreased the dissolution rate by measuring the amount of zinc ions released in an electrolytic solution at pH 7 \(\pm\) 0.04. The Zn\({}^{2+}\) kinetics were evaluated by measuring the nitric acid amount required to maintain the solution pH at the fixed value. The results showed that, by increasing the amount of doping, the dissolution of the nanoparticles decreased accordingly (Figure 7). This behavior was attributed to the stronger chemical bonds between the iron dopant and the host ZnO lattice, which resulted in a higher difficulty for the Zn\({}^{2+}\) ions to be released in the environment. First principle calculations confirmed these experimental evidences [108]. The computed energetics evaluated for different iron doping levels showed that iron can stabilize the ZnO nanoparticles. Moreover, in the same work, it was also proven that the coordination state of iron was mainly Fe\({}^{2+}\) and that the inclusion of the dopant did not damage the crystal lattice in a considerable way. In another work, Fe-doped ZnO nanoparticles, synthesized by flame spray pyrolysis, were tested in terms of cytotoxicity [204]. The authors analyzed different Fe doping levels to find the best one, which allowed the selective killing of tumoral cells. The cytotoxicity assays were performed on normal murine mesenchymal stem cells (MSCs), human bronchial epithelial cells (Beas-2B), cancer murine lung squamous carcinoma cells (KLN-205) and human cervical cancer cells (HeLa) cells. The experimental results revealed that NPs with low doping levels were more efficient at damaging tumoral cells rather than the healthy cells. Furthermore, the amount of Zn\({}^{2+}\) released, measured by FluoZin-3 staining coupled with fluorescence microscopy (Figure 8), was reduced by increasing the Fe doping. In detail, 2 wt.% Fe doping was considered the best choice to achieve selectivity between tumoral and normal cells. This finding was further confirmed by co-culture cells experiments, finding an increased toxicity against cancer cells with respect to healthy ones.