## 3 Results and Discussion
### Biodistribution Studies  
Ex vivo biodistribution studies were carried out in CFW mice after intravenous injection for the determination of the in vivo kinetics of the radiolabeled AuNPs at four different concentrations (100 \(\mu\)L per injection, leading to final administered doses of 31.25, 31.25 \(\times\) 10\({}^{-1}\), 31.25 \(\times\) 10\({}^{-2}\) and 31.25 \(\times\) 10\({}^{-3}\)\(\mu\)g per mouse). The results for uptake in the RES organs (lung, liver and spleen) are summarized in Figure 3. Furthermore, the full sets of biodistribution results are provided in Tables 6-8. As shown in Figure 3, as well as in Tables 6-8, the nanoparticles for the three lowest concentrations were accumulated mostly in three organs, liver, spleen and lungs, due to the phenomenon of opsonization which leads to high uptake of the nanoparticles by phagocytic-rich organs (liver, spleen and lungs) [27]. This high accumulation is also normal for nanoparticles of that size since larger nanoparticles are more likely to attach to blood vessels' walls in the case of flow than smaller ones [28]. On the other hand, it has been observed that the most concentrated sample was almost exclusively distributed in lungs. This can be explained due to the fact that cationic lipophilic compounds accumulate extensively in lung tissues due to trapping mitochondria and lysosomes (as presented in  
Figure 3: Ex vivo biodistribution results of [99 mTc]Tc-AuNPs for the main organs of interest: (**A**) lung, (**B**) liver and (**C**) spleen. Mean values (\(n=3\)) and SD (bars) are shown (\(x\) axis not in scale). Asterisks indicate the statistical significance of the differences between the results (* \(p<0.05\), **\(p<0.01\), *** \(p<0.001\) and *** \(p<0.0001\)). The absence of an asterisk denotes a non-significant statistical difference. Table 1, the most concentrated sample presents a positive zeta potential, while the other samples present negative zeta potentials). On the contrary, this distribution is minor for anionic and neutral compounds [29]. The positive charge provokes rapid adsorption of plasma proteins after intravenous administration, leading to particle aggregation. These aggregates are trapped in small capillaries, especially in lungs, or taken up by phagocytosis by macrophages [27]. It is very important to highlight that the nanoparticles, once entering the bloodstream, are covered by the so-called protein corona which could alter their physicochemical characteristics. As a result, a nanoparticle's unique properties could be different during the biodistribution experiment and can therefore lead to diverse biological behavior. The adsorption of the corona protein by the nanoparticle surface depends on its size, charge, shape and lipophilicity, as well as the environment it has moved through. More specifically, regarding their size, it has been demonstrated that the smaller gold nanoparticles tend to form larger protein coronas when compared to larger gold nanoparticles [30]. Furthermore, several studies reveal that increasing the surface charge of the nanoparticle leads to higher protein adsorption. The serum proteins are mostly negatively charged and tend to adsorb on positively charged nanoparticles. The formation of protein corona causes changes in the hydrodynamic diameter and zeta potential of the nanoparticle [31], which in turn affects in vivo biodistribution.