## 3 Results and Discussion
### MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolzolium Bromide] Assay  
The MTT assay was conducted in order to evaluate the toxicity of both bare and functionalized AuNPs against the 4T1 cell line that is used as a model of TNBC (Triple Negative Breast Cancer) [24]. The 4T1 tumor resembles human stage IV breast cancer, with metastatic behavior [25]. Therefore, this cell line provides an interesting tumor model with spontaneous metastases to various sites (e.g., lung, bone, etc.) which can be very useful when evaluating the therapeutic potential of radiolabeled nanoparticles with alpha- or beta-emitting isotopes [26].  
The samples were treated with different concentrations of AuNPs for 24 h. The results of the MTT assay are summarized in Figure 2 and indicate that bare AuNPs revealed very low toxicity at the highest examined concentration, with cell viability reducing gradually from 92.41 \(\pm\) 4.75% at the lowest concentration (0.5 \(\upmu\)g/mL) to 79.71 \(\pm\) 2.68% at the highest concentration (50 \(\upmu\)g/mL). In the case of the nanoparticles functionalized with the thiol ligand, cytotoxicity studies also indicated low toxicity at the highest examined concentration, with cell viability reducing from 100.64 \(\pm\) 2.37% at the lowest concentration (50 \(\times\) 10\({}^{-2}\)\(\upmu\)g/mL) to 80.06 \(\pm\) 5.87% at the highest concentration (50 \(\upmu\)g/mL). It is also worth mentioning that the cell viability after incubation with the ligand was \(\sim\)70% at all tested concentrations, indicating that the presence of AuNPs did not have an important impact on the biological behavior of the ligands on the cells [18].  
Functionalization and Radiolabeling of AuNPs with [99 mTc][Tc(H\({}_{2}\)O)\({}_{3}\)(CO)\({}_{3}\)]\({}^{+}\)  
The functionalization of AuNPs with the ligand and the following radiolabeling procedures are described above. The main purpose of this functionalization was to provide an effective chelator for the successful and robust radiolabeling of AuNPs with \({}^{99m}\)Tc carbonyls. After functionalization, strong Au-S bonds were formed between the AuNPs and  
\begin{table}
\begin{tabular}{c c c} \hline \hline \begin{tabular}{c} **Concentration of** \\ **Functionalized AuNPs** \\ \end{tabular} & **Hydrodynamic Diameter (nm)** & **Zeta Potential (mV)** \\ \hline \hline \begin{tabular}{c} 312.5 \(\upmu\)g/mL \\ \end{tabular} & 198.0 & +18.0 \(\pm\) 0.4 \\ \hline \begin{tabular}{c} 312.5 \(\times\) 10\({}^{-1}\)\(\upmu\)g/mL \\ \end{tabular} & 168.8 & \(-\)34.0 \(\pm\) 0.6 \\ \hline \
\begin{tabular}{c} 312.5 \(\times\) 10\({}^{-2}\)\(\upmu\)g/mL \\ \end{tabular} & 140.9 & \(-\)17.3 \(\pm\) 0.4 \\ \hline
\begin{tabular}{c} 312.5 \(\times\) 10\({}^{-3}\)\(\upmu\)g/mL \\ \end{tabular} & 112.8 & \(-\)11.0 \(\pm\) 0.5 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Hydrodynamic diameters of functionalized AuNPs.  
Figure 2: MTT assay of 4T1 cells treated with different concentrations of bare and ligand-functionalized AuNPs.  
the thiol group of the ligand. For radiolabeling, functionalized AuNPs of all concentrations were incubated at 70 \({}^{\circ}\)C for 2 h with 100 \(\upmu\)L of [99 mTc]Tc][Tc(H\({}_{2}\)O)\({}_{3}\)(CO)\({}_{3}\)]\({}^{+}\). The precursor [99 mTc]Tc][Tc(H\({}_{2}\)O)\({}_{3}\)(CO)\({}_{3}\)]\({}^{+}\) carries three labile water molecules which are easily substituted by the three nitrogen atoms of the tridentate thiol ligand to afford the desired 99 mTc. The radiolabeling yield was approximately 85%. After purification by centrifugation, radiochemical purity for all samples was >95%, and it remained stable for at least 24 h post-purification, as determined by HPLC analysis (Table 2). Furthermore, the formed radiolabeled nanoparticles present a lipophilic nature, as was demonstrated by our previous work, due to the lipophilic character of the ligand used in the present study.