## 4 Discussion  
This study shows that MNPs and LHRH-MNPs can be used in imaging human TNBC in xenograft tumors as negative (T2) MRI contrast agents. Specifically, in T2-weighted MR imaging, the signal intensity in each voxel is given by  
\[\text{SI}=\text{S}_{0}\text{e}^{-\text{TE/T2}} \tag{4}\]  
where S\({}_{0}\) is proportional to both the spin density and T1-weighting factor in the voxel, TE is the echo time, and T2 is the transverse relaxation time of a given tissue [35]. Hence, when the gray scale is darker, the SI is smaller and the relaxation time T2 is shorter. With the presence of nanoparticles in tumor, an additional magnetic field (caused by the nanoparticles) is introduced and induces local field inhomogeneity that increases the speed of proton transverse relaxation and shortens the T2 relaxation time, resulting in a hyperintense change in MRI signals. The negative agents or T2 agents are so-called since they cause darkening of images on their sites. Our results showed that the targeted LHRH-MNPs accumulated in various vital organs in the non-tumor bearing mice, including the liver, spleen, kidney and spleen (Figures 5 and 6). The remarkable darkening of liver and spleen is most likely because both are macrophage-rich organs, which take up nanoparticles and finally remove them by common elimination pathways in the reticuloendothelial system [36]. For instance, Kupffer cells in the liver [37; 38] can uptake LHRH-MNPs, which lead to a faster T2 relaxation in liver and the resultant darkening effect. The negative signals in heart and kidney are probably due to the presence of LHRH-MNPs in the blood pool [39]. Non-targeted MNPs caused negative signals in tumors by T2-weighted MRI imaging immediately following intratumoral and intravenous injections. This may be attributed to the enhanced permeability and retention (EPR) effect in solid tumors as a passive uptake pathway for nanoparticle accumulation to facilitate the specific MR imaging of tumor sites [40]. In addition, MNPs have positively-charged surfaces, as revealed by zeta potentials, and thus they are able to adhere more easily to the negatively-charged surfaces of cell membranes by electrostatic interactions. Positively-charged nanoparticles are generally taken up to a greater degree under faster cell phagocytosis rates, compared  
Figure 10: Immunohistochemistryistochemistry staining of excised breast tumor tissue 4-weeks post tumor induction, showing expressed LHRH receptors (green). Cell nuclei were labeled by DAPI (blue). to their neutral or negatively-charged counterparts [41]. It appears that the tumor T2 signal intensity of MNP-treated animals started to revert to the pre-injection intensity after 24 h, for both intratumoral and intravenous injections (Figures 8 and 9). It is likely that MNPs undergo gradual metabolic processes, thereby reducing the amount and distribution of MNPs in the tumors. Compared to MNPs, targeted LHRH-MNPs exhibited excellent lesion-detecting ability and longer imaging windows in T2-weighted MR imaging for TNBC (Figures 8 and 9). In intratumoral injections, the negative contrast in the tumor remained constant for 14 d, whereas the relative contrast started to recover in the MNP-treated group at 24 h, following injection. In intravenously injected mice, infused LHRH-MNPs caused significant signal attenuation, with an increase of over 2.5-fold at 24 h compared to 2 h, indicating continuous darkening of the tumor. Specifically, after 2 h, the edge of the tumor began to show enhancement, and the intensification became more obvious at 24 h, where the majority of the tumor were darkened compared to the pre-injection state (Figure 9). Such enhancement was not significant in MNP treated animals. These results suggest the specificity of LHRH-MNPs in the targeted imaging of TNBCs.