## 3 Results
### In Vivo MRI of Tumor-Bearing Mice through Intratumoral Injections  
To evaluate the applicability of LHRH-MNPs for breast cancer imaging, T2-weighted MRI scans were performed in a MDA-MB-231 xenograft tumor model. This TNBC tumor model was established in-house by the subcutaneous injection of breast cancer cells into back flanks of non-tumor-bearing nude mice, and the tumor volume was monitored by MRI on a weekly basis for durations up to 28 d (Figure 7). All of the mice tolerated the procedure well without any noticeable complications.  
Figure 5: In vivo T1-weighted MRI scans of LHRH-MNPs into non-tumor-bearing nude mice (no tumors), showing darkening effect. (**A**) Image slice depth highlighting liver (I), heart (h) and spleen (s). (**B**) Image slice depth highlighting kidney (k).  
Figure 6: In vivo T2-weighted MRI scans of LHRH-MNPs injected into non-tumor-bearing nude mice (no tumors), showing a darkening effect. (**A**) Image slice depth highlighting the liver (I). (**B**) Image slice depth highlighting the kidney (k), liver (I), spleen (s) and major blood vessels (v).  
Figure 8 shows T2-weighted MR scans obtained from mice with subcutaneous xenograft tumors at 4 weeks post induction. Each tumor was injected directly with either MNPs or LHRH-MNPs at a dose of 20 mg of Fe\({}_{3}\)O\({}_{4}\) per kg mouse body weight. Before intratumoral injections, the positions of the xenograft tumors were visible, as indicated by yellow dashed ovals (Figure 8). The immediate post-contrast MRI scans showed significant signal voids in the tumors (indicated by the yellow arrows in Figure 8), owing to the presence of injected nanoparticles. MRI scans were used to monitor the mice at 2 h, 24 h and 2 weeks post intratumoral injection (Figure 8). For MNPs, the contrast enhancement was present immediately after nanoparticle injection, and then the signal started to recover after 24 h (Figure 8A,C). In contrast to MNPs, the tumors injected with LHRH-MNPs maintained a negative signal for durations up to two weeks (Figure 8B,D).  
To quantify the contrast level, the tumors were identified as the region of interest (ROI), and signal-to-noise ratio (SNR) and relative contrast (%) were calculated from the MRI scans using Equations (1)-(3) presented in Section 2.6. Specifically, the signal intensity (SI) of the tumor at each scanning time point after injection of MNPs or LHRH-MNPs injections were compared to the SI value of the tumor before nanoparticle administration. The injection of MNPs led to tumor enhancement immediately after injection with a relative contrast of \(32\pm 6\%\) and \(27\pm 10\%\) at 2 h (\(p>0.05\)). Whereas after 24 h, the contrast level was reduced to half (\(14\pm 9\%\)), which was maintained for two weeks (\(15\pm 10\%\)) (\(p>0.05\)). Compared to injection of MNPs, injection of LHRH-MNPs resulted in overall higher relative contrast that was preserved at \(\sim\)60% (59 \(\pm\) 22%, 58 \(\pm\) 21%, 59 \(\pm\) 23%, and 54 \(\pm\) 25%, for immediate, 2 h, 24 h and 2 weeks post injection) through a period of 2 weeks. No significant difference was observed between these values (\(p>0.05\)), suggesting that long-term detectability of LHRH-MNPs could be achieved in vivo under T2-weighted MR imaging for at least 14 d after intratumoral injection.  
Figure 7: Tumor volume growth profile up to 28 d post tumor implantation as measured by MRI.