## 3 Results and Discussion  
In a typical iodination reaction involving the radiolabeling of proteins or antibodies, the benzene ring of tyrosine is labeled at the ortho position relative to the hydroxyl group [45]. Given the average number of hydroxyl groups per f-Gd\({}_{3}\)N@C\({}_{80}\) (-26) following the functionalization procedure [34], it is likely that the \({}^{124}\)I attaches directly to the fullerene cage but the position of attachment is unclear. A possible iodination position is illustrated in the reaction schematic (Figure 3); however further studies involving iodination with non-radioactive NaI at higher I to f-Gd\({}_{3}\)N@C\({}_{80}\) ratios will be required to fully elucidate the iodination position. In the radiolabeled product prepared in this study, the I to f-Gd\({}_{3}\)N@C\({}_{80}\) ratio was low due to the high specific activity of \({}^{124}\)I used, and the low radioactivity (or number of radioactive molecules) required for PET detection relative to the concentration of f-Gd\({}_{3}\)N@C\({}_{80}\) required for MRI. Therefore, at these low ratios, I labeling would not be expected to significantly alter the physical and chemical characteristics of the f-Gd\({}_{3}\)N@C\({}_{80}\). Indeed, Figure 4 (A) shows no detectable differences in the UV-vis absorption spectra; likewise, MRI relaxivity was unchanged following iodination. Following the iodination reaction, the radiolabeled product (\({}^{124}\)I-f-Gd\({}_{3}\)N@C\({}_{80}\)) was purified by gel chromatography during which 0.5 mL fractions were collected and radioactivity measured using a dose calibrator. UV absorbance measurements were also collected for each fraction at 263 nm to monitor the elution of the f-Gd\({}_{3}\)N@C\({}_{80}\). Radioactivity and UV absorption elution profiles are presented in Figure 4 (B). In order to determine the labeling efficiency, the total activity of the 3 fractions containing the \({}^{124}\)I-f-Gd\({}_{3}\)N@C\({}_{80}\) was divided by the activity of \({}^{124}\)I  
literature [34, 35, 36, 37, 41, 43, 44], it was possible to obtain the radiochemical purity by HPLC separation. This was due to the high purity of the f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\) starting material which would only yield three possible products following the iodination reaction, namely f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\), \({}^{124}\)I-f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\), and free \({}^{124}\)I. Using size exclusion separation methods, the radiolabeled and non-labeled f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\) will have similar retention, leaving the free \({}^{124}\)I to be the only separable species. As seen in Figure 5, the peak in the UV trace at 14.5 min (red line--right axis), due to UV absorbance by the f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\), was accompanied by a corresponding peak in the radioactive trace at 14.9 min (black line--left axis) which indicated the formation of the labeled \({}^{124}\)I-f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\) product. The 0.4 min offset in retention time is due to the placement of the radioactive detector in series after the UV detector in our HPLC setup and is a factor of tube length and flow rate. The radioactive peak in the iodination product at 19.5 min is free \({}^{124}\)I. This is corroborated by the 19.5 min retention time of \({}^{124}\)I alone in solution (black dashed line--left axis). Integration of the peak areas of the iodination product yields 95% radiochemical purity. Rats bearing orthotopic T9 brain tumors were imaged day 10 post tumor implantation with commercial gadodiamide in order to determine the proper depth of cannula placement for infusion (images from two rats: Figure 6(A,D)). On day 12, 24 \(\mu\)L of \({}^{124}\)I-f-Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\) (\(7.77\times 10^{-2}\) MBq \({}^{124}\)I, 0.04 mM Gd\({}_{3}\)N(\(\circled{\rm C}_{80}\)) was infused intratumorally; bright contrast (due to the endohedral Gd) can be seen around the infusion site on the T\({}_{1W}\) image (Figure 6(B,E)).