Experiment 3 (n = 5–7/group) was performed to determine whether GF or GF + Lys could affect the specific tumor uptake of 64Cu-cyclam-RAFT-c(-RGDfK-)4 in addition to their effects on the kidneys, using tumor-bearing mice. It should be noted that throughout this study, each injectate was adjusted to a 0.2 mL volume with NS to avoid any possible effect due to the injected volume. At 3 and/or 24 h post-injection (p.i.), check details the mice were sacrificed and their blood was drawn. The kidney, tumor, and other major organs of interest were dissected and weighed, and the radioactivity was measured using a gamma counter with decay correction. Radioactivity concentration was expressed

as a percentage of the injected dose Y-27632 solubility dmso per gram of tissue (%ID/g) normalized to a body weight of 20 g. Tumor-bearing mice (n = 4/group) received an i.v. injection of ∼18.5 MBq 64Cu-cyclam-RAFT-c(-RGDfK-)4 with or without co-injection of 80 mg/kg GF ± 400 mg/kg Lys. Using a small-animal PET system (Inveon; Siemens Medical Solutions USA, Inc., Malvern, PA), dynamic PET imaging for a duration of 60 min (12 scans of 5 min each) was performed immediately p.i., followed by 30-min static imaging

at 3.5 and 24 h p.i. During scanning, the mice in prone position were anaesthetized with 1–1.5% isoflurane, while maintaining normal body temperature. Images were reconstructed using a 3D maximum a posteriori (MAP) method (18 iterations with 16 subsets; β = 0.2) without attenuation

correction. Image analysis was performed using the ASIPro VM™ Micro PET Analysis software (Siemens Medical Solutions, USA, Inc.). The total injected dose was calculated by decay correction of total activity present at the time of injection (t = 0). For radioactivity quantification in the tumor, both kidneys, and urinary bladder, regions of interest (ROIs) encompassing the whole tissue area on each of coronal slices were drawn manually, and all ROIs were linked to form a 3D volume of interest (VOI) using the 3D (VOI) Terminal deoxynucleotidyl transferase dimensionality tool. For each VOI, the percentage of the total injected dose (%ID) was calculated to represent the total activity accumulation in the urinary bladder and both kidneys and the mean %ID/g to represent tumor uptake, assuming a tissue density of 1 g/mL. To quantify the radioactivity in the renal cortex, ROIs encompassing the cortex were drawn from 3 coronal slices, the mean %ID/g of each slice was recorded, and the average value of mean %ID/g from the 3 slices was calculated. To estimate the radioactivity in the blood pool, a ROI with a fixed size of 0.1 cm2 was placed over the heart, and the blood radioactivity was quantified as the mean %ID/g. Normal mice (n = 3/group) were treated with the same injection schedule as in the aforementioned PET study. At 1 and 24 h p.i., the mice were sacrificed and urine, blood, kidney, and liver were sampled.