Page 108 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 5
the in vivo stability and the biodistribution of [111In]SB3 without/with (-/+) PA, the latter by performing both biodistribution and SPECT/MRI studies in mice bearing GRPR-expressing PC-3 tumor xenografts.
Materials and Methods
Radiolabeling
SB3 (Fig. 2) was radiolabeled with 111In (Covidien) as previously described [31]. Small volume reactions were performed in conical vials. Sodium acetate was used to control pH, resulting in a final pH value of 4-4.5 and quenchers (ascorbic- and gentisic acid 3.5 mM and methionine 10 mM as final concentration) were added to prevent radiolysis. A mixture of 1 nmol SB3 and 125 MBq equivalent 111InCl3 and quenchers in a final volume of 140 μL were heated for 15 min at 80°C. To complex non-incorporated 111In, after cooling down to room temperature, 5 μL of 4 mM DTPA was added. To obtain a molar activity of 12.5 MBq/nmol, after the labelling 9 nmoles of SB3 was added. The incorporation was determined by instant thin layer chromatography (ITLC) silica gel using sodium citrate (0.1 M, pH 5) as solvent [32] and radiochemical purity (RPC) was measured by a HPLC system (Breeze, Waters), containing a 2690 quaternary pump, and radioactivity was monitored with a NaI detector, digital multichannel analyzer and dedicated software (MetorX B.V). A Symmetry C18 column (5 μm x 4.6 mm x 250 mm, Waters) was used with a gradient profile of: 0–5 min 100% A, 5-5.01 min 60% B, 5.01–20 min 80% B, 20–20.01 min 100% B, 20.01–25 min 100% B, 25.01–30 min (flow 1 mL/min), where A was 0.1% trifluoroacetic acid (TFA) in H2O and B was LC-MS grade methanol. No further purification was required.
A molar activity of 12.5 MBq/nmol and 125 MBq/nmol was used for the biodistribution and SPECT/MRI studies, respectively. For the in vivo stability studies both molar activities were used. To determine the affinity of SB3 for the human GRPR in a competition-binding assay, Tyr4-Bombesin (Sigma-Aldrich) was radiolabeled with iodine-125 (125I) based on the Iodo Beads® (Pierce) method, adapted with 5 min pre-incubation before addition of the peptide, as previously described [33, 34]. In short, 100 μL of PBS 50 mM Pi pH 7.5, sodium iodide solution (10-20 MBq Na125I in 0.01 M NaOH) and pre-rinsed Iodo Beads® were pre-incubated for 5 min, and Iodo Beads® were removed. Then,
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