Radiochemical synthesis of 225Ac-DOTA-c(RGDyK), Quality Control, and in vitro Serum Stability

The complexation of ²²⁵Ac with DOTA-c(RGDyK) was achieved by reacting DOTA-c(RGDyK) (5-10 μg (5-10 μL,1.0 mg/mL in water)) with ²²⁵Ac(NO₃)₃ (3.4 MBq) that was diluted in 100 μL of water containing 10 μL of 20% L-ascorbic acid. The pH of the resulting solution was adjusted to 5.5-6 using 1 M Tris buffer (10-12 μL), and then incubated at 60ºC for 1 h. Reaction progress and radiochemical purity of ²²⁵Ac-DOTA-c(RGDyK) were measured without further purification using ITLC with gamma counting, radio-TLC, gamma counting of radio-HPLC fractions, or CLI. In vitro serum stability was carried out by adding 50 µL of ²²⁵Ac-DOTA-c(RGDyK) (2 MBq) to 900 µL of human serum. The solutions (n = 4) were incubated at 37 °C for 10 days and were analyzed daily using radio-TLC, ITLC with gamma counting or size exclusion chromatography using a Superdex 200 10/300 GL^TM column (GE Healthcare Life Sciences, Piscataway, NJ) and phosphate buffered saline (PBS) as eluent with a flow rate of 0.5 mL/min.