*3.3. Preparation of <sup>225</sup>Ac-DOTA-Benzene-p-SCN*

DOTA-benzene-p-SCN (pDOTA-Bz-SCN) (1 mg) was dissolved in 50 µL of 0.01 M NaOH, adjusting to a final volume of 2 mL with 1 M acetate buffer (pH 5.0). The <sup>225</sup>Ac progeny in the decay chain have γ emissions. Therefore, to quantify the <sup>225</sup>Ac activity, under secular equilibrium, a CRC-55tR radioisotope calibrator setting was used (Capintec Inc., Mirion Technologies, Florham Park, NJ, USA) (calibration # 775 with a 5× multiplier), which mainly considers the 213Bi 440 keV γ emission [12,31]. To 200 µL of the DOTAbenzene-p-SCN, 50 µL of <sup>225</sup>AcCl<sup>3</sup> (18 MBq in 0.01 M HCl) were added. Finally, the mixture was incubated at 95 ◦C for 30 min. The radiochemical purity of <sup>225</sup>Ac-DOTA-benzenep-SCN was determined using an HPLC reverse phase chromatography system (Waters Corporation, Milford, MA, USA). The separation of the samples was performed with a Waters µBondapak-C18 column at a flow rate of 1 mL/min. A linear gradient of H2O with 0.1% TFA (A)/CH3CN with 0.1% TFA (B), from 100% to 10% of A in 20 min, was used. Fractions of 0.5 mL (40 fractions) were collected and the activity measured in a well-type scintillation NaI(Tl) detector (Auto In-v-tron 4010; NML Inc., Houston, TX, USA). The retention times of <sup>225</sup>AcCl<sup>3</sup> and <sup>225</sup>Ac-DOTA-benzene-p-SCN were 3 min and 12.5 min, respectively. The <sup>225</sup>Ac-DOTA-benzene-p-SCN was obtained with radiochemical purity greater than 99%.
