Abstract
<title>Abstract</title> <p> Purpose Radiolabeled nanoparticles (NPs) are investigated for preclinical imaging and nano-brachytherapy, where accurate in vivo localization is essential. Here, we present a benchtop 2D multi-pinhole X-ray imaging system for characteristic X-ray imaging using an energy-resolving 2 × 2 cadmium zinc telluride detector. Procedures: Polyethylene glycol (PEG)-coated iodine-125 ( <sup>125</sup> I)-labeled gold (Au) core and silver (Ag) shell NPs (hereafter <sup>125</sup> I-NPs) were used. The multi-pinhole X-ray imaging system was calibrated using <sup>125</sup> I-NPs containing solutions, and the detection limit was estimated. Three CT26 tumor-bearing BALB/c mice received intratumoral injections of 500 µCi <sup>125</sup> I-NPs. In vivo imaging was performed with 10-min scan times at 0, 3, 6, 24, 96, and 120 h post-injection, followed by endpoint ex vivo imaging of the excised tumor and major organs. Results The multi-pinhole X-ray imaging system showed linearity between activity and characteristic X-ray photon counts with R <sup>2</sup> = 0.9992, and the detection limit was 0.05 µCi. In vivo imaging quantified intratumoral retention kinetics, showing a marked decline within 24 h followed by a slower phase through 120 h. Endpoint ex vivo imaging supported high tumor selectivity at 120 h, with 26.41 ± 2.36% of the injected dose and 172.65 ± 15.45% of the injected dose per gram retained in the tumor. The liver and thyroid uptake remained low at 1.17 ± 0.90% of the injected dose and 0.77 ± 0.60% of the injected dose per gram in the liver and 1.92 ± 0.17% of the injected dose and 94.22 ± 8.15% of the injected dose per gram in the thyroid. The difference between in vivo and ex vivo measurements ranged from 0.03 to 3.51 µCi. Conclusions The multi-pinhole X-ray imaging system enables assessment of retention and biodistribution of <sup>125</sup> I-NPs, providing guidance for preclinical optimization when tumor retention and normal tissue safety are key design criteria. </p>